ABSTRACTS
ABSTRACTS OF THE 60TH ANNUAL MEETING OF THE SCIENTIFIC AND
STANDARDIZATION COMMITTEE OF THE INTERNATIONAL SOCIETY
ON THROMBOSIS AND HAEMOSTASIS JUNE 23–26, 2014
Animal, Cellular, and Molecular
Models
ACM01
Early gene expression biomarkers sensitive to a micro
volume of blood in the joint
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Objectives: Hemarthrosis is the hallmark of severe hemophilia A, and
results in synovial hypertrophy and destruction of cartilage and bone,
leading to hemophilic arthropathy and crippling disability. The identification and application of sensitive and specific biochemical markers
associated with joint bleeding would enhance the ability of early detection, providing an opportunity for timely and more effective intervention. The objective of this study was to identify early gene expression
biomarkers for detection of a micro volume of blood in the mouse joint.
Methods: Syngeneic whole blood or saline was microinjected into the
knee joint of F8/ mice pretreated with human recombinant factor
VIII and into wild-type mice. Mice were euthanized and joint tissues
collected 2, 4, 6, and 24 h after injection. Expression of genes in the
joint tissue of mice injected with blood was compared to that of control mice (injected with saline or untreated) using TaqMan real-time
PCR assay. Correlation was analyzed between changes in gene expression and the presence of blood in the joint assessed histologically (HE
stains) for each time point.
Results: The biochemical markers (cxcl1, csf2, csf3, IL-1b, IL6, etc.),
in which we have previously shown significant changes in mouse
plasma and joint tissue 1–3 days after massive hemarthrosis, were now
investigated in mouse joint tissue 2–24 h following intra-articular
injection of a micro volume of blood. Most genes showed an increase
in expression (> 4-fold, P < 0.05) as early as 2 h following injection of
blood compared to joints injected with saline. The maximum expression of these genes varied from 2 to 6 h after injection of blood and
decreased nearly to the control level 24 h following blood injection.
Conclusion: This study identified a number of genes significantly upregulated in response to a minimal amount of blood in the mouse joint
and verified the specificity of candidate markers by comparison with
presence of blood in joint tissue.
Disclosure of Interest: N. Hakobyan: none declared, L. Cong: none
declared, X. Song: none declared, C. Enockson: none declared, J.
O’Brien: none declared, L. Valentino: since the preparation of this
abstract, L.Valentino became an employee of Baxter Bioscience.
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Hakobyan N, Cong L, Song X, Enockson C, O’Brien J and
Valentino LA
Pediatrics, Rush University Medical Center, Chicago, IL, USA
specific receptors on phagocytes. In this study we found that urokinase
plasminogen activator receptor (uPAR) plays an important role in
internalization of apoptotic cells, and also characterized the underlying mechanisms.
Methods: The uPAR/ and wild-type mice were challenged by injection of NBD-labeled PS beads and apoptotic cells. The phenotype of
efferocytosis was observed by using in vitro and in vivo phagocytosis
assay. Binding of HK to liposomes was analyzed using Western blot.
Results: In a flow cytometry–based phagocytic assay, uPAR-deficient
macrophages displayed significant defect in internalization but not tethering of apoptotic cells. When uPAR/ mice were challenged with
apoptotic cells, they exhibited pronounced splenomegaly resulting from
accumulation of abundant apoptotic cells in spleen. Overexpression of
uPAR in HEK-293 cells enhanced efferocytosis, which was inhibited by
annexin V and phosphatidylserine liposome, suggesting that uPARmediated efferocytosis is dependent on PS. In serum lacking high
molecular weight kininogen (HK), a uPAR ligand, uPAR-mediated efferocytosis was significantly attenuated, which was rescued by replenishment of HK. As detected by flow cytometry, HK selectively bound
to apoptotic cells but not viable cells. In purified systems, HK was specifically associated with PS liposome. HK binding to apoptotic cells
induced its rapid cleavage to two-chain HKa and bradykinin. Both
heavy chain and light chain HKa were associated with PS liposome and
apoptotic cells. HKa has higher binding affinity than HK to uPAR.
Overexpression of Rac1/N17 cDNA inhibited uPAR-mediated efferocytosis. HK plus PS liposome stimulated a complex formation of CrkII
with p130Cas and Dock-180 and Rac1 activation in uPAR-293 cells.
Conclusion: HK opsonizes uPAR-mediated efferocytosis, suggesting a
novel function of contact activation system.
Disclosure of Interest: None declared.
ACM02
High molecular weight kininogen binds phosphatidylserine and opsonizes urokinase plasminogen activator
receptor–mediated efferocytosis
Xie Z1, Yang A1, Colman RW2 and Wu Y2
1
Cyrus Tang Hematology Center, Soochow University, Suzhou,
China, Suzhou, China; 2The Sol Sherry Thrombosis Research Center,
Temple University School of Medicine, Philadelphia, PA, USA
Objectives: Phagocytosis of apoptotic cells is essential for the regulation of immune responses and tissue homeostasis and is mediated by
ACM03
Thrombin-induced podocyte injury is protease activated receptor (PAR)-3 and -4 dependent
Sharma R1,2, Waller A2, Guess AJ2, Agrawal S2, Isermann B3,
Smoyer WE2, Nieman MT4 and Kerlin BA2
1
Hematology/Oncology and BMT, Nationwide Childrens
Hospital; 2Center for Clinical and Translational Research, The
Research Institute at Nationwide Children’s Hospital, Columbus,
USA; 3Clinical Chemistry and Pathobiochemistry, Otto-vonGuericke University, Magdeburg, Germany; 4Pharmacology, Case
Western Reserve University, Cleveland, USA
Objectives: Nephrotic syndrome (NS), the most common form of glomerular disease, is associated with a severe complex hypercoagulopathy and increased endogenous thrombin potential, which may
contribute to progressive glomerular injury. This is suggested by studies showing that thrombin may injure podocytes in vitro. The molecular mechanisms by which thrombin induces podocyte injury are not
yet known. Thrombin is known to signal through protease activated
receptors (PARs) in other cell types. Thus, we hypothesized that
thrombin exacerbates glomerular injury by enhancing podocyte apoptosis in a PAR-dependent manner.
Methods: Experiments were performed with differentiated, conditionally immortalized human podocytes. Podocyte apoptosis was determined after 36 h of thrombin (20 nmol L–1) exposure by TUNEL
assay. Specific PAR antibodies were utilized to determine which PARs
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
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ABSTRACTS
mediate thrombin-induced podocyte apoptosis. One-way ANOVA and
t-tests were used to determine statistical significance (SigmaPlotTM).
Results: Thrombin exposure induces an increase in apoptosis
(P < 0.05). Blockade of PAR-3 or PAR-4 decreased apoptosis by
approximately 30% (P < 0.05). However, blockade of PAR-1 or PAR2 did not significantly alter apoptosis. Inhibition of thrombin enzymatic
activity with hirudin significantly decreased apoptosis (P < 0.05).
Results: There was a significant correlation between proteinuria severity and
clinically relevant markers such as ETP and clot formation time and firmness. Importantly, plasma [TAT] did not change with worsening proteinuria,
suggesting that massive proteinuria alone is not sufficient to activate coagulation in vivo. In contrast, when an active thrombotic episode was induced by
IVC ligation, proteinuric rats exhibited significantly higher plasma [TAT],
which ultimately translated into greater thrombus formation (Fig 1).
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Conclusion: Thrombin-induced podocyte injury is mediated in a PARdependent fashion. Specifically in this in vitro model, PAR-3 and
PAR-4 appear to mediate thrombin induced podocyte injury. It is not
yet known if this finding is mediated in a manner dependent on PAR3/ 4 heterodimerization, which would be a novel mechanism not previously described in human cells. Interrupting thrombin-mediated podocyte injury may provide a novel therapeutic approach for NS.
Disclosure of Interest: None declared.
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ACM04
Proteinuria severity is directly correlated with ex vivo
and in vivo markers of thrombotic potential in an
experimental model of nephrotic syndrome
Waller AP1, Sharma R1, Smoyer WE1, Chanley MA1, Nieman MT2
and Kerlin BA1
1
Center for Clinical and Translational Research, Nationwide
Children’s Hospital, Columbus; 2Pharmacology, Case Western
Reserve University, Cleveland, USA
Objectives/Introduction: Nephrotic syndrome (NS) is characterized by
glomerular injury and massive urine protein loss, which leads to severe
blood coagulation derangements and a high prevalence of life-threatening thrombotic complications. Unfortunately, the net effect of proteinuria on thrombotic potential remains unknown; thus, proteinuria
as a biomarker lacks appropriate validation. We hypothesized that
proteinuria severity is directly correlated with hypercoagulopathy.
Methods: Using the puromycin aminonucleoside (PAN)-induced
nephrosis model, we compared established markers of thrombin generation (ETP) and global hemostasis (ROTEM) in blood collected from
the inferior vena cava (IVC) of anesthetized male Wistar rats exhibiting a range of proteinuria levels (morning spot urines analyzed for
[protein:creatinine]).To further delineate the in vivo significance of the
relationship between proteinuria severity and thrombin activation,
IVC ligation was performed in NS and sham rats (6/group), with subsequent analysis of inducible thrombus formation (weight) and plasma
thrombin/antithrombin complex (TAT; ELISA).
Conclusion: Proteinuria severity is directly proportional to hypercoagulability as assessed by both ex vivo thrombin generation and global hemostasis and by an in vivo thrombosis model. The physiologic relevance
of this relationship provides key evidence for the biologic relevance of
proteinuria as an easily measured biomarker for thrombotic risk in NS.
Disclosure of Interest: None declared.
ACM05
Tissue factor–expressing tumor cells can bind to tissue
factor pathway inhibitor under static and shear conditions in vitro
Che S, DeLeonardis C, Shuler M and Stokol T
Cornell University, Ithaca, USA
Objectives: Many epithelial tumors overexpress tissue factor (TF),
which is associated with tumor progression and metastasis. Endothelial cells constitutively express tissue factor pathway inhibitor (TFPI),
which inhibits TF-initiated coagulation by binding to factor VIIa
(FVIIa) and/or FXa complexed with TF. We hypothesized that
tumor-expressed TF can mediate tumor adhesion to endothelial cells
via interactions with TFPI.
Methods: High (MDA-MB-231) and low (MCF-7) TF-expressing
breast cancer cells were pretreated with FVIIa (1–100 nmol L–1) and/
or FX (10 or 150 nmol L–1) before incubation with recombinant TFPI
under static and shear conditions in microfabricated wells and microfluidic channels, respectively. The numbers of adherent cells were
counted using phase contrast microscopy.
Results: We found that MDA-MB-231, but not MCF-7, cells bound to
recombinant TFPI (50 lg mL–1) and that binding was abolished with
an anti-TF antibody. Binding was dependent on the FVIIa concentration and shear, with no binding being observed beyond a shear rate of
1.0 dyn cm–2.
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© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
ABSTRACTS
Conclusion: We show, for the first time, that TF-expressing tumor cells
can be captured by purified TFPI, a ligand constitutively expressed on
the endothelium (without a priori activation) under low shear. Our results
suggest that TFPI could be a novel ligand that helps mediate the arrest
of high TF-expressing tumor cells in the vasculature during metastasis.
Disclosure of Interest: None declared.
Conclusion: This study provided evidence in platelets at static or flow
condition that two calpain-cleavage relevant sequences integrin b3
T755NITYRGT762 and R760GT762 were critical to bidirectional signaling and outside-in signaling, respectively, consistent with the previous observation in CHO cell model.
Disclosure of Interest: None declared.
ACM06
Effect of truncations on integrin b3 mimicking calpain
cleavage on integrin signal transduction in platelets of
b3-transgenic mice
ACM07
The effect of UC-MSC homing on the immune inflammatory microenvironment and targeted improvement
of prothrombotic state
Shi X, Cui X, Huang J, Yang J, Liu P, Zhou Y, Ruan Z, Long Z and
Xi X
Shanghai Institute of Hematology, Ruijin Hospital, Shanghai
Jiaotong University, School of Medicine, Shanghai, China
Yin J, Gu W, Lin C, Ren M, Cai X, Ni J, Wu W and Gu J
Hematology Department, Clinical Medical College of Yangzhou
University; Yangzhou Institute of Hematology, Yangzhou, China
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Objectives: We transplanted umbilical cord mesenchymal stem cells
(UC-MSCs) to collagen typeII–induced arthritis rats (CIA rats) and
then preliminarily implored the effect of UC-MSCs transplantation on
inflammatory microenvironment and the adjustment of immune
related prothrombotic state in CIA rats.
Methods: One hundred twenty rats were randomly divided into a control (D) group, the CIA model (M) group, a superparamagnetic iron
oxide nanoparticles (SPION)-labeled UC-MSC treatment (SU) group,
and an AMD3100-blocking SPION-labeled UC-MSC treatment
(ASU) group. The number of UC-MSCs in the involved joint were
analyzed by magnetic resonance imaging (MRI). Inflammatory factors
and thrombosis indexes were investigated by ELISA in serum, and
those in joints were examined by Western blot.
Results: The labeled UC-MSCs gathered in inflammatory joints of the
CIA rats but not in other tissues and the ASU group. Compared with
group D, 7 days after transplantation, SDF-1 significantly increased
with the number of UC-MSCs in the inflammatory knee joints of
group SU. With time, the number of MSCs decreased with SDF-1 in
the inflammatory joints of group SU. In group M, IL-6, TNF alpha,
and IFN-gamma increased significantly in serum and knee joint while
anti-inflammatory factor IL-10 obviously decreased. One week after
transplantation, IL-6, TNF alpha, and IFN-gamma were significantly
lower in group SU than in group M. However, IL-10 increased in
group SU, compared with group M. Similar changes were also
observed in thrombotic indexesTF, D-dimer, and VWF.
Conclusion: Local inflammatory microenvironment attracted UCMSCs specifically homing to injured tissue. SDF-1/CXCR4 facilitated
UC-MSC homing, and its specific blocker, AMD3100, interrupted
UC-MSCs homing to the inflammatory part. UC-MSCs transplantation could improve the inflammatory microenvironment and immunerelated prothrombotic state of CIA rats, and further treated CIA rats.
Disclosure of Interest: None declared.
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Objectives: Our previous study in CHO cell model showed the calpain
cleavage of b3 cytoplasmic domain at sites of F754 and Y759 regulates
the integrin signaling. However, whether this regulation mechanism
exists in platelets needs to be determined. This study aims to observe
the effect of truncations at the two sites on integrin signal transduction
in platelets through transgenic mice.
Methods: Transgenic mice with b3 truncations at sites of F754 and
Y759 (b3-D754, b3-D759) were established through transplanting b3
deficient mouse bone marrow cells infected by MSCV MigR1 retrovirus
containing different b3 truncation mutants into lethally radiated wildtype mice. The platelets were harvested for the function assays including
soluble fibrinogen binding, the event typical for inside-out signaling,
and platelet spreading, for outside-in signaling. Platelet adhesion to
fibrinogen or collagen-coated surface under flow also was tested to
assess the ability of the platelets to resist shear force.
Results: A complete defect in soluble fibrinogen binding was observed
in b3-D754 platelets, while the capability of b3-D759 platelets to bind
soluble fibrinogen remained almost unaffected, similar to wild-type b3
(Fig. 1 A). A nearly complete defect in spreading was present in b3D754 platelets, as well as in b3-D759 platelets (Fig. 1B). At shear rates
of 125 s, the adhesion on the fibrinogen-coated surface was completely
defective in b3-D754 platelets but partially reduced in b3-D759 platelets
(Fig. 1C), while at high shear rates of 1500 s, the adhesion on the collagen-coated matrix was significantly impaired in both of them (Fig. 1D).
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ACM08
Spatial dynamics of fibrin clot growth investigation:
measurement of frozen plasma samples and evaluation
of the assay sensitivity to the different concentration
of trisodium citrate added during blood collection
Korotina N1, Sokolova L1, Panteleev M1,2,3,4,5 and Ataullakhanov
F1,2,3,4,5
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HemaCore LLC; 2Center for Theoretical Problems of PhysicoChemical Pharmacology, RAS; 3National Research Center for
Hematology, Health Ministry RF; 4Federal Research and Clinical
Center of Pediatric Hematology, Oncology and Immunology;
5
Lomonosov Moscow State University, Moscow, Russian Federation
Objectives: Parameters of spatial fibrin clot growth―thrombodynamics―for frozen samples were measured with aim to study intra- and
extra-group differences.
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
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ABSTRACTS
redness and swelling aggravated. Skin ulcerates and joint obstacles
with weight were seen in the serious state. One week after the CIA rats
model were made, levels of serum inflammatory factors IL-6, TNF
alpha, IFN-gamma increased significantly, while anti-inflammatory
factor IL-10 significantly decreased, compared with control group. TF
and VWF were significantly higher in CIA rats group than those in
control group.
Disclosure of Interest: None declared.
ACM10
Inferior vena cava (IVC) branch variations in C57BL/6
mice impact thrombus size in IVC ligation model
Diaz JA, Farris D, Wrobleski S, Myers D and Wakefield T
Surgery, Vascular Surgery, University of Michigan, Ann Arbor,
USA
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ACM09
The relation of inflammatory factors and immunerelated prothrombotic state in CIA rats
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Gu J1, Ren M2, Gu W3, Qiu H2, Li Z2, Cai X3, Wang Y2 and Sun X2
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Yangzhou Institute of Hematology; 2Medical College of
Yangzhou University, Yangzhou; 3Xuzhou Medical College,
Xuzhou, China
Objectives: There are limitations to studying venous thrombosis (VT)
in humans. Therefore, animal models of VT are key tools for investigators in understanding the VT mechanism. Recently, IVC branches
have been subject to debate, causing controversy in the field. The
objective of this work is to understand, for the first time, how the variability of IVC branches in commonly used C57BL/6 impact thrombus
formation.
Methods: C57BL/6 male mice (n = 96), 20–25 g, were subjected to a
laparotomy, the infrarenal IVC was visualized and photographed, and
the IVC ligation model was performed including various interruptions
of the IVC branches (Table 1). Two days after IVC ligation, thrombus
weight (TW), as a parameter of thrombus size, was assessed. For controls, the IVC ligation model was used, with all back and side branches
interrupted.
Results: We found four different anatomical patterns (Table 1). Back
branches were the most consistent vessels. Two back branches were
present in 98% of the mice. Side branches were more prevalent on the
mouse’s right side (34%), compared to the left (20%). In mice where
side branches were absent (21%), back branches appeared larger.
Also, 25% of mice had both side branches. Complete ligation of all
branches, controls, had the most consistent and largest TW
(0.0334 g 0.0008 g), while groups that had no branches interrupted
had the smallest TW (0.0036 0.0004 g), a 9-fold decrease. All
groups with open back branches had smaller TW (P < 0.05) than controls. Results are shown in Table 1
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Both 3.2% and 3.8% trisodium citrate as available in routine blood
collection were tested regarding influence on spatial fibrin clot growth
parameters in a specified range of calcium ion concentration.
Methods: Recalcified platelet-free plasma (frozen or fresh) samples
were put in contact with the surface-immobilized tissue factor. Light
scattering changes due to fibrin clot growth were processed by the
thrombodynamics analytical system.
Results: A series of measurements on four frozen plasma pools (not
less than four healthy donors each) were performed using the standard
thrombodynamics procedure (not less than n = 120 measurements for
each pool). Statistically significant difference of parameters for different pools was shown (Kruskal–Wallis, P = 0.05). The coefficient of
variation for the parameters measured did not exceed 6% within the
pool series. To investigate the sensitivity of the assay to the anticoagulant (3.2% or 3.8% trisodium citrate) added into the tube during the
blood collection, there were two titrations of calcium ions done (10
and 50 mmol L–1 added calcium chloride, each concentration quarter
measured) for samples prepared on 3.2% or 3.8% trisodium citrate anticoagulated blood. There were no statistically significant difference of
measured parameters observed for standard calcium ions concentration range used in thrombodynamics (Kruskal–Wallis, P = 0.05; ANOVA, P = 0.05).
Conclusion: Thrombodynamics parameters for different frozen plasma
pools samples statistically differ from each other but have good reproducibility characteristics within the pool. Thus, it is necessary to use
one’s own range of feasible values as far as reliable difference of the
values measured for different samples series was observed. The standard thrombodynamics procedure allows both 3.2% and 3.8% of trisodium citrate anticoagulated blood use.
Disclosure of Interest: N. Korotina is an employee of HemaCore LLC,
L. Sokolova is an employee of HemaCore LLC, M. Panteleev is an
employee of HemaCore LLC, F. Ataullakhanov is an employee of HemaCore LLC.
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Objectives: Rheumatoid arthritis (RA) is systemic autoimmune disease
characterized by immune inflammatory damage of small peripheral
joints. Much data have shown the risk of vein thrombosis in RA is on
the rise. Type II collagen–induced arthritis (CIA) rats have similarities
in pathogenesis with humans with RA. We investigated the inflammatory factor and immune-related prothrombotic state in CIA rats.
Methods: Forty-eight Sprague –Dawley (SD) rats were randomly
divided into the CIA rat group and the control group. Inflammatory
factors IL-10, IL-6, TNF-alpha, and IFN-gamma and thrombus
indexes TF and von Willebrand factor (VWF) were detected by ELISA
at 1, 3, and 5 weeks after creation of the CIA rat model.
Results: All CIA rats manifested decreased activity and slow weight
growth. Two days after the first immunization, swelling and ecchymosis in ankle and toe of rats were observed. Six days later, redness and
swelling aggravated. Skin ulcerates and joint obstacles with weight
were seen in the serious state. One week after the CIA rat model were
made, levels of serum inflammatory factors IL-6, TNF-alpha, and
IFN-gamma increased significantly, while anti-inflammatory factor
IL-10 significantly decreased, compared with control group. TF and
VWF were significantly higher in the CIA rats than those in the control group.
Conclusion: All CIA rats manifested decreased activity and, slow
weight growth. Two days after the first immunization, swelling and
ecchymosis in ankle and toe of rats were observed. Six days later,
Conclusion: Using the IVC ligation model, variations in TW was
observed, for the first time, based on different branch interruption patterns, compared to the fully ligated controls. Two back branches had
the most consistent anatomy and open back branches had the largest
negative impact on thrombus size. This work supports that the IVC
branches significantly affect thrombus burden in C57BL/6 mice, and
further studies should be conducted in order to standardize this and
other animal models of VT.
Disclosure of Interest: None declared.
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© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
ABSTRACTS
Results: In comparison to saline control (6.8 1.1 s), apixaban
(300 lg kg–1) prolonged bleeding time (17.1 5.6; P = 0.024). Beriplex at doses ≤ 10 U kg–1 did not shorten bleeding time, while 10 U
kg–1 Profilnine did. FEIBA prolonged bleeding beyond that seen with
apixaban alone (22.6 11.4 and 37.5 5.2 min for 5 and 10 U kg–1
doses; P < 0.001 apixaban + 10 U kg–1 FEIBA vs. apixaban). 100 and
300 lg kg–1 rivaroxaban prolonged bleeding time (20.8 2.5,
P = 0.002 vs. saline; 25.0 10.0 min, P < 0.001 vs. saline). Profilnine
was more effective than Beriplex or FEIBA in reversing rivaroxabaninduced bleeding. Dabigatran-induced bleeding was prevented by Beriplex or Profilnine but enhanced by FEIBA (50.3 8.3 vs.
15.9 1.2 min; P < 0.001). Treatment of apixaban- or rivaroxabananticoagulated rats with FEIBA + 10 mg kg–1 epsilon aminocaproic
acid, produced bleeding times comparable to those of non-anticoagulated rats (apixaban: 6.7 5.0 min; rivaroxaban: 7.7 6.4 min). Ex
vivo analysis of blood samples did not show any relevance with the
observed bleeding or its modulation.
Conclusion: PCCs appear useful for neutralizing bleeding induced by
direct factor Xa and thrombin inhibitors, but each PCC exhibits a distinct neutralization profile. Co-administration of a fibrinolytic inhibitor may enhance effectiveness of hemorrhage reversal by PCCs.
Disclosure of Interest: None declared.
ACM11
Organ-specific tissue factor activity is significantly
altered in experimental malaria
Moore J, Bracken TC, Sarr D, Doyle A, Ghobrial M, Jenkinson T
and on behalf of Vascular Biology
Infectious Diseases, University of Georgia, Athens, USA
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ACM13
Equid herpes virus type-1 activates platelets through
the procoagulant effects of tissue factor
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Stokol T1, Yeo WM1, Burnett D2 and Catalfmo JL1
1
Population Medicine and Diagnostic Science; 2Population
Medicine and Diagnostic Sciences, Cornell University, Ithaca,
USA
Objectives: Equid herpes virus type I (EHV-1) is a major infectious
pathogen of horses, causing outbreaks of abortion and myeloencephalopathy worldwide. These two clinical syndromes syndromes are
caused by thrombosis in placental and spinal vessels. The mechanisms
by which EHV-1 causes thrombosis in some infected horses are
unknown. We hypothesized that EHV-1 binds to and activates equine
platelets.
Methods: Platelet-rich plasma (PRP) was derived by low-speed centrifugation of platelet-leukocyte–rich plasma obtained from citrate-anticoagulant equine blood. PRP was incubated for 10 min with two
EHV-1 strains, abortifacient RacL11 and neuropathogenic Ab4, at
various multiplicities of infection (MOI). Virus was purified from rabbit kidney (RK) cell lysates by sucrose gradient centrifugation. Thrombin and convulxin-stimulated platelets and vehicle or RK lysatetreated platelets were used as positive and negative controls, respectively. P selectin expression and platelet-derived microparticle release
were used as markers of platelet activation and were quantified by flow
cytometry.
Results: Both EHV-1 strains induced P selectin expression and platelet
microvesiculation in an MOI-dependent manner. P selectin expression
required exogenous calcium and was inhibited by hirudin. Platelet activation was abolished in human factor VII–deficient plasma and was
restored by addition of 1 nmol L–1 recombinant human factor VIIa.
EHV-1 glycoprotein B DNA was amplified from platelets and platelets
could transfer virus to RK cells. Glycoprotein B was also amplified
from platelets isolated from horses after experimental in vivo infection
of EHV-1.
Conclusion: EHV-1 activates equine platelets through tissue factor–
mediated thrombin generation. Activation is likely mediated through
direct binding of EHV-1 to platelets. Platelets may become activated
after binding EHV-1 in vivo during viremia, which could contribute to
pathological thrombus formation in some infected horses.
Disclosure of Interest: None declared.
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Objectives: Severe malaria is associated with sequestration of Plasmodium falciparum–infected erythrocytes in the brain, causing cerebral
malaria (CM), or in the placenta (PM). Extensive fibrin deposition
and tissue factor (TF) expression in affected tissues characterize both
CM and PM, but the extent to which dysregulated hemostasis drives
pathogenesis remains incompletely understood.
Methods: C57BL/6J mice were infected with 106 P. berghei ANKA, a
species capable of inducing CM, or 106 P. chabaudi AS, a non-CM
strain, and serially sacrificed from days 3 to 6 postinfection. Mice were
monitored for neurological symptoms, including seizures, tremor, and
impaired motor skills. TF activity was assessed in homogenized brain,
liver, and lung samples from these mice using a modified one-step clotting assay.
Results: TF activity was significantly higher in brain (> 1400-fold;
P < 0.01) and liver (> 5-fold; P < 0.01), but not lung, of mice with
CM relative to uncomplicated malaria cases. In mice sacrificed prior
to the onset of CM, these organs tended to have reduced TF activity
relative to uninfected mice, most notably in the brain (50-fold
decrease). Concurrently, profound reductions in TF activity were
observed in brain (> 360-fold, P = 0.0571), lung (> 1200-fold,
P = 0.0159), and liver (> 4400-fold, P = 0.0571) with P. chabaudi AS
infection.
Conclusion: Increased TF activity in the brains of mice that succumb
to CM suggests a significant role for coagulation in the pathogenesis
of experimental CM. The biological significance of reduced TF activity
during early, ascending infection with both parasite strains remains to
be determined. Ongoing experiments with TF-floxed/cre mice seek to
elucidate the critical cellular sources of TF in P. berghei ANKAinduced CM and P. chabaudi AS-induced PM. The importance of the
proinflammatory cytokine tumor necrosis factor (TNF) in malariaassociated TF activity is being assessed with TNF and TNF receptor
null mutant mice.
Disclosure of Interest: None declared.
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ACM12
Neutralization of hemorrhage induced by direct factor
Xa and thrombin inhibitors by prothrombin complex
concentrates in a rat model
Jeske W1, Escalante V1, McGeehan E2, Walenga JM1, Kalodiki E3,
Fareed J2 and Bakhos M1
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Thoracic and Cardiovascular Surgery; 2Pathology, Loyola
University Medical Center, Maywood, USA; 3Imperial College,
London, UK
Objectives: Concerns remain over bleeding with direct factor Xa and
thrombin inhibitors in cases of overdose or medical emergency. This
study tested the ability of PCCs to reverse bleeding induced by apixaban, rivaroxaban, or dabigatran in a standardized rat tail resection
model.
Methods: Individual groups of anesthetized rats (8–10) were anticoagulated with apixaban, rivaroxaban, or dabigatran and subsequently
treated with vehicle, three-factor (Profilnine), four-factor (Beriplex), or
activated four-factor PCC (FEIBA). Five minutes post-PCC administration, a tail snip was performed and bleeding time was measured.
Upon cessation of bleeding, blood samples were collected for ex vivo
analysis and the rat was humanely euthanized.
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© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
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ABSTRACTS
ACM14
Development of cell lines producing fully active recombinant vitamin K–dependent (VKD) clotting factors
without vitamin K epoxide reductase (VKOR) coexpression
Methods: To comply with the WHO guideline, candidate material of
national standard was produced about 7300 vials from final bulk product of human tetanus immunoglobulin by Green Cross. Manufacturing
process, safety tests, and the analysis of candidate characteristics were
examined and confirmed to meet the acceptance criteria. Then, collaborative study of three laboratories including KFDA was designed to estimate and assign the potency of candidate material. All laboratories
executed potency tests according to the in vivo toxin neutralization test of
Minimal Requirements of Biologics by comparing candidate material
with the first international standard of human tetanus immunoglobulin.
Results: We analyzed the variability and proficiency of experimental
results statistically, to show that there was no significant difference in
inter- and intra-laboratory variability. In addition, the robust Z-score
of all laboratories did not exceed 1, so proficiency was sufficient to
guarantee the consistency of collaborative study. Geometrical mean of
estimates from 49 tests was 32.74 IU vial–1. Assigning it as an official
potency, the candidate material was listed in the Korean National Biological Standard Reference Materials.
Conclusion: Based on the results from this collaborative study, the registered national standard of human tetanus immunoglobulin provides
a stable supplement and reliability for the relevant manufacturers’
quality control and national lot release test.
Disclosure of Interest: None declared.
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Biorheology
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ACM15
Establishment of anti-tetanus human immunoglobulin
national standard for potency test
Kang YS1, Ryu DH1, Kim J-H1, Choi KS2, Nam K1, Lee YK3, Oh
HJ4, Ahn C-Y1 and Ban SJ5
1
Blood Products Division; 2Cell and Gene Therapy Products
Division; 3Biologics Division; 4National Lot Release Center;
5
Biologics Research Division, NiFDS, Ministry of Food and Drug
Safety, Cheongwon-gun, Korea
Objectives: The World Health Organization (WHO) has manufactured
and established a global biological standard via an international collaborative study and recommends that national authorities establish
their own reference standard, which is calibrated with international
standard. We planned to establish a national standard for anti-tetanus
human immunoglobulin, which is administered for the prophylaxis or
treatment of tetanus.
or
BR01
Platelets from premature neonates have increased
platelet affinity for von Willebrand factor (VWF) under
arterial shear compared to platelets from term neonates
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Objectives: Vitamin K–dependent (VKD) proteins, including prothrombin, factor VII, factor IX, factor X, protein C, protein Z, and
protein S, play a central role in blood coagulation. Complex posttranslational modifications such as c-carboxylation of glutamates and
glycosylation are essential for these proteins to function. For the treatment of blood disorders, recombinant VKD coagulation factors are
produced in mammalian cell lines. Co-expression of vitamin K epoxide
reductase (VKOR), an enzyme involved in c-carboxylation, supports
the cellular protein processing machinery. We assessed whether it is
feasible to express fully active VKD proteins in the absence of VKOR
in mammalian cell lines.
Methods: Two VKD coagulation proteins were expressed in Chinese
hamster ovary (CHO) cells with and without VKOR co-expression.
Protein function was determined using functional activity assays standardized to plasma-derived protein analogues. Single cell–derived
clones were screened according to cell-specific productivity, specific
activity, and electrophoresis pattern.
Results: Analytical assays were established to detect impaired c-carboxylation by functional and antigen assays as well as Western blotting. Characterization of cell lines revealed a clone-specific relationship
between high activity and reduced cell specific productivity. Insufficient
c-carboxylation and activation were identified as root causes for this
effect. No distinct differences in productivity and activity of the two
different coagulation factors were detected in clones either with or
without VKOR co-expression. Protein activity also depended on environmental factors such as nutrient limitation at higher cell densities,
regardless of whether VKOR was co-expressed or not.
Conclusion: Clone-specific properties appeared to be more relevant
than the beneficial effects of VKOR co-expression in yielding active
protein. Co-expression of VKOR was not essential to retrieve full
activity.
Disclosure of Interest: F. Horling is an employee of Baxter Innovations
GmbH, E. B€
ohm is an employee of Baxter Innovations GmbH, S.
Knappe is an employee of Baxter Innovations GmbH, J. Koehn is an
employee of Baxter Innovations GmbH, J. Lengler is an employee of
Baxter Innovations GmbH, S. Till is an employee of Baxter Innovations GmbH, F. Scheiflinger is an employee of Baxter Innovations
GmbH, M. Dockal is an employee of Baxter Innovations GmbH.
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€hm E, Knappe S, Koehn J, Lengler J, Till S,
Horling F, Bo
Scheiflinger F and Dockal M
Baxter Innovations GmbH, Vienna, Austria
Cowman J1, Dunne E1, Quinn N2, Geoghegan S2, Molloy E2,
Ricco AJ1 and Kenny D1
1
Biomedical Diagnostics Institute, Royal College of Surgeons in
Ireland; 2Department of Neonatology, National Maternity
Hospital, Holles Street, Dublin, Ireland
Objectives: It is unclear if platelet function is different between premature and full-term infants. This is in part due to the fact that only small
volumes of blood are available. We sought to characterise platelet
behaviour using a novel flow-based assay that quantifies platelet function in lL volumes of blood under arterial shear
Methods: Blood from premature infants < 30 weeks gestation
(N = 13) and full term infants (N = 13) was pumped through custom
made parallel plate flow chambers with channel dimensions of 50 lm
height and 2 mm width coated with von Willebrand factor (VWF)
under arterial shear (1500 s). Platelet adhesion events such as platelet
rolling and translocation (stop/start motion of the platelet) were
recorded. Customized platelet tracking software, which can accurately
track individual platelet movement from frame to frame (30 frames
s–1) was used to measure platelet behaviour.
Results: There was a significant increase in the number of platelets that
interacted with VWF in premature compared to full-term infants
(339 21 vs. 240 16 SEM, P = 0.004). Higher numbers of platelets
from premature infants translocated on VWF (232 14 vs. 157 9
SEM, P ≤ 0.0001) and platelet translocation distances (7.1 0.3 vs.
6.3 0.2 lm SEM, P = 0.0425) were greater. Since our results showed
a significant increase in the number of platelets interacting with VWF in
premature vs. term infants we therefore examined glycoprotein Ib
(GPIb) expression on platelets from these two groups. Premature
infants (N = 6) had higher GPIb receptor counts compared to our full
term (N = 5) cohort (48,805 1433 vs. 38,598 2109 SEM
P = 0.0026).
Conclusion: Our modified parallel plate chambers allow us to monitor
platelet behaviour with < 100 lL of blood. Our results demonstrate a
12/06/2014
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
ABSTRACTS
significant increase in platelet interaction with VWF under arterial
shear in preterm infants compared to term infants and this interaction
is associated with an increase in expression of GPIb.
Disclosure of Interest: None declared.
BR02
Simultaneous measurement of thrombin generation
and fibrin formation in plasma and whole blood applying continuous flow
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Objectives: Thrombin is the key enzyme converting fibrinogen into
fibrin. Assays measuring thrombin generation (TG) and fibrin formation (FF) are often used separately. Factor XIIa (FXIIa) has been
shown to strengthen the fibrin network independently of thrombin,
illustrating the need to simultaneously detect TG and FF. In vivo a clot
must resist the shear stress at the injury. Increasing shear rates augment platelet adhesion, but decrease fibrin deposition. Therefore, we
aimed to develop an assay that simultaneously measures TG and FF,
while applying venous/arterial flow conditions.
Methods: We have redesigned an air-bearing rheometer, to measure
FF based on changes in viscosity. Resulting variables include lag time,
maximum clot strength, and time to maximum clot strength. Introduction of a fluorescent camera allows the measurement of TG based on
the conversion of a thrombin-sensitive substrate, as in the Calibrated
Automated Thrombogram (CAT) assay. The rheometer enables application of laminar flow rates ranging from 100 to 1200 s–1.
Results: For all variables related to FF and TG, the intra-/inter-assay
variations were below 10% in PPP, PRP, and whole blood. Increasing
flow rates resulted in reduced endogenous thrombin potential (ETP)
and peak values. Accordingly, maximum fibrin clot strength was inversely related to increased flow rates. In defibrinated plasma, an increase
in fibrinogen was accompanied with a dose-dependent elevated ETP
and maximum clot strength. We confirmed that an increase in FXIIa
enhances the clot strength without influencing TG. Our system allowed
us to study fibrinolysis using tissue plasminogen activator. We clinically validated the system in patients undergoing cardiothoracic surgery and compared it with existing methods including CAT and
rotational thromboelastometry.
Conclusion: We have developed and clinically validated a new method
capable of measuring TG, FF, and fibrinolysis in plasma/whole blood
under flow, bringing coagulation experiments one step closer to physiology.
Disclosure of Interest: None declared.
the anticoagulant activity of FXa inhibitors during epiodes of serious
uncontrolled bleeding or before urgent/emergent surgery. AnXa is catalytically inactive but retains high-affinity binding to direct FXa inhibitors as well as ATIII-dependent inhibitors such as enoxaparin. Here
we report the first demonstration in healthy subjects that AnXa can
rapidly reverse enoxaparin-induced anti-FXa activity and restore normal hemostatic mechanisms.
Methods: Enoxaparin was administered at 40 mg SQ QD for 6 days to
steady state and AnXa or vehicle was administered as an IV bolus
(30 mg min–1) utilizing a 6:3 ratio of AnXa to placebo-treated subjects.
Results: From two dose cohorts (210 and 420 mg), mean anti-FXa
activity at 3 h after the last enoxaparin dose (~ Cmax: just prior to
AnXa administration) on day 6 was 0.36 0.08 IU mL–1
(mean SD). Immediately after bolus administration (2 min), antiFXa activity was reduced to 0.12–0.13 IU mL–1 (below the established
enoxaparin therapeutic anticoagulation threshold) and inhibition of
thrombin generation was reversed to normal baseline levels. The
nearly complete reversal of anti-FXa activity and restoration of normal thrombin generation was maintained for 2–3 h after bolus administration of AnXa. AnXa was well tolerated (no thrombotic events,
serious, or severe adverse events). Adverse events related to AnXa or
placebo occurring in ≥ 10% of subjects were infusion-related reactions
(n = 4, all mild). One subject discontinued prior to receiving AnXa
due to rash from enoxaparin.
Conclusion: In conclusion, results from this ongoing study demonstrate
that AnXa is able to rapidly reverse the anticoagulant effects of the
ATIII-dependent fXa inhibitor enoxaparin, as assessed by PD markers. AnXa is well-tolerated and is a promising, universal antidote for
both direct and indirect fXa inhibitors.
Disclosure of Interest: M. Crowther is a consultant for Portola Pharmaceuticals, G. Levy is an employee of Portola Pharmaceuticals, G.
Lu is an employee of Portola Pharmaceuticals, P. Conley is an
employee of Portola Pharmaceuticals, J. Castillo is an employee of
Portola Pharmaceuticals, S. Hollenbach is an employee of Portola
Pharmaceuticals, J. Leeds is an employee of Portola Pharmaceuticals,
J. Lin is an employee of Portola Pharmaceuticals, L. Barron is an
employee of Portola Pharmaceuticals, S. Russell is an employee of
Portola Pharmaceuticals, S. Connolly is a consultant for Portola Pharmaceuticals, J. Curnutte is an employee of Portola Pharmaceuticals.
R
Pelkmans L1,2, Bouwhuis A3, Ninivaggi M1,2, al Dieri R1,2, Hemker
C1,2, Lance M3, de Laat B1,2 and Hilde K1,2
1
Department of Biochemistry; 2Synapse BV; 3Department of
Anaesthesiology, CARIM, Maastricht University Medical Center,
Maastricht, The Netherlands
7
Control of Anticoagulation
COA01
Reversal of enoxaparin-induced anticoagulation in
healthy subjects by andexanet alfa (PRT064445), an
antidote for direct and indirect fXa inhibitors―a phase
2 randomized, double-blind, placebo-controlled trial
Crowther M1, Levy G2, Lu G2, Conley PB2, Castillo J2, Hollenbach
S2, Leeds J2, Lin JP2, Barron L2, Russell S2, Connolly S1 and
Curnutte JT2
1
Medicine, McMaster University, Hamilton, Canada; 2Portola
Pharmaceuticals, South San Francisco, USA
Objectives: Andexanet-alfa (AnXa) is a modified, recombinant human
factor Xa (FXa) molecule developed as a specific antidote to reverse
COA02
Statin use reduces incidence of adverse events in
patients with atrial fibrillation: analysis in ACTIVE-W
& A and AVERROES studies
Lauw MN1,2, Vanassche T1, Shestakovska O1, Pais P3, Dans AL4,
Eikelboom JW1 and Connolly SJ1
1
Population Health Research Institute, McMaster University and
Hamilton Health Sciences, Hamilton, Canada; 2Department of
Vascular Medicine, Academic Medical Center, Amsterdam, The
Netherlands; 3Department of Medicine, St. John’s Medical
College, Bangalore, India; 4College of Medicine, University of
the Philippines, Manila, Philippines
Objectives: Atrial fibrillation (AF), the most common cardiac arrhythmia, independently increases the risk of cardioembolic stroke. Anticoagulants are effective for stroke prevention in patients with AF but are
limited by their high bleeding risk. Previous studies have shown that
statins reduce incidence of cardiovascular events and recurrent venous
thrombosis. We explored the effect of statin use on the incidence of
adverse events in patients with AF, treated in the ACTIVE-W,
ACTIVE-A, and AVERROES studies.
Methods: For all patients in the ACTIVE-W & A and AVERROES
studies, statin use on baseline was documented. Cox proportional hazard models were used to estimate the association between statin use
and the risk of a subsequent adverse event, adjusted for the covariates
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
12/06/2014
8
ABSTRACTS
(6.3%) developed 2+ apixaban restrictions during a mean follow-up of
1.5 1.2 years. Patients who developed a CrCl < 50 mL min–1 had a
higher initial CHADS2 score (2.3 1.1 vs. 1.8 1.2, P < 0.001) and
a higher initial HAS-BLED score (3.0 1.1 vs. 2.4 1.2, P < 0.001).
In the regression analysis, development of a CrCl < 50 mL min–1 was
associated with a history of liver disease (HR 6.75, P = 0.001), heart
failure (HR 1.59, P = 0.008), female gender (HR 1.5, P = 0.011), and
older age (HR 1.08 yr–1, P < 0.001).
Conclusion: From 6.3% to 14.5% of patients taking warfarin for AF
develop worsening renal dysfunction below thresholds that would
require on-treatment dosing changes if treated with apixaban, dabigatran or rivaroxaban instead of warfarin. Specific comorbidities may be
useful in identifying who should have periodic monitoring of renal
function if TSOAC therapy is initiated.
Disclosure of Interest: G. Barnes: none declared, S. Kaatz had grant/
research support from: Boehringer-Ingelheim, Bristol Myer Squibb,
Bayer/Jansen/Johnson and Johnson, Eisai, Iverson Genetics Diagnostics/Medicare, National Institutes of Health, Canadian Institute of
Health Research, Blue Cross/Blue Shield of Michigan, consultant for:
Boehringer Ingelheim, Bristol Myer Squibb/Pfizer, Jansen/Johnson
and Johnson, Daiichi Sankyo, Speakers Bureau: Jansen/Johnson and
Johnson, Boehringer-Ingelheim, GlaxoSmithKline, X. Gu: none
declared, M. Heung: none declared, B. Haymart: none declared, E.
Kline-Rogers: none declared, S. Almany: none declared, J. Kozlowski:
none declared, D. Beasley: none declared, G. Krol: none declared, S.
Ahsan: none declared, J. Froehlich Grant/Research support from: sanofi-aventis; Blue-Cross/Blue Shield of Michigan; Mardigian Foundation; Fibromuscular Disease Society of America, consultant for:
sanofi-aventis, Ortho-McNeil, Merck, Boeringer Ingelheim.
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Conclusion: Our data indicate that statin use was consistently associated with a reduced incidence of adverse events in patients with AF,
independent of the antithrombotic therapy. These results provide
rationale for future studies to determine the additional value of statins
for stroke prevention in AF.
Disclosure of Interest: None declared.
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age, sex, heart failure, diabetes, prior stroke or transient ischemic
attack, and use of hypertensive drugs. A pooled analysis was performed for all patients, stratified by study treatment group. Analyzed
adverse events were stroke or systemic embolism (SE), ischemic or
unspecified stroke or SE, vascular death, and major bleeding.
Results: Of 20,260 included patients with AF, 6445 had statin use on
baseline documented and 13,391 had not. Statin use was associated
with a reduced incidence of stroke or SE (adjusted hazard ratio [aHR]
0.81; 95% CI 0.71–0.93; P = 0.002), ischemic or unspecified stroke or
SE (aHR 0.86; 95% CI 0.75–0.99; P = 0.04), and vascular death (aHR
0.87; 95% CI 0.77–0.97; P = 0.01; Table 1). No association of statin
use with major bleeding was observed (aHR 1.01; 95% CI 0.86–1.19).
Consistent estimates were found on separate analyses for patients with
antiplatelet or anticoagulant therapies.
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COA03
Development of renal dysfunction in atrial fibrillation
patients: the Michigan Anticoagulation Quality
Improvement Initiative (MAQI2) experience
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Barnes G1, Kaatz S2, Gu X1, Heung M3, Haymart B1, Kline-Rogers
E1, Almany S4, Kozlowski J5, Beasley D6, Krol G7, Ahsan S7 and
Froehlich J1
1
Cardiovascular Center, University of Michigan, Ann Arbor;
2
Hurley Medical Center, Flint; 3Division of Nephrology,
University of Michigan, Ann Arbor; 4Division of Cardiology,
Beaumont Health System, Royal Oak; 5Huron Valley-Sinai
Hospital, Commerce Township; 6West Michigan Heart, Grand
Rapids; 7Henry Ford Hospital, Detroit, USA
Objectives: To explore the stability of renal function in patients treated
with warfarin for stroke prevention in atrial fibrillation (AF) as an
estimate of potential safety concern if target-specific oral anticoagulants (TSOAC) were used in place of warfarin.
Methods: AF patients initiating warfarin therapy at one of seven anticoagulation management clinics in Michigan, USA were included.
Patients developing CrCl < 50 mL min–1, < 30 mL min–1 (CockroftGault) or creatinine (Cr) ≥ 1.5 with age ≥ 80 and/or weight ≤ 60 kg at
any time during follow up were used as endpoints given their association with necessary dose changes for rivaroxaban, dabigatran and
apixaban, respectively. Logistic regression analysis was used to generate odds ratios for renal dysfunction (CrCl < 50 mL min–1) on anticoagulation therapy.
Results: Of the 1470 AF patients in the study, 1093 had a
CrCl > 50 mL min–1, 1365 had a CrCl > 30 mL min–1, and 1290 had
a Cr < 1.5 with ≤ 1 apixaban weight/age restrictions at the time of
warfarin initiation. Also, 159/1093 (14.5%) developed CrCl < 50 mL
min–1, 96/1365 (7.0%) developed CrCl < 30 mL min–1, and 81/1290
COA04
Vitamin K antagonists vs. heparin for the treatment of
splanchnic vein thrombosis in the isth registry: results
of 12-month follow-up and a propensity score analysis
Riva N1, Ageno W1, Schulman S2, Bang SM3, Sartori MT4,
Grandone E5, Beyer-Westendorf J6, Barillari G7, Di Minno MND8,
Duce R9, Malato A10, Santoro R11, Poli D12, Verhamme P13,
Martinelli I14, Kamphuisen P15, Alatri A16, Oh D17, D’Amico EA18,
Rezende SM19, Becattini C20, Bucherini E21, Dentali F1 and on
behalf of IRSVT study investigators
1
Department of Clinical and Experimental Medicine, University
of Insubria, Varese, Italy; 2Department of Medicine, McMaster
University, Hamilton, Ontario, Canada; 3Department of Internal
Medicine, Seoul National University, Seoul, Korea; 4Clinical
Medicine II, University Hospital of Padova, Padova; 5IRCCS Casa
Sollievo della Sofferenza, S. Giovanni Rotondo, Italy; 6Center for
Vascular Medicine and Department of Medicine III, Division of
Angiology, University Hospital ‘Carl Gustav Carus’, Dresden,
Germany; 7Center for Hemorrhagic and Thrombotic Diseases,
University Hospital, Udine; 8Department of Clinical Medicine
and Surgery, Federico II University, Naples; 9Thrombosis Center,
Galliera Hospital, Genoa; 10Cattedra ed UO di Ematologia con
Trapianto, Policlinico Universitario di Palermo, Palermo;
11
Haemophilia Center, Azienda Ospedaliera Pugliese-Ciaccio,
Catanzaro; 12Thrombosis Center, Careggi Hospital, Florence,
Italy; 13Leuven University, Leuven, Belgium; 14Department of
Internal Medicine and Medical Specialties, Fondazione IRCCS
Ca’ Granda - Ospedale Maggiore Policlinic, Milan, Italy;
15
University of Groningen, Groningen, The Netherlands; 16Centro
Emostasi e Trombosi, A.O. Istituti Ospitalieri di Cremona,
Cremona, Italy; 17Department of Internal Medicine, Pochon CHA
University, Seoul, Korea; 18Hospital das Clınicas da Faculdade de
Medicina, Universidade de Sao Paulo, Sao Paulo;
12/06/2014
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
ABSTRACTS
Departamento de Clınica M
edica, Universidade Federal de
Minas Gerais, Belo Horizonte, Brazil; 20Department of Internal
and Vascular Medicine, University of Perugia, Perugia;
21
Ospedale di Faenza, Faenza, Italy
19
indigestion by the 2 weeks. In these 9 cases, the symptoms were mild.
Prescription of antacids/analgesia allowed patients to remain on the
NOACs. One of 11 Dabigatran patients was appropriately changed at
the first appt to rivaroxaban for recurrent VTE prophylaxis; 35% of
patients prescribed NOACs required input by their 1-month appt.
Conclusion: A significant number of patients being prescribed NOACs
experience early adverse effects that may lead to discontinuation or
complications of the drug, suggesting the need for monitoring.
Patient-centered follow-up allows safe discontinuation if required, and
data capture of adverse events and patient adherence, while these new
drugs remain in their infancy.
Disclosure of Interest: None declared.
COA06
Defining time in therapeutic range for clinicians: frequency of dose changes is a good surrogate marker for
identifying patients with suboptimal anticoagulation
with warfarin
Vijenthira A1, Le Gal G1,2,3 and Carrier M1,2,3
Faculty of Medicine, University of Ottawa; 2Hematology, The
Ottawa Hospital; 3Clinical Epidemiology, Ottawa Hospital
Research Institute, Ottawa, Canada
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Objectives: Patients on warfarin with low time-in-therapeutic range
(TTR) are more likely to have adverse events (arterial or venous events,
major bleeding, etc.). Previous studies have also demonstrated that new
oral anticoagulants have less efficacy and safety benefits compared to
warfarin when the TTR is > 65%. Therefore, many governmental agencies and insurance companies will only financially cover new oral anticoagulants following a trial of warfarin and if patients have a TTR
≤ 65%. We aimed to determine a simple, sensitive, and specific predictor of TTR ≤ 65% during the initial 3 months of warfarin initiation.
Methods: Cross-sectional study of patients newly initiated on warfarin
for any indication with uninterrupted therapy for 3 months. TTR was
calculated using the Rosendaal method. Patients were divided by TTR
≤ 65% or > 65%. The association and performance of the number of
dose changes, INR tests, and INRs ≤ 1.7 or ≥ 4.0 in predicting TTR
≤ 65% were assessed with the Student’s t-test, Pearson’s correlation,
and receiver operating characteristics (ROC) curve analysis.
Results: There were 670 patients, with a median age of 58 years. The
most common indication for anticoagulation was venous thromboembolism (93%). The mean TTR in the first 3 months was 68 21%
(range 10–100%). The number of dose changes was significantly and
strongly associated with TTR ≤ 65% (6 3 vs. 2 2, P < 0.0001,
r = 0.70). On ROC curve analysis, three dose changes showed 90%
sensitivity and 56% specificity for a TTR ≤ 65%. Three INRs ≤ 1.7
had 98% specificity in ruling in TTR ≤ 65%.
Conclusion: Following initial stabilization of the INR, three dose
changes or three INRs ≤ 1.7 in the first 3 months following warfarin
initiation might help clinicians to identify patients with TTR ≤ 65%.
Disclosure of Interest: None declared.
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Objectives: Splanchnic vein thrombosis [SVT] is a challenging disease,
because of the concurrent increased risk of bleeding and potentially
life-threatening complications. We aimed to explore the actual management of SVT in a large prospective cohort and to report clinical
outcomes during follow-up.
Methods: Consecutive SVT patients were enrolled in a multicenter
international registry, from 2008 to 2012. Clinical outcomes (major
bleeding; vascular events, defined as venous or arterial thrombosis;
mortality) were collected. A propensity score (PS), created from baseline characteristics of patients receiving the two main treatments (different dosages of parenteral anticoagulants vs. vitamin K antagonists
[VKAs]), was used to estimate the effect of therapeutic strategies. We
here report the results of the 12-month follow-up.
Results: There were 613 patients enrolled: mean age 53.1 14.8 years;
62.6% males. Initially, 143 patients were not anticoagulated; 175
received
parenteral
anticoagulants
only
(mean
duration
6.28 4.21 months) and 295 started VKAs (9.67 3.53 months).
Major bleeding occurred in 25 patients, 16 on treatment (4.53/100
patient-years [pt-y]) and 9 off treatment (5.45/100 pt-y); vascular
events in 47 patients, 27 and 20 (7.93/100 pt-y vs. 11.85/100 pt-y);
death in 79 patients, 44 and 35 (12.35/100 pt-y vs. 20.30/100 pt-y).
Solid cancer, ascites, anemia, and thrombocytopenia were inversely
associated with the prescription of VKAs in a binary logistic regression. Using PS based on these variables, we matched 102 patients treated with VKAs with 102 patients treated with parenteral
anticoagulants. Major bleeding occurred in 5.88% vs. 0.98% for
VKAs and heparin, respectively; vascular events in 2.94% vs. 8.82%;
and mortality in 5.88% vs. 18.63%.
Conclusion: In our cohort of SVT patients, the incidence of vascular
and bleeding events at 1-year follow-up was relevant. Although the PS
matching did not allow for a complete balance of heterogeneity, in designated patients VKA treatment appeared to be sufficiently safe.
Disclosure of Interest: None declared.
9
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COA05
Safe implementation of the new oral anticoagulants
(NOACs) requires patient monitoring
Loizou E, Toh C-H and Dutt T
Haematology, The Royal Liverpool University Hospital,
Liverpool, UK
Objectives: While a consensus exists for patient follow-up on warfarin,
there is no guidance as to whether or how patients on NOACs should
be monitored. While NOACs are being prescribed in practice, in the
absence of any planned follow-up, there is no system for capturing
data on adverse events or patient adherence.
Methods: This was a prospective study to assess the value of a followup system for patients prescribed NOACs in a hospital. Patients were
asked to attend the Anticoagulation Clinic at 2 weeks and 1 and
6 months. At the first appointment (appt), specific drug, dose, and
indication were recorded, along with any adverse effects and patient
adherence at subsequent appts.
Results: There were 37 patients prescribed NOACs over a 12-month
period: 26/37 rivaroxaban and 11/37 dabigatran. All attended the first
appt, 34/37 the 1 month appt and 20/23 due the 6 month appt. 1/11
stopped dabigatran by the 1 month due to severe nausea and vomiting. Also, 2/26 discontinued rivaroxaban; 1 due to severe tiredness and
hair loss by the 2 weeks, drug discontinued at 1 month due to persistent tiredness and hallucinations; 1 complained of fatigue and emotional lability at 2 weeks, drug discontinued at 1 month due to
suicidal ideation. And 2/11 on dabigatran and 1/26 on rivaroxaban
experienced headaches by 2 weeks. Three on each drug experienced
COA07
What is the best time sampling for the two direct oral
anticoagulants (DOACs), dabigatran and rivaroxaban?
Douxfils J1, Baudar J2, Devalet B3, Samama MM4, Chatelain B2,
Dogn
e J-M1 and Mullier F2
1
Department of Pharmacy, University of Namur, Namur;
2
Hematology Laboratory; 3Department of Hematology, CHU
Dinant-Godinne UCL Namur, Yvoir; 4Department of
^tel-Dieu University Hospital, Paris, Belgium
Hematology, Ho
Objectives: The objective of this study is to evaluate the interindividual
response to direct oral anticoagulants (DOACs) at different time
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
12/06/2014
10
ABSTRACTS
points in order to identify which interval between the drug intake and
the blood sampling should be preferred for the assessment of DOACs.
Methods: Patients treated with rivaroxaban (n = 33) or dabigatran etexilate (DE) (n = 18) were included in the study. For each patient,
blood was taken at three different intervals:
● At Ctrough (22–26 h [h] and 11 to 13 h after the last intake of
rivaroxaban and DE, respectively).
● At Cmax: 2 and 3 h after drug ingestion.
For rivaroxaban samples, PT was performed with Triniclot PT Excel
Sâ, RecombiPlasTin 2Gâ and Innovinâ and plasma concentrations
estimated with the Biophen Direct Factor Xa Inhibitorâ. For DE samples, aPTT was perfomed with STAâ-C.K.Prest and SynthasILâ and
plasma concentrations estimated with the Hemoclot Thrombin Inhibitorâ. Wilcoxon analyses were performed. Test for effective pairing was
computed with the Spearman non-parametric correlation coefficient.
Results: Figure 1 provides the box-plot and the min to max of the different assays for rivaroxaban and DE. Our results confirm the interindividual variation in drug levels at similar time points. When
comparing intervals, we can see that results at Ctrough clearly differ
from results obtained 2 or 3 h after the intake of the drug. For rivaroxaban, the median after 2 or 3 h is closely the same with similar 25–
75th percentile values. For dabigatran, the plasma concentration is
higher at 3 than at 2 h. These data are consistent with initial pharmacokinetic studies.
Conclusion: We can assert that it seems preferable to take the blood
3 h after the last intake of DE to avoid missing the Cmax. For rivaroxaban, the results are similar. Thus, we recommend to systematically
performing blood sampling at 3 h to assess the maximal response.
Sampling at Ctrough is also required to evaluate the drug clearance.
Disclosure of Interest: None declared.
COA08
Presence of direct factor Xa inhibitors leads to a
protracted thrombin generation peak
Bloemen S1,2, Huskens D1, de Laat B1,2, Hemker HC1 and
Al Dieri R1
1
Synapse BV; 2Department of Biochemistry, CARIM, Maastricht,
The Netherlands
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Objectives: Several studies have shown a protraction of the thrombin
peak when direct factor Xa (FXa) inhibitors are present in plasma during thrombin generation (TG) tests (in vitro and ex vivo). Until now
the mechanism behind the strong inhibition of the peak and protraction of the curve was not explained. The aim of this study was to elucidate this mechanism.
Methods: TG was assessed in platelet-poor plasma (PPP) and plateletrich plasma (PRP) with and without rivaroxaban. TG was also measured in control plasma and FVII-, FVIII-, FIX-, and FXI-deficient
plasmas, activated with tissue factor (TF), FIXa, or kaolin. The effect
on FXa generation was tested in a reconstituted system triggered with
either TF/FVIIa or FIXa.
Results: Rivaroxaban inhibits the peak of TG curves significantly
(83%, P < 0.01), whereas the endogenous thrombin potential (ETP) is
not inhibited to that extent (34%), leading to a protracted curve (Figure 1). When the intrinsic pathway is bypassed (e.g, FIX-deficient
plasma), the first part of the peak is no longer present. When the
extrinsic pathway is evaded (e.g, triggering with FIXa), the second part
of the peak disappears. In PRP, the protracted shape is absent and is
only found after freezing as to have phospholipids (PL) present at the
start of the reaction. Inducing FXa generation with TF/FVIIa results
in a delayed response compared to triggering with FIXa. The inhibition by rivaroxaban on the intrinsic pathway is stronger.
Conclusion: When direct FXa inhibitors are present we found a protracted TG curve in which the first part is due to reactions in the Josso
loop (FVIII and FIX) and the second part to TF dependent reactions.
Based on the experiments in PRP, we hypothesize that the effect of rivaroxaban is based on the amount of FXa bound to PL. The protracted
thrombin formation, a strongly inhibited peak but relatively high
ETP, might explain a lower bleeding rate in patients compared to
other anticoagulants that inhibit peak and ETP to the same extent.
Disclosure of Interest: None declared.
12/06/2014
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
ABSTRACTS
COA09
Clinical outcome of major bleeding in patients on oral
anticoagulant treatment: results from the CLIMBING
Study
cians, general practitioners, and cardiology registrars. Data was analyzed using interpretative phenomenological analysis, a qualitative
methodology.
Results: Study 1: Three overarching themes comprised patients’ experiences: the initial consultation, life after the consultation, and patients’
reflections. Patients commented on the reassurance experienced during
the consultation, but they perceived the decision-making process
mostly led by the physician. Lack of education and take-home materials during the initial consultation were highlighted. Patients’ uptake of
information was influenced by past experiences and knowledge of
stroke and/or bleeding. Study 2: Two overarching themes covered physicians’ experiences: communicating information and challenges with
warfarin prescription for AF. Physicians’ approach to the consultation
style shifted through a continuum of compliance-adherence-concordance during the consultation. Time and the perceived patient trust in
them as the expert led to physicians to adopt a paternalistic approach.
Guideline adherence and the need to adopt a multidisciplinary
approach were pointed out as current challenges.
Conclusion: There is a need to target patients’ and physicians’ ability
to communicate with each other in a comprehensible way. This project
has illustrated the benefit of using a qualitative approach to understand the lived experience of the physician–patient consultation.
Disclosure of Interest: None declared.
R
Franco L1, Becattini C1, Lorenzini G2, Sbrojavacca R3, Nitti C4,
Cappelli R5, Cattinelli S6, Masotti L2 and Agnelli G1
1
Internal and Cardiovascular Medicine-Stroke Unit, University of
Perugia, PERUGIA; 2Internal Medicine, Cecina Hospital, Cecina;
3
Emergency Medicine, AOU Santa Maria della Misericordia,
Udine; 4Emergency Medicine, Ospedali Riuniti Umberto I Lancisi- Salesi, Ancona; 5Internal medicine, University of Siena,
Siena; 6Emergency Medicine, Ospedale Cattinara, Trieste, Italy
or
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COA11
Vitamin K antagonist therapy induces high plasma levels of factor VIII and fibrinogen and reduces thickness
of fibrin fibers
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Bloemen S1,2, de Laat M1,3, ten Cate-Hoek A4, ten Cate H4, de
Laat B1, Hemker HC1 and Al Dieri R1
1
Synapse BV; 2Department of Biochemistry, CARIM, Maastricht;
3
Department of Cardiology, Atrium Medical Centre, Heerlen;
4
Department of Internal Medicine, CARIM, Maastricht, The
Netherlands
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Objectives: Major bleeds (MBs) in non-surgical patients include events
with a wide spectrum of clinical severity and outcome. Whether MBs
occurring in patients on oral anticoagulants should be further categorized for the estimated risk of death is undefined.
Methods: Patients on oral anticoagulants admitted to the emergency
department for MB were included in a prospective, cohort study
(CLIMBING). The primary outcome of the study was death during
the hospital stay.
Results: As for February 1, 2014, 312 patients with MBs while on
treatment with vitamin K antagonists were included in this study, 184
with intracranial hemorrhage (ICH). Death occurred in 67 patients
(21%). ICH (OR 4.28; 95% CI 1.90–9.66; P < 0.001), trauma (OR
2.09; 95% CI 1.06–4.15; P < 0.03), and re-bleeding (OR 4.13; 95% CI
1.99–8.58; P < 0.001) were independent predictors of death, while
INR at admission, time to INR normalization, and HAS-BLED (or
any of its items) were not. Among patients with ICH, age (OR 1.10,
95% CI 1.04–1.17, P < 0.001), Glasgow Coma Scale ≤ 8 (OR 11.09,
95% CI 3.92–31.35, P < 0.001), lobar localization (OR 3.78, 95% CI
1.32–10.81, P < 0.01), and re-bleeding (OR 8.27, 95% CI 2.53–27.05,
P < 0.001) were independent risk factors for death, while INR at
admission, time to INR normalization, history of renal or heart failure, and treatment with fresh frozen plasma (FFP) or prothrombin
complex concentrates (PCCs) were not. Among patients with nonICH MB, age (OR 1.10, 95% CI 1.01–1.21, P < 0.049) and shock at
presentation (OR 11.16, 95% CI 2.59–47.99, P < 0.001) were independent risk factors for death, while site of bleeding, INR at admission,
time to INR normalization, re-bleeding, hemoglobin decrease ≥ 2 g/
dL, transfusion ≥ 2 units of packed red blood cells, and treatment with
FFP or PCC were not.
Conclusion: Among patients with MB while on oral anticoagulants,
the risk for death is higher in those with ICH. Risk factors for death
differ between patients with ICH and those with non-ICH MB.
Disclosure of Interest: None declared.
11
COA10
The experiences of consultations on oral anticoagulation for AF: the importance of qualitative research in
cardiovascular sciences
Xuereb CB1,2,3, Shaw RL2, Lip GYH2,3 and Lane DA3
1
Psychology, University of Malta, Msida, Malta; 2School of Life &
Health Sciences, Aston University; 3Centre for Cardiovascular
Sciences, University of Birmingham, Birmingham, UK
Objectives: Multiple-perspective qualitative designs can aid researchers
to develop a more multifaceted account of a phenomenon and as a
form of triangulation of data. Two interlinking studies aimed to
explore patients’ and physicians’ experiences of atrial fibrillation (AF)
and warfarin.
Methods: Audio-recorded semistructured individual interviews were
used. Study 1: Three AF patient subgroups were interviewed (n = 11);
accepted, refused, or discontinued warfarin. Study 2: Four physician
subgroups (n = 16): consultant cardiologists, consultant general physi-
Objectives: Vitamin K antagonists (VKAs) attenuate factors II (FII),
FVII, FIX, and FX, as well as the anticoagulants protein C (PC) and
protein S (PS). The prothrombin time used for monitoring the effect of
VKA only evaluates FII, FVII, and FX. This study aimed to investigate the function of all factors as well as their overall effect on thrombin generation (TG), not only in plasma but also in whole blood (WB).
Methods: Blood samples were collected from 150 consenting patients
on VKA and from 20 consenting healthy volunteers. TG was measured in WB, platelet-rich plasma, and platelet-poor plasma. The international normalized ratio (INR) and the levels of FII, FV, FVII,
FVIII, FIX, and FX were assessed as well as antithrombin (ATIII),
fibrinogen, PC, and PS. Fibrin formed on paper disks during TG in
WB was fixated for scanning electron microscopy (SEM), and the
fibrin thickness was determined.
Results: All TG parameters in WB correlated significantly (P < 0.01)
with the parameters in plasma. Vitamin K–dependent clotting factor
levels decreased as the INR increased. FVII, FX, and PC decreased
more and FIX and PS decreased less, when comparing these factors to
FII. Interestingly, patients had a significantly higher FVIII level compared to the healthy donors (184% vs. 118%, P < 0.01) and higher
fibrinogen levels (4.0 g/L vs. 3.2 g/L, P < 0.01). Levels of ATIII and
FV were similar between the two groups. In the WB clot structure, the
fibrin fibers were thicker in the donors compared to the patients.
Conclusion: Thrombin generation data in whole blood correlate well
with plasma TG parameters. The fibrin fibers of patients were thinner
than those of the donors, which is surprising since thin fibers are usually observed at high thrombin levels rather than at low ones. This
observation can be explained by the finding of higher fibrinogen levels
in patients. The increased levels FVIII and fibrinogen might be a risk
factor for recurrent thrombosis after cessation of therapy.
Disclosure of Interest: None declared.
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
12/06/2014
12
ABSTRACTS
COA12
Interest of thrombin time in the periprocedural management for patients on dabigatran etexilate
period from January 1, 2006, through December 31, 2006. Individuals
for whom the indication for testing was listed as a neurologic sign or
symptom were included in our analysis. We recorded demographic
information, testing indication, test results, decisions in medical management ensuing from the test results, and documentation that
patients were notified of the test results and the implications.
Results: During the study period, 479 patients underwent thrombophilia screening, of whom 169 (35%) had a neurologic sign, symptom,
or diagnosis listed as the study indication. We identified no patients in
this dataset whose test results influenced physician decisions about
dose, type, or duration of antithrombotic treatment.
Conclusion: Evidence-based guidelines discourage routine testing for
hereditary and acquired thrombophilia in patients suffering arterial
thrombotic events such as stroke or TIA. Similarly, there is no scientific evidence to support screening for thrombophilia in individuals
with non-thrombotic neurologic conditions. Nevertheless, we found
the practice to be common at our institution despite the lack of impact
of test results on patient care and the potential for harm. Our data
point to a critical need for educating clinicians about the appropriate
use of thrombophilia screening tests.
Disclosure of Interest: None declared.
Douxfils J1, Remacle S2, Lessire S2, Dincq A-S2, Gourdin M2,
Chatelain B3, Dogne J-M1 and Mullier F3
1
Department of Pharmacy, University of Namur, Namur;
2
Department of Anesthesiology; 3Hematology Laboratory, CHU
Dinant-Godinne UCL Namur, Yvoir, Belgium
Objectives: The French Working GIHP proposed that a dabigatran
plasma concentration ([]) ≤ 30 ng mL–1 is safe to carry out an invasive
procedure. Some authors make proposals based on Thrombin Time
(TT). However, TT is difficult to standardize.
The objectives of the present study were:
● To determine the optimal [thrombin] with a variety of instruments and reagents.
● To assess the repeatability of TT at optimized conditions.
● To compare the sensitivity and linearity of TT at residual [dabigatran] (DA) with those of APTT and hemoclot thrombin inhibitor (HTI).
Methods: DA was spiked at increasing [] in pooled citrated normal
human platelet-poor plasma. The following [DA] were prepared: 0, 5,
10, 20, 30, 40, and 50 ng ml–1. Bovine thrombin (HemosILâ TT) and
human thrombin (STAâ-TT) were tested on four instruments: STA-R
Evolutionâ, ACLTOPâ, CS2000iâ, and KC10â.
Results: Except for STAâ-TTon STA-Râ, the [optimized] is not the
one recommended by the manufacturer (Table 1).
ACLTOP
KC10â
CS2000iâ
1.5
3.8
3.8
5.0
1.5
5.0
1.4
3.8
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STAâ-TT
HemosILâ TT
STA-Râ
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APTT is not sensitive enough in low [DA], whereas HTI is not suitable
in [DA].
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Conclusion: TT may be more informative than aPTT and HTI to provide guidance to carry out an urgent procedure or surgery. Each laboratory should optimize its TT procedure according to its
coagulometer-reagent combination.
Disclosure of Interest: None declared.
COA13
Impact of thrombophilia testing on diagnosis and
treatment of patients with neurologic disorders: a single-center experience
Wasp G1, Shatzel J1, Whyman J1, Harbin D2, Lane M3, Marehbian
J3 and Ornstein D1,4,5
1
Internal Medicine, Dartmouth-Hitchcock Medical Center,
Lebanon; 2Geisel School of Medicine, Hanover; 3Neurology;
4
Pathology; 5Hematology, Dartmouth-Hitchcock Medical Center,
Lebanon, USA
Objectives: Screening for thrombophilia in patients with ischemic
stroke and other neurologic disorders is not supported by scientific evidence. Nevertheless, we have observed that such screening is commonly performed. We undertook the current study to review the
practice of thrombophilia screening in patients with stroke and other
neurologic disorders at our institution. We reviewed all thrombophilia
test panels performed over a 1-year period to determine the number
and type of abnormal tests results and the effect of the results on treatment decisions
Methods: We reviewed the medical records of all patients who underwent screening for thrombophilia at our institution during the 1-year
s A1,2, Bautista M1,
Quintero J1, Cardenas L1, Bonilla G1, Llina
1
1
Navas M , Gomez M and on behalf of The Clinical Care Program
in Joint Replacement Surgery
1
n Santa Fe Bogot
Orthopaedics, Hospital Universtario Fundacio
a;
2
Universidad de los Andes, Bogota, Colombia
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Table 1 Optimized [thrombin] (NIH mL–1) on eight combinations
reagent/instrument
COA14
Major orthopedic surgery: is the risk of major bleeding
higher in elderly patients?
Objectives: The use of pharmacological prophylaxis for venous thromboembolism is highly recommended for the vast majority of patients
undergoing major orthopedic surgery. It has been documented that in
the general population the use of these agents increases the risk of
major bleeding. However, the frequency of this complication has not
been studied in the subpopulation of elderly patients. Our purpose
with the present study is to determine the risk of major bleeding in
patients > 70 years old undergoing major orthopedic surgery compared to those operated at a younger age.
Methods: In a retrospective cohort study, patients who underwent
total hip or total knee arthroplasty during 5 consecutive years were
included. Patients with other possible causes of bleeding were
excluded. All medical records were reviewed in order to determine the
occurrence of major bleeding. The risk of major bleeding in patients
70 years or older was compared to that of patients < 70 years old. Relative risks (RRs) and confidence intervals (CIs) were calculated, and a
multivariate analysis was performed.
Results: A total of 1048 patients were analyzed (56% hip arthroplasties, 44% knee arthroplasties). At the time of surgery, 553 (53%)
patients were 70 years or older, while 495 (47%) were younger than
70 years. Patients who were ≥ 70 years old showed an increased risk
of major bleeding: RR 2.42 (95% CI 1.54–3.81). During total hip
arthroplasty, the RR was 2.61 (95% CI 1.50–4.53) and during total
knee arthroplasty was 2.25 (95% CI 1.03–4.94). After the multivariate
analysis, age continued to be independently associated with a higher
risk of major bleeding.
Conclusion: Patients who are 70 years or older are at a higher risk of
major bleeding during major orthopedic surgery. Therefore, the use of
appropriate strategies to mitigate the risk in this group of patients is
encouraged.
Disclosure of Interest: J. Quintero: none declared, L. Cardenas: none
declared, G. Bonilla Consultant for: Boehringer Ingelheim, BristolMyers-Squibb, Pfizer, Sanofi, Speakers Bureau: Boehringer-Ingelheim,
Bristol-Myers-Squibb, DePuy orthopaedics, Pfizer, Sanofi, A. Llinas is
a consultant for: 3M, Bayer, Zimmer, Speakers Bureau: 3M, Baxter,
Bayer,Boehringer-Ingelheim, Bristol-Myers-Squibb, Covidien, Glaxo,
12/06/2014
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
ABSTRACTS
Novonordisk, Pfizer, Zimmer, M. Bautista: none declared, M. Navas:
none declared, M. Gomez: none declared.
Results:
COA15
Effect of eculizumab therapy on the coagulation status
of patients with paroxysmal nocturnal hemoglobinuria
Gribkova I1,2, Tsvetaeva N1, Seregina E3, Nikulina O1, Balandina
A3, Gorbatenko A1 and Sinauridze E1,2
1
National Research Center For Hematology; 2Center of
Theoretical Problem of Physico-Chemical Pharmacology; 3Center
of Pediatric Hematology, Oncology and Immunology, Moscow,
Russian Federation
Diagnosis
No. of
patients
Target
INR PT
INR PT in target
zone (%)
DVT/PE
Z95.x
I 42, 48, 49
Malignancy with DVT/PE
Ischemic stroke, TIA
Other (i73.x, D68, etc.)
Total
152
29
89
8
2
16
296
2–3
2.5–3.5
2–3
2–3
1.5–2.5
2–3
–
53
82
56
48
100
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There are 62.5% of patients in target zone. In 2009 (February), 39.4%
had an interval of 1.8–3.5, and in 2014 (February), 69.9% had an
interval of 1.8–3.5. Changes that we made are that each patient
received a leaflet with instructions and an explanation about the content of leaflet, given face to face. The patient is kindly asked to read
and understand what is written and told to feel free to ask questions
concerning any doubts about the contents. In July 2013, the Ministry
of Health introduced a project ‘admission time’; each patient has a
reserved scheduled time of about 10–15 min to spend with his physician, to allow sufficient time to spend with each patient to provide education and better communication.
Conclusion: Many factors affect and influence the success of treatment
and patients’ compliance. We can influence some but not all. Macro
and micro factors and subjective and objective issues will be always
present and influence positively or negatively.
Disclosure of Interest: None declared.
COA17
Streptokinase and low molecular weight heparin affect
platelet aggregation in Ukrainian patients with atherothrombotic and cardioembolic ischemic stroke
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Objectives: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare,
generally acquired, life-threatening disease of the blood characterized
by complement-induced intravascular hemolytic anemia, red urine and
thrombosis. The monoclonal antibody eculizumab is indicated for the
treatment of patients with PNH to reduce hemolysis. So it is reasonable to suppose that eculizumab is effective for thrombosis reduction
also. Currently the majority of patients (63%) receive concomitant
anticoagulant therapy. The effect of anticoagulant withdrawal during
eculizumab treatment has not been studied. The aim of this study was
to compare thrombin generation in patients with PNH with or without
anticoagulant therapy before and after infusion of eculizumab.
Methods: Three patients with PNH were under observation over
approximately 1 year. Serum lactate dehydrogenase (LDH) levels were
measured to estimate eculizumab effects on hemolysis. Thrombin generation test was measured before and after eculizumab administration
to estimate coagulation status (endogenous thrombin potential (ETP)
was used as parameter).
Results: All patients achieved a reduction in serum LDH levels (from
1874–7106 to 380–612 U/L) during eculizumab therapy, which indicated the reduction of hemolysis. ETP levels during eculizumab therapy were nearly normal ranges (900–1414 nmol L min–1) but
sometimes prolonged periods of hypercoagulation were observed
(1580–3900 nmol L min–1). When 2 patients received concomitant
anticoagulant therapy with enoxaparin ETP levels decreased significantly (190–332 nmol L min–1).
Conclusion: Eculizumab reduces hemolysis―the main cause of serious
PNH health problems. But investigation by thrombin generation test
makes it clear that risk of hypercoagulation events remains during eculizumab therapy without anticoagulants.
Disclosure of Interest: None declared.
13
COA16
Oral anticoagulation treatment and PT INR response in
our department; comparison between 2009 and 2014
Jovanoska V1 and Jovanoski I2
Department of Transfusiology Prilep, ITM Republic of
Macedonia; 2Ortopedic and Traumatology, General Hospital
Prilep, Prilep, Macedonia, The Former Yugoslav
1
Objectives and Aims: To analyze the patient compliance and INR
response of OAT and to achieve the goals of OAT treatment, to compare the PT INR response of oral anti-coagulation treatment (OAT)
between 2009 and 2014 in our department, and to define the critical
points and areas of improvements that might affect compliance to
OAT during this period.
Material and Methods: Retrospectively, from the medical records we
analyzed patient data and PT INR response to OAT during February
2014. INR PT was analyzed for patients who takes OAT > 1 month. A
total of 296 patients were analyzed. Only patient with an INR
response in target zone was selected; the others, out of target zones,
are excluded in calculations.
Burlova-Vasylieva M, Kravchenko N, Katrii T and Savchuk O
Biochemistry, Educational and Scientific Centre, ‘Institute of
Biology’ of Taras Shevchenko National University of Kyiv, Kyiv,
Ukraine
Objectives: Platelets play an important role in pathogenesis of cardiovascular disease and secrete numerous bioactive factors that are
involved in the coagulation processes. The aim of our study was to
investigate the platelet reactivity under non-protein (ADP) and protein
(low molecular weight heparin [LMWH]) and streptokinase (SK)
agents in patients with atherothrombotic ischemic stroke and cardioembolic ischemic stroke with atrial fibrillation (AF).
Methods: The study involved 16 atherothrombotic ischemic stroke
patients, 11 cardioembolic ischemic stroke patients with AF, and 15
healthy donors. ADP-induced platelet aggregation was assessed in
platelet-rich plasma using a photo-optical aggregometer, before and
after LMWH treatment of patients with ischemic stroke. SK was used
as an additional in vitro reagent.
Results: It was shown that platelet response to ADP in patients with
atherothrombotic ischemic stroke was 26% higher relative to donors
and was not increased in cardioembolic ischemic stroke patients with
AF. SK caused elevation of ADP-induced platelet aggregation in 30%
of donors, 36% of atherothrombotic stroke patients, and 67% of cardioembolic ischemic stroke patients with AF. After LMWH administration, a tendency to elevation of platelet response to ADP in patients
with cardioembolic ischemic stroke with AF was observed. Addition
of SK to the medium significantly stimulated ADP-induced aggregation of patients with cardioembolic ischemic stroke with AF.
Conclusion: Platelet response to ADP was significantly increased in
patients with atherothrombotic ischemic stroke. Cardioembolic ischemic stroke with atrial fibrillation was characterized by normal platelet
response to ADP and increased response to streptokinase and LMWH.
Disclosure of Interest: None declared.
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
12/06/2014
14
ABSTRACTS
COA18
Coumadin-induced skin necrosis: case presentation
levels and percentages of patients with a negative D-dimer were compared between three subgroups according to glomerular filtration rate
(GFR): ≥ 90 mL min–1 (normal renal function), 60–89 mL min–1 (mild
renal impairment), and 30–59 mL min–1 (moderate renal impairment).
Results: D-dimer levels increased and the proportion of negative Ddimer decreased significantly according to renal status: 46% negative
D-dimer in patients with normal GFR, 31% in patients with mild
renal impairment, 11% in those with moderate renal impairment, corresponding to number of patients needed to test to obtain one negative
test of 2.2, 3.2, and 9, respectively.
Conclusion: The clinical usefulness of D-dimer decreases with renal
impairment. However, PE can still be ruled out by negative D-dimer in
a substantial proportion of patients with non-high clinical probability,
avoiding exposure to contrast media.
Disclosure of Interest: None declared.
Jovanoska V1 and Jovanoski I2
Department of Transfusiology Prilep, ITM Republic of
Macedonia; 2Orthopedic and Traumatology, General Hospital
Prilep, Prilep, Macedonia, The Former Yugoslav
1
COA20
Efficacy and safety outcomes of treatment of acute
venous thromboembolism: a systematic review and
network meta-analysis
Castellucci LA1,2,3, Cameron C2, Le Gal G1,3 and Carrier M1,2,3
The Ottawa Hospital; 2University of Ottawa; 3Ottawa Hospital
Research Institute, Ottawa, Canada
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COA19
Effects of impaired renal function on levels and performance of D-dimer in patients with suspected pulmonary embolism
Robert-Ebadi H1, Bertoletti L2, Combescure C3, Le Gal G4,
Bounameaux H5 and Righini M5
1
Division of Angiology and Hemostasis, Geneva University
Hospitals and Faculty of Medicine, Geneva, Switzerland;
2
Thrombosis Research Group, University Jean Monnet, SaintEtienne, France; 3Division of Clinical Epidemiology, Geneva
University Hospital and Faculty of Medicine, Geneva,
Switzerland; 4Thrombosis Program, Division of Hematology,
Department of Medicine, University of Ottawa, Ottawa, Canada;
5
Division of Angiology and Hemostasis, Geneva University
Hospital and Faculty of Medicine, Geneva, Switzerland
or
Objectives: Many different anticoagulant strategies have been evaluated for the treatment of acute venous thromboembolism (VTE), yet
little guidance exists regarding which drug is most effective and safe.
We sought to summarize and compare the efficacy and safety of various management options (unfractionated heparin [UFH] and vitamin
K antagonist [VKA], low molecular weight heparin [LMWH] and
VKA, LMWH/dabigatran, LMWH/edoxaban, rivaroxaban, apixaban, and LMWH alone) for treatment of acute VTE.
Methods: A systematic literature search was conducted using MEDLINE, EMBASE, and the EBM reviews. Randomized controlled trials
reporting rates of recurrent VTE and major bleeding were included.
Odds ratios (95% credible interval [CrI]) for these outcomes were generated. Each treatment strategy was compared to LMWH/VKA.
Results: A total of 45 RCTS (44,989 patients) were included in our
analysis. Pairwise comparisons are depicted in the Figure. Compared
to LMWH/VKA, a treatment strategy using UFH/VKA was associated with an increased risk of recurrent VTE (OR 1.5; 95% CrI 1.2–
1.9). The risk of major bleeding was lower with rivaroxaban (OR 0.6;
95% Crl 0.4–0.9) and apixaban (OR 0.3; 95% CrI 0.2–0.6) compared
to LMWH/VKA.
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Objectives and Aim: To present a patient with Coumadin-induced skin
necrosis (CISN) diagnosed only on the clinical history, clinical presentation, and response to treatment. CISN refers to a rare condition in
which there is paradoxical blood clotting. It affects 0.01–0.1% of
patients treated with oral anticoagulants.
Methods: Diagnosis of CISN is made clinically. Blood tests for protein
C and protein S are important to assess the likely predisposing causes.We present a patient with CISN diagnosed only on the clinical history, clinical presentation, and response to treatment.
Results: The patient was a 72-year-old woman diagnosed with deep
vein thrombosis on her right limb. Recommended treatment was
LMWH and tablets of Acenokumarol at 4 mg (only vitamin K antagonist available in our country) to start. Patient was not able to provide
the LMWH because of financial reasons and only started treatment
with tablets Acenokumarol as previously scheduled. After 5 days, she
complained of pain, sensation of pressure, and paresthesia. On the left
thigh, the external portion under the knee, there was redness. Erythematosus flush without edema was inspected. That condition was first
not recognized. After 2 days, she came with more complaints of pain.
The situation of her skin condition was worsening, with petechiae and
early necrosis; small necrotic eschar were seen. We suspected CISN.
Immediately the tablets Acenokumarol were stopped and treatment
with LMWH was started. Local cleansing of the wound and dressing
application were done. The patient’s condition was improved, and
after 3 weeks the wound was completely healed.
Conclusion: An erly diagnosis of CISN, based on clinical history and
presentation and immediate appropriate treatment, is crucial. Because
anticoagulation is a component of therapy for many major acute and
chronic illnesses, recognition of this condition is crucial for prompt
intervention in a clinical practice.
Disclosure of Interest: None declared.
Objectives: Clinical probability and D-dimer measurement play an
essential role in the noninvasive diagnostic strategies for pulmonary
embolism (PE). PE can be ruled out without further imaging in
patients with non-high clinical probability and negative D-dimer. Ddimer level is increased in patients with renal impairment. Whether its
diagnostic usefulness is maintained in these patients is not well determined. We thus aimed to evaluate the effects of renal impairment on
diagnostic performances of D-dimer in patients with suspected PE.
Methods: A retrospective analysis of 1625 patients with suspected PE
included in a multicenter prospective study was performed. D-dimer
12/06/2014
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
ABSTRACTS
anticoagulation. We evaluated characteristics of patients that are not
switched from VKA to NOACs in daily care.
Methods: The prospective Dresden NOAC registry stopped enrolment
of dabigatran and rivaroxaban patients on February 28, 2013. Participating physicians were asked to enrol all VKA patients seen for regular INR testing, who remained on VKA at that day and for whom
switching to NOAC was not anticipated. VKA patients were followed
for 1 year.
Results: In total 50 physicians registered both patients on NOAC-therapy (n = 831) and on VKA therapy (n = 533). VKA indication was
arterial fibrillation (AF) in 430 (80.7%) cases, venous thromboembolism (VTE) in 62 (11.6%), mechanical heart valves in 21 (3.9%) and
other indications in 20 (3.8%) cases. Patients had a mean age of
72.5 years (range 28–93 y) and 317 (59.5%) patients were male. Mean
duration of VKA pre-treatment was 70.7 months (range 1–432 m).
Reasons for non-transition to NOAC are given in table 1, as stated by
the attending physician. Most frequent reason was ‘stable INR’
(93.4%).
As of January 31st 2014, completed FU correlated to 230.25 patient
years. At 6 months-FU (n = 460), 445 patients (96.7%) were still taking VKA, 11 patients (2.4%) were switched to other anticoagulants
and the remaining 4 patients (0.9%) stopped taking anticoagulants
completely.
Conclusion: All treatment strategies have similar efficacy and safety
profiles compared to LMWH/VKA. UFH/VKA was the least effective
regimen, whereas rivaroxaban and apixaban were associated with the
greatest risk reduction for safety.
Disclosure of Interest: None declared.
COA21
Effectiveness, safety and management of novel direct
oral anticoagulants in daily care – updated results of
the prospective Dresden noac registry (NCT01588119)
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Beyer-Westendorf J, Michalski F, Thieme C and Tittl L
Center for Vascular Diseases, University Hospital ‘Carl Gustav
Carus’, Technical University Dresden, Dresden, Germany
Objectives: Novel oral anticoagulants (NOAC) have been introduced
for long-term anticoagulant treatment in VTE and AF. However, the
effectiveness and safety of NOAC as well as management of peri-interventional and bleeding situations in daily care remain to be evaluated.
Methods: The prospective NOAC registry was initiated in November
2011. A network of more than 230 physicians in the district of Saxony,
Germany, enrol up to 2500 NOAC patients, which are prospectively
followed by the central registry office for up to 36 months.
Results: Until January 31st 2014, 2448 patients were enrolled into the
registry. of these, 1790 patients received rivaroxaban (564 for VTE,
1204 for SPAF, 22 off-label), 350 received dabigatran (342 for SPAF
and 8 off-label) and 308 received apixaban (302 for SPAF and 6 offlabel). About 30% of patients were switched from VKA, the remaining
were newly anticoagulated patients.
Currently, 13814 completed FU visits correlate to 5541.1 patientyears. We will present updated data on the rates, management and
outcome of major cardiovascular events, major and NMCR bleeding
events, scheduled and unscheduled surgical or interventional procedures and NOAC discontinuation rates. For instance, in the valid-for
safety-analysis of rivaroxaban patients, rates of major bleeding per
100 patient-years were 3.4 (95% CI 2.6–4.4) for all patients, 3.1 (95%
CI 2.2–4.3) for SPAF patients and 4.1 (95% CI 2.5–6.4) for VTE
patients. Bleeding-related mortality 90 days after major bleeding associated with rivaroxaban exposure was 5.1% only. Similar data for dabigatran and apixaban will be presented.
Conclusion: Our data are the first prospectively collected data on the
management and outcome of NOAC patients treated in daily care and
indicate that NOAC therapy is effective and safe with low rates of
major or fatal outcome events. Surgical or interventional procedures
are common and can usually be managed with short NOAC interruptions without LMWH bridging.
Disclosure of Interest: J. Beyer-Westendorf has grant/research support
from: Research grant from Bayer, Boehringer Ingelheim, Pfizer,
Speakers Bureau: Speakers honoraria from Bayer, Boehringer Ingelheim, Pfizer, F. Michalski: none declared, C. Thieme: none declared,
L. Tittl: none declared.
15
COA22
Reasons for not-switching from VKA to NOAC and
persistence on VKA – a VKA subgroup analysis of the
prospective Dresden NOAC registry (NCT01588119)
Beyer-Westendorf J, Michalski F, Thieme C and Tittl L
Center for Vascular Diseases, University Hospital ‘Carl Gustav
Carus’, Technical University Dresden, Dresden, Germany
Objectives: Currently, novel oral anticoagulants (NOAC) are replacing
vitamin-K antagonists (VKA) in many patients receiving long-term
Conclusion: NOAC-experienced physicians in private practises keep
around 40% of anticoagulated patients on VKA, mostly due to the
fact INR is considered to be stable or NOAC to be expensive. During
the following 6 months, < 5% of these patients need to discontinue
VKA treatment, indicating that the subjective assessment of the
attending physician identifies patients with acceptable VKA treatment
adherence.
Disclosure of Interest: J. Beyer-Westendorf has grant/research support
from: Research grants from Bayer, Pfizer, Boehringer Ingelheim,
Speakers Bureau: Speakers honoraria from Bayer, Pfizer, Boehringer
Ingelheim, F. Michalski: none declared, C. Thieme: none declared, L.
Tittl: none declared.
COA23
Centrally adjudicated cause of death during anticoagulant treatment – results of the prospective Dresden
NOAC registry (NCT01588119)
Beyer-Westendorf J, Michalski F, Thieme C and Tittl L
Center for Vascular Diseases, University Hospital ‘Carl Gustav
Carus’, Technical University Dresden, Dresden, Germany
Objectives: Novel oral anticoagulants (NOAC) have been approved
for acute and extended treatment of venous thromboembolism (VTE)
or long-term anticoagulation in atrial fibrillation (AF). A major concern of physicians is the fear of uncontrolled bleeding or cardiovascu-
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
12/06/2014
16
ABSTRACTS
prothrombin concentrates (PCC). We therefore evaluated the applicability of potential appropriate coagulation assays in analyzing the
reversal of NOAC anticoagulation by PCC.
Methods: PCC was titrated into plasma or whole blood spiked with
increasing concentrations rivaroxaban, apixaban or dabigatran and
samples were subjected to the following tests: prothrombin time (PT),
modified PT (mPT), activated partial prothrombin time (aPTT), thrombin generation (TGA) and thromboelastography (ROTEM, TEG).
Results: Although all examined coagulation assays showed sensitivity
towards NOACs, only the mPT and the parameters lag time (TGA)
and reaction time R (TEG triggered with tissue factor, TEG-TF)
revealed an in vitro effective concentration within the therapeutic range
for all three inhibitors examined. Assays triggered by contact activation
(aPTT, INTEM) did not show inhibitor reversal by PCC. Assays triggered by tissue factor showed NOAC type and NOAC concentration
dependent anticoagulation reversal effects of PCC ranging from partial
normalization to overcorrection of the following parameters: clotting
or reaction time (PT, mPT, TEG-TF, EXTEM, FIBTEM); angle in
thromboelastography (TEG-TF); TGA lag time, area under the curve
(AUC) and peak thrombin. Extent of reversal was tissue factor concentration dependent. TGA-AUC (5 pmol L–1 tissue factor, 4 lmol L–1
phospholipids) was the only parameter showing complete reversal of
anticoagulation by PCC for all NOACs ranging from 200 to 800 lg/L.
Conclusion: TGA-AUC fits with the concept that reversal assessment
of NOAC anticoagulation by PCC should be based on measurements
on the clotting potential or thrombin generating potential of the
plasma or whole blood patient sample. Low sensitivity of TGA-AUC
for NOACs and its correlation with bleeding are issues that remain to
be resolved.
Disclosure of Interest: H. J. Brinkman is an employee of: Sanquin,
manufacturer of the PCC used in the study, J. Dinkelaar: none
declared, S. Patiwael is an employee of: Sanquin, manufacturer of the
PCC used in the study, J. Harenberg has grant/research support from:
Dietmar Hopp Foundation for determination of anticoagulant effects
of DOAC in different matrices, A. Leyte: none declared.
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lar events during NOAC therapy resulting in fatal outcomes. We evaluated causes of death in a large prospective registry of NOAC
patients.
Methods: The prospective NOAC registry was initiated in November
2011. A network of more than 230 physicians in the district of Saxony,
Germany, enrol up to 2500 patients in the registry, which are prospectively followed by the central registry office for up to 36 months. Every
death was centrally adjudicated and categorized according to standard
definitions.
Results: Until January 31st 2014, 2448 patients were enrolled into the
registry. of these, 1790 (73.1%) patients received rivaroxaban, 350
(14.3%) received dabigatran and 308 (12.6%) received apixaban.
NOAC indication was arterial fibrillation (AF) in 1848 (75.5%) cases,
venous thromboembolism (VTE) in 568 (23.2%), and other indications in 32 (1.3%) cases. Patients had a mean age of 71.9 years (range
14–100y) and 1294 (52.9%) patients were male.
13814 completed FU correlated to 5541.1 patient years. In total 105
patients died (1.9 per 100 patient years). Causes of death are presented
in table 1.
Cardiovascular death was the most common cause of death but mainly
consisted of sudden cardiac death and chronic heart failure and rarely
consisted of acute thromboembolic events. Fatal bleeding accounted
for 5.7% of all fatalities and only occurred during anticoagulant therapy. More than 35% of deaths could be attributed to acute infections
or terminal malignant disease.
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Conclusion: In patients receiving NOAC therapy mortality mainly
relates to age, sudden cardiac death, malignant disease or acute infections. Thromboembolic events and fatal bleeding together account for
only 11.4% of all fatalities.
Disclosure of Interest: None declared.
COA24
How should we measure the reversal of direct factor
XA or thrombin inhibitors by prothrombin complex
concentrate?
Brinkman HJM1, Dinkelaar J2, Patiwael S1, Harenberg J3 and
Leyte A2
1
Plasma Proteins, Sanquin; 2Haematology and Clinical Chemistry
Laboratory, OLVG, Amsterdam, Netherlands; 3Clinical
Pharmacology Mannheim, Ruprecht-Karls-University Heidelberg,
Heidelberg, Germany
Objectives: Controversial data are reported on the usefulness of methods to determine the reversal of dabigatran, rivaroxaban and apixaban
(non-vitamin K direct oral anticoagulants (NOAC)) by 3 or 4 factor
COA25
No effect of platelet supplementation to reverse the
P2Y12 inhibitor ticagrelor: an in vitro study
Martin A-C1,2, Berndt C1, Gaussem P1,3, Gouin-Thibault I1,4,
Siguret V1,3, Le Bonniec B1, Bachelot-Loza C1 and Godier A1,5
1
INSERM, UMR-S1140; 2Cardiologie, HIA Val de gr^
ace, Paris;
3
Hematology, AP-HP, European Georges Pompidou Hospital;
4
Hematology, AP-HP, Cochin Hospital; 5Anesthesia and Critical
Care, Fondation Rotschild, Paris, France
Objectives: Ticagrelor, a reversible P2Y12 inhibitor, is recommended
for the treatment of acute coronary syndrome. There is currently no
available antidote in case of bleeding event. Platelet transfusion, the
current strategy to reverse other antiplatelet drugs, has been suggested
to be ineffective in this setting, although no data are available. We thus
assessed the potential of platelet supplementation to restore in vitro
platelet function inhibited by ticagrelor.
Methods: Whole blood (WB) from 18 blood donor volunteers was
spiked with ticagrelor (3.25 lM, equivalent to the peak concentration
after a 180 mg loading dose) or aspirin (25 lM), used as positive control. Platelet aggregation was performed by impedance aggregometry
on WB and light transmission (LTA) on platelet rich plasma (PRP)
using adenosine diphosphate (ADP) or arachidonic acid (AA) as spe-
12/06/2014
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
ABSTRACTS
cific agonists for ticagrelor and aspirin, respectively. Platelet supplementation was defined as the addition of washed platelet suspension,
corresponding to at least 60% of WB platelet count. Results are
expressed in ohms or maximal percentage of aggregation for impedance and LTA, respectively.
Results: Ticagrelor strongly inhibited ADP-induced platelet aggregation compared to control either in WB (1.8 Ω vs.8.8 Ω, P < 0.05,
n = 6) or in PRP (14% vs.77% P < 0.05, n = 6). As well, aspirin inhibited AA-induced WB aggregation (1.3 Ω vs.6 Ω, P < 0.05, n = 6).
Platelet supplementation of aspirin-treated samples fully restored AAinduced platelet aggregation (9.8 Ω vs. 1.3 Ω, P = 0.008). In contrast,
platelet supplementation failed to correct the antiplatelet effects of ticagrelor on ADP-induced aggregation both in WB (1.5 Ω vs. 1.3 Ω,
P > 0.05) and PRP (13% vs. 14%, P > 0.05).
Conclusion: This in vitro study shows that platelet supplementation
fails to restore platelet aggregation in the presence of ticagrelor. Our
results support the hypothesis for an inefficacy of platelet transfusion
to control bleeding in patients receiving ticagrelor.
Disclosure of Interest: None declared.
17
Conclusion: Coadministration of EDX with RIF results in decreased
EDX exposure, but increased active metabolite exposure and minimal
change in anticoagulant effects, presumably due to increased contribution from active metabolites. Thus, no change in dosing of EDX is
required with RIF.
Disclosure of Interest: J. Mendell is an employee of: Daiichi Sankyo
Pharm Development, S. Chen is an employee of: Daiichi Sankyo
Pharm Development, L. He is an employee of: Daiichi Sankyo Pharm
Development, M. Desai is an employee of: Daiichi Sankyo Pharm
Development, D. Parasrampuria is an employee of: Daiichi Sankyo
Pharm Development.
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COA26
The effect of rifampin on the pharmacokinetics (PK)
and pharmacodynamics (PD) of edoxaban in healthy
subjects
COA27
Healthcare cost of recurrent venous thromboembolism
and bleeding events among patients with acute venous
thromboembolism
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Objectives: Edoxaban (EDX) is a direct oral FXa inhibitor. It undergoes limited metabolism via carboxylesterases and cytochrome P450
(CYP) 3A4 and is a substrate for the efflux transporter, P-glycoprotein
(P-gp). This drug interaction study evaluated the effect of multiple
doses of rifampin (RIF), a CYP and P-gp inducer and organic anion
transporting polypeptides (OATP) inhibitor on the single dose PK and
PD of edoxaban and its active metabolites, M4 and M6.
Methods: This was an open-label, fixed-sequence study in healthy subjects, who received a single oral dose of EDX 60 mg on 2 occasions –
prior to administration of RIF and concomitantly with RIF on Day 7,
after RIF 600 mg BID was administered for 7 days. Plasma concentrations of EDX, M4, and M6 were measured, along with PD markers
PT and aPTT.
Results: Thirty-four subjects, ages 21 to 44 y were enrolled, and 32
completed the study. The PK parameters of EDX and metabolites are
presented in the Table 1. Concurrent with a decrease in edoxaban
exposure, the exposure to active metabolites, M4 and M6, increased.
Thus, overall PD effects associated with anticoagulant activity showed
minimal (<10%) change (Figure).
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Daiichi Sankyo Pharma Development, Edison, USA
Table 1 PK parameters of EDX and its metabolites
Edoxaban
Parameter
Cmax
(ng mL–
1
)
tmax (h)a
AUC∞
(ng.h
mL–1)
t1/2 (h)
CL/F (L/
h)
M4
M6
EDX
N = 34
EDX + RIF
N = 32
EDX
N = 34
EDX + RIF
N = 32
EDX
N = 34
EDX + RIF
N = 32
243 100
257 61.8
23.1 13.2
108 33.0
8.55 4.10
44.5 12.3
1.08 (0.50,
4.00)
1835 442
1.00 (0.50,
3.00)
1192 214
1.81 (1.00,
3.05)
161 69.8
2.00 (1.00,
3.00)
449 140
1.49 (0.983,
3.00)
91.1 28.2
1.00 (0.617,
3.00)
261 57.4
13.6 6.06
34.8 9.22
6.54 4.24
52.0 9.76
14.3 6.0
NC
4.23 3.78
NC
14.3 5.58
NC
10.1 3.81
NC
Shorr A1, Johnston S2, Jing Y3, Schaiff R4, Trocio J4, Thomson E2,
Juneau P2 and Graham J3
1
Pulmonary & Critical Care Medicine Service, Washington
Hospital Center, Washington; 2Truven Health Analytics,
Bethesda; 3Bristol-Myers Squibb, Plainsboro; 4Pfizer Inc., New
York, USA
Objectives: Outcomes and costs associated with acute venous thromboembolism (VTE) have been well described; however, little information exists illustrating the costs associated with recurrent VTE events
and with bleeding complications following the acute anticoagulation
period.
Methods: We conducted a retrospective cohort study using U.S.
health insurance claims data. Recurrent VTE (hospitalizations/emergency department [ED] visits) and bleeding over a 1-year period after
acute VTE served as our co-primary endpoints. We identified recurrent VTE events based on ICD-9-CM coding and defined bleeding
based on previously published criteria to identify either major bleeding (MB) or clinically-relevant non-major bleeding (CRNMB). We
included patients who were ≥ 18 years, had experienced a non-fatal
hospitalization or ED visit with a principal diagnosis of VTE between
1-Jan-2007 and 31-Mar-2013 (first visit = index) and had ≥ 1 claim
for anticoagulation (warfarin, enoxaparin, rivaroxaban) or placement
of an inferior vena cava filter ≤ 30 days after index. Per-patient permonth (PPPM) total healthcare costs were compared between
patients with vs. without recurrent VTE or bleeding (separately) using
multivariable regressions.
NC, not calculated.
Data presented as arithmetic mean SD.
a
Median (Min, Max).
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
12/06/2014
ABSTRACTS
therapy (RR 0.83; 95% CI 0.51 to 1.36; P = 0.0008 for NI), whereas
major bleeding occurred in 5 patients (0.19%) and 27 patients
(1.00%), respectively (P < 0.0001 for superiority).
Conclusion: Compared with conventional therapy, apixaban was noninferior for efficacy at 7 and 21 days, and produced less major bleeding. These findings indicate that (i) the use of a higher apixaban dose
for the initial 7 days did not compromise the safety of apixaban and
(ii) the good overall benefit-to-risk profile of this all-oral apixaban regimen begins early in treatment.
Disclosure of Interest: G. Raskob Consultant for: Served as a consultant and has received honoraria from Pfizer and BMS, A. Gallus Consultant for: Served as a consultant and has received honoraria from
Pfizer and BMS, G. Agnelli Consultant for: Served as a consultant and
has received honoraria from Pfizer and BMS, H. Buller Consultant
for: Served as a consultant and has received honoraria from Pfizer and
BMS, A. Cohen Consultant for: Served as a consultant and has
received honoraria from Pfizer and BMS, S. Chaudhuri is an employee
of: Pfizer Ltd, P. Sanders is an employee of: Pfizer Ltd, J. Thompson is
an employee of: Pfizer Inc, J. Weitz Consultant for: Served as a consultant and has received honoraria from Pfizer and BMS.
Results: The final cohort included 123,809 patients (mean age 59 years,
50% male). Recurrent VTEs, MB, and MB/CRNMB occurred in
16,992, 9571, and 22,446 patients, respectively, over 1 year of follow up.
PPPM costs for claims associated with recurrent VTE, MB, and MB/
CRNMB equaled $4224, $4110, and $1846, respectively. Adjusted excess
PPPM total healthcare costs related to recurrent VTEs, MB, and MB/
CRNMB equaled $4055, $4650, and $1974, respectively (all P < 0.01).
Conclusion: Recurrent VTEs and/or bleeding may complicate the course
of VTE, and the costs related to these events are substantial. Interventions which reduce these outcomes may confer substantial cost offsets.
Disclosure of Interest: A. Shorr Consultant for: Truven Health Analytics, S. Johnston is an employee of: Truven Health Analytics, Y. Jing is
an employee of: Bristol-Myers Squibb, R. Schaiff is an employee of:
Pfizer Inc., J. Trocio is an employee of: Pfizer Inc., E. Thomson is an
employee of: Truven Health Analytics, P. Juneau is an employee of:
Truven Health Analytics, J. Graham is an employee of: Bristol-Myers
Squibb.
COA28
Time course of recurrent venous thromboembolic and
major bleeding events during anticoagulant therapy
with apixaban or enoxaparin/warfarin: analysis of the
amplify data using landmark method
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Objectives: The AMPLIFY trial, which compared a 6-month
course of oral apixaban (10 mg bid for 7 days, then 5 mg bid)
with conventional therapy (enoxaparin followed by warfarin INR
2.0–3.0) in 5395 patients with symptomatic venous thromboembolism (VTE), demonstrated that apixaban is non-inferior for efficacy, but produces significantly less bleeding. Because recurrent
events cluster in the initial 3 weeks of treatment, we performed a
landmark analysis to compare the incidences of recurrent VTE/
VTE-related death and bleeding at two time points, 7 and
21 days, to determine whether apixaban was non-inferior to conventional therapy for efficacy and produced less bleeding at these
early time points.
Methods: This analysis used the same adjudicated outcomes of symptomatic recurrent VTE/VTE-related death and major bleeding, as in
the primary trial analysis, and applied the same non-inferiority (NI)
margin (relative risk [RR] 1.8).
Results: At 7 days, recurrent VTE/VTE-related death occurred in 18
of 2661 patients (0.68%) given apixaban and in 23 of 2676 patients
(0.86%) given conventional therapy (RR 0.79; 95% confidence interval [CI] 0.43–1.46; P = 0.0033 for NI), whereas major bleeding
occurred in 3 of 2676 patients (0.11%) and 16 of 2689 patients
(0.60%), respectively (P = 0.0029 for superiority). At 21 days, recurrent VTE/VTE-related death occurred in 29 of 2652 patients (1.09%)
given apixaban and 35 of 2667 patients (1.31%) given conventional
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Korte W1, Marlar R2, Krause M3 and Rathbun S2
Zentrum F€
ur Labormedizin, St. Gallen, Switzerland; 2Health
Sciences Center, University of Oklahoma, Oklahoma City, USA;
3
Deutsche Klinink Diagnostik GmbH, Wiesbaden, Germany
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Raskob GE1, Gallus AS2, Agnelli G3, Buller HR4, Cohen A5,
Chaudhuri S6, Sanders P7, Thompson J8 and Weitz JI9
1
College of Public Health, University of Oklahoma Health
Sciences Center, Oklahoma City, USA; 2Department of
Haematology, Flinders Medical Centre & Flinders University,
Adelaide, Australia; 3University of Perugia, Perugia, Italy;
4
Department of Vascular Medicine, Academic Medical Center,
Amsterdam, Netherlands; 5King’s College Hospital, London;
6
Pfizer Ltd, Sandwich; 7Pfizer Ltd, Tadworth, UK; 8Pfizer Inc,
Groton, USA; 9McMaster Univeristy and Thrombosis and
Atherosclerosis Research Institute, Hamilton, Canada
COA29
The prothrombinase induced clotting time test (PICTâ),
a robust alternative to aptt in the management of ufh
therapy
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Objectives: To validate PefakitâPICTâ as alternative to aPTT in the
managment of patients undergoing UFH therapy.
Methods: 377 samples distributed over 3 centers were obtained from
informed consented patients undergoing UFH therapy. Each sample
was analyzed making use of aPTT (HemosIL aPTT SP, IL), anti-Xa
(Biophen Heparin 6, Aniara) and PefakitâPICTâ (DSM Pentapharm).
Dose response curves were obtained by plotting aPTT or PiCTâ clotting times vs. UFH concentration as determined by anti-Xa activity.
Therapeutic ranges were established as the clotting time spanning a
concentration of 0.3–0.7 U mL–1 calculated from regression analyses.
Incorrect diagnosis is defined as incorrect in or out therapeutic range
classification by respective clotting time-based assay compared to antiXa results. A 15% is used as model for clinically significant clotting
time variation (Adcock et al 1998 9:463)
Results: The correlation between anti-Xa activity and PiCTâ is significantly higher than between aPTT and anti-Xa activity and higher
specificity and lower overall error in diagnosis. The proportion of samples with a clinically relevant error (outside a 15% margin of the
expected clotting time) is significantly reduced with PiCTâ
Parameter/Test
Pefakitâ PiCTâ
aPTT
P
r (95% CI)
0.88
(0.86–0.90)
16
0.76
(0.71–0.80)
26
<0.0001
0.0002
29.7
66.8
<0.0001
Incorrect
Diagnosis%
% D ≥ 15%
calculated CT
12/06/2014
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
ABSTRACTS
19
patients treated with apixaban, rivaroxaban, edoxaban, and dabigatran, respectively vs. patients treated with standard therapy over the
trial durations (Table). The medical cost avoidance associated with use
of NOACs vs. standard therapy were similar in the sensitivity analysis
when differences in treatment durations were taken into account.
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COA31
Incremental healthcare burden of bleeding among
patients with venous thromboembolism in the US
Amin A1, Jing Y2, Trocio J3, Lin J4, Lingohr-Smith M4 and Graham J2
University of California, Irvine; 2Bristol-Myers Squibb,
Plainsboro; 3Pfizer, New York; 4Novosys Health, Flemington, USA
iza
Conclusion: Pefakitâ PiCTâ shows a stronger level of correlation with
anti-Xa than aPTT, with singificantly more samples correctly identified when used as anti-Xa surrogate in three different clinical centers.
In this sense, Pefakitâ PiCTâ appears as a more robust and reliable
test when managing patients under UFH therapy. Therefore, it might
represent the long time sought for alternative to the aPTT for this
application.
Disclosure of Interest: W. Korte has grant/research support from: Clinical Study DSM Pentapharm, R. Marlar has grant/research support
from: Clinical Study DSM Pentapharm, M. Krause has grant/research
support from: Clinical Study DSM Pentapharm, S. Rathbun has
grant/research support from: Clinical Study DSM Pentapharm.
Conclusion: When any of the four NOACs are used instead of standard
therapy for VTE treatment medical costs are reduced. Apixaban is
associated with the greatest reduction in medical costs, which is driven
by medical cost reductions associated with both efficacy and safety
endpoints. Further evaluation may be needed to validate these results
in the real-world setting.
Disclosure of Interest: A. Amin Consultant for: Bristol-Myers Squibb,
Pfizer, Y. Jing is an employee of: Bristol-Myers Squibb, J. Trocio is an
employee of: Pfizer, J. Lin is an employee of: Novosys Health, M.
Lingohr-Smith is an employee of: Novosys Health, J. Graham is an
employee of: Bristol-Myers Squibb.
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COA30
Evaluation of medical costs associated with use of new
oral anticoagulants compared with standard therapy
among patients with venous thromboembolism based
on clinical trial results
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Graham J2
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University of California, Irvine; 2Bristol-Myers Squibb,
Plainsboro; 3Pfizer, New York; 4Novosys Health, Flemington, USA
Objectives: This study evaluated differences in medical costs associated
with clinical endpoints from randomized trials that compared the new
oral anticoagulants (NOACs), dabigatran, rivaroxaban, apixaban, and
edoxaban to standard therapy for treatment of patients with venous
thromboembolism (VTE).
Methods: Event rates of efficacy and safety endpoints, as defined in
clinical trials (AMPLIFY, EINSTEIN-Pooled, RECOVER-1, RECOVER II, Hokusai-VTE trial), were obtained from published trial
data. One year incremental costs among patients with clinical events
from a US payer perspective were obtained from the literature or
healthcare claims database and inflation adjusted to 2013 costs. Differences in medical costs for clinical endpoints associated with use of each
of the NOACs vs. standard therapy were then estimated. A sensitivity
analysis was also conducted in which all drug treatment duration was
normalized to 6 months.
Results: A lower rate of major bleedings was associated with use of any
of the NOACs vs. standard therapy. Except for dabigatran, use of any
of the NOACs was also associated with a lower rate of recurrent VTE/
death. As a result of the reduction in clinical event rates overall medical costs were reduced by $918, $482, $344, and $146 for VTE
1
Objectives: This study evaluated healthcare resource utilization and
costs associated with major bleeding (MB) and clinically relevant nonmajor bleeding (CRNMB) among patients with venous thromboembolism (VTE).
Methods: Patients (≥ 18 years of age and continuously insured) with a
diagnosis of VTE (1/1/2008–12/31/2011) were identified from MarketScan claims databases. Patients who did not have any bleeding during
the study period were grouped into a no bleeding cohort and a random
date after VTE diagnosis was selected as the index date. VTE patients
who experienced MB within 1 year of the initial VTE diagnosis were
grouped into a MB cohort and patients without MB, but with CRNMB
were grouped into a CRNMB cohort. All cause and bleeding related
healthcare resource utilization and payments (inflation adjusted to
2013 level) during a 12-month follow-up period after the index date
(included in follow-up period) of the initial bleeding event were measured and compared. Multivariable generalized linear models were
used to evaluate incremental healthcare payments after adjusting for
key patient characteristics.
Results: Among the 112,885 patients identified with a VTE diagnosis,
14% (n = 15,897) had MB and 14% (n = 15,842) had CRNMB; 72%
(n = 81,146) had neither of these events occur during the study period.
For patients in MB and CRNMB cohorts the total mean bleeding
related inpatient and outpatient medical claim payments during the
follow-up period were $49,779 [Std Error: $820] and $2,187 [$89]
respectively. After adjustment for key patient characteristics the estimated mean differences in total bleeding related medical payments
were $45,367 [$1,853] for patients with MB and $2,140 [$88] for
patients with CRNMB vs. patients with no bleeding.
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
12/06/2014
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ABSTRACTS
COA33
Appropriateness of management of target specific anticoagulants in an inner-city population
Rash C, Cheng WH, Galanter W, Hellenbart E, Shapiro N and
Nutescu E
University of Illinois at Chicago, Chicago, USA
Conclusion: Among patients with VTE diagnosis in the US, approximately 28% have a bleeding event within 1 year of VTE diagnosis and
about half of these patients experience MB. Patients with MB have
greatly elevated healthcare costs.
Disclosure of Interest: A. Amin Consultant for: Bristol-Myers Squibb,
Pfizer, Y. Jing is an employee of: Bristol-Myers Squibb, J. Trocio is an
employee of: Pfizer, J. Lin is an employee of: Novosys Health, M.
Lingohr-Smith is an employee of: Novosys Health, J. Graham is an
employee of: Bristol-Myers Squibb.
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COA32
Sensitivity of prothrombin time to the presence of rivaroxaban varies substantially between reagents and
subjects
Jones R1, Woolley A1, Maclean R2, van Veen J2 and Kitchen S1
Coagulation; 2Haematology, Royal Hallamshire Hospital,
Sheffield, UK
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Objectives: Several studies have reported that the sensitivity of the
prothrombin time (PT) to rivaroxaban depends on the reagents used
for PT determination. Some guidelines therefore recommend that
laboratories should know the sensitivity of their PT reagent to the
presence of rivaroxaban. We have evaluated the effects of rivaroxaban on the PT with different reagents including some where data
are lacking.
Methods: PTs were performed on plasma samples from 10 normal volunteers spiked with rivaroxaban to attain 8 concentrations in the range
0–800 ng mL–1 (based on Anti Xa assay) using the following reagent/
instruments: Innovin/CS2100; Thromborel S/CS2100; PT Fib HS Plus
/IL TOP; Recombiplastin 2G/IL TOP: TriniClot XLS/Destiny Plus.
Results in the presence of drug were divided by the results in the
absence of drug to derive PT ratios
Results: The mean PT ratio in the presence of 120 ng mL–1 were as follows; Innovin – 1.14, Thromborel S, 1.13; Recombiplastin – 1.36; PT
Fib HS+ – 1.51; Triniclot XLS- 1.62. For the sample containing
275 ng mL–1 PT ratios were 1.3 (Innovin and Trel S), 1.8 with recombiplastin, 2.0 with PT Fib HS + and 2.22 with Triniclot XLS. When
the sample contained 800 ng mL–1 rivaroxaban PT ratios were on
average 1.7 and 1.8 with Innovin and Thromborel S compared to 3.0
and 3.3 with Recombiplastin and PT Fib HS plus and 3.8 with triniclot
XLS. For all reagents there was considerable variability between individuals in the sensitivity to presence of drug.
Conclusion: Sensitivity of the PT to the presence of rivaroxaban was
reagent dependant and varies between individual subjects. Triniclot
XLS, PT HFib HS plus and Recombiplastin 2G were more sensitive
than Innovin or Thromborel S.
Disclosure of Interest: R. Jones: none declared, A. Woolley: none
declared, R. Maclean Speakers Bureau: lecture fees, J. van Veen
Speakers Bureau: lecture fees, S. kitchen Speakers Bureau: lecture
fees.
Objectives: This study’s aim was to assess the appropriateness of anticoagulation (AC) management in patients treated with target specific
anticoagulants (TSOAC).
Methods: This was a prospective, cross-sectional cohort study in an
inner-city population treated with warfarin or a TSOAC for more than
3 months. Appropriateness was defined as documented assessments of
laboratory parameters, drug-drug interactions, AC adherence, and
performance of patient education at least every 12 months (6 months
if CrCl ≤ 50 mL/min) for TSOACs and at least every 2 months for
warfarin. Social-demographic characteristics, AC related outcomes,
and appropriateness of AC management between two treatment
groups were compared by two-sample independent t-test for continuous variables, and chi-square test for categorical variables.
Results: A total of 138 patients were included, 73 on warfarin and 65
on TSOAC. Patients on warfarin had an average of 15 clinic visits in
12 months, with the same amount of drug-drug interactions, adherence, patient education, and laboratory test assessments. In contrast,
patients on TSOAC averaged two clinic visits in 12 months, with no
documented assessments for drug-drug interactions, 0.3 adherence
assessments, 1.8 occurrences of patient education, and 1 assessment of
laboratory tests (P < 0.05 for each, compared to warfarin). Among 20
patients with CrCl ≤ 50 mL/min on warfarin, 100% received one or
more assessments in each of the four areas in the preceding 6 months.
However, none of the 7 patients on a TSOAC with CrCl ≤ 50 mL/min
received all four appropriateness assessment components during either
the preceding 6 or 12 months. Appropriateness of management for
renal impairment patients was significantly worse in TSOAC group
compared with warfarin (P < 0.001).
Conclusion: Appropriateness of AC management was worse in patients
treated with TSOAC compared to warfarin, especially in the domains
of adherence and drug-interaction and in patients with renal impairment.
Disclosure of Interest: None declared.
COA34
Spinart trial 3-year results: improved quality of life in
adults using prophylaxis with Bayer’s sucrose-formulated recombinant factor VIII
Pocoski J, Silberman C, Humphries T, Walker D and Hong W
Bayer HealthCare Pharmaceuticals, Whippany, USA
Objectives: Health-related quality of life (HRQoL) was assessed in the
3-year SPINART study, which compared routine prophylaxis vs. ondemand treatment with Bayer’s sucrose-formulated recombinant
FVIII (rFVIII-FS) in adults with severe hemophilia A. Here, we report
patient perspective on HRQoL, an important component for measuring treatment effectiveness, using a hemophilia adult specific QoL
instrument (Haemo-QoL-A) and a generic QoL instrument (EuroQoL
5D [EQ-5D]).
Methods: SPINART, an open-label, randomized, controlled, multinational study enrolled previously treated patients aged 12–50 years with
severe hemophilia A and no prophylaxis for > 12 consecutive months
in the past 5 years. Patients received rFVIII-FS on demand or as prophylaxis (25 IU/kg 3 times weekly). Haemo-QoL-A assessments (6
domains) were performed at baseline, month 6, and years 1, 2, and 3
(higher Haemo-QoL-A scores indicate better HRQoL). Betweengroup comparison was made using constrained longitudinal data
analysis. The mean distribution-based clinically important difference
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© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
ABSTRACTS
21
bd dosing), and were within the normal reference range at < 479 ng
mL–1. At 91 and 479 ng mL–1 the PT results with PTHS + (most sensitive method) were 1.8 and 9.0 s greater than baseline respectively.
APTT with the least (Actin FS/CS2100i analyzer) and most (APTT
SP/IL TOP analyzer) sensitive method were 2.1 and 3.2 s prolonged
respectively at 91 ng mL–1. At 479 ng mL–1 APTTs were 4.4 and 7.9 s
greater than baseline respectively.As expected the Clauss fibrinogen
and Thrombin Time were unaffected at all concentrations.
Conclusion: Sensitivity of PT and APTT is reagent dependant and prolongation beyond baseline results was minor even with therapeutic levels of Apixaban. Therefore PT and APTT are not clinically useful tests
for measuring the anticoagulant effect of Apixaban
Disclosure of Interest: None declared.
(MCID) for Haemo-QoL-A ranged from 5.2–7.2 for the total score
and 6.2–9.1 for the physical functioning domain). EQ-5D assessments
included the utility index scores (MCID, 0.074) and the visual analog
scales (VAS).
Results: of the 84 patients (42 prophylaxis, 42 on demand) in the
intent to treat population, Haemo-QoL-A data were available for
41 and 42 patients, respectively. The least squares (LS) mean change
from baseline to year 3 for Haemo-QoL-A (total and domain-specific) for the 2 treatment groups is shown in the Table. The mean
change in EQ-5D VAS score from baseline to year 3 for patients
receiving prophylaxis and on-demand treatment was 1.80 and
10.49, respectively; corresponding utility index scores were 0.01
and 0.06, respectively.
COA36
Determinants of anticoagulation adherence using Morisky medication adherence scale
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Carrier M1,2,3
1
The Ottawa Hospital; 2University of Ottawa; 3Ottawa Hospital
Research Institute, Ottawa, Canada; 4Maastricht University,
Maastricht, Netherlands
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Conclusion: HRQoL showed improvement over 3 years with prophylaxis compared with on-demand use in adults with severe hemophilia
A as reflected by Haemo-QoL-A and EQ-5D assessments.
Disclosure of Interest: J. Pocoski is an employee of: Bayer HealthCare
Pharmaceuticals, C. Silberman is an employee of: Bayer HealthCare
Pharmaceuticals, T. Humphries is an employee of: Bayer HealthCare
Pharmaceuticals, D. Walker is an employee of: Bayer HealthCare
Pharmaceuticals, W. Hong is an employee of: Bayer HealthCare Pharmaceuticals.
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Objectives: Poor adherence to prescribed medications is a common
challenge among patients with chronic illness. The efficacy and safety
of anticoagulation therapy may be reduced in patients with poor drug
adherence. Direct oral anticoagulants (DOAC) have a shorter half-life
compared to vitamin-K antagonists (VKA), raising theoretical concerns
of higher risk of recurrent venous thromboembolism or systemic embolism in patients with poor adherence to DOACs. Self-reported adherence of patients on anticoagulant therapy is seldom reported in the
literature. This study aimed to assess self-reported adherence profiles of
patients prescribed oral anticoagulants and to compare adherence of
patients on VKA and DOACs (rivaroxaban, dabigatran, apixaban).
Methods: A cross-sectional study was conducted in an outpatient anticoagulation clinic. Adult patients taking oral anticoagulants completed an anonymous survey (22 questions) including basic
demographic data and self-reported medication adherence. The outcome measure was the 4-item Morisky score to assess self-reported
adherence. Non-adherence was defined as patients with ≥ 1 positive
answers to at least one question from the Morisky scale.
Results: of 500 patients, 277 (56.5%) had good adherence to oral anticoagulation. Multivariate logistic regression was conducted: female
gender (P = 0.01), older age (P < 0.01), and use of other medications
(P < 0.01) were associated with good anticoagulant adherence. No difference in adherence with use of VKA vs. DOACs was identified.
Conclusion: Patients requiring long-term anticoagulation self-report poor
drug adherence. There are no major differences in self-reported rates of
adherence between VKA and DOACs. Factors contributing to good
adherence are female gender, older age and use of additional medications.
Disclosure of Interest: None declared.
COA35
The effects of apixaban on routine coagulation tests
including prothrombin time and activated partial
thromboplastin time
Lowe A1, Hickey K1, Maclean R2, van Veen J2 and Kitchen S1
Coagulation; 2Haematology, Royal Hallamshire Hospital,
Sheffield, UK
1
Objectives: Apixaban is a direct FXa inhibitor with predictable pharmacokinetics and pharmacodynamic properties with no required laboratory monitoring in clinical trials. We have evaluated the effects of
Apixaban on routine coagulation tests using a range of drug concentrations.
Methods: Prothrombin Time (PT) (Innovinâ, ThromborelâS, Recombiplastin 2Gâ and PTHS+â), Activated Partial Thromboplastin Time
(APTT) (Synthasilâ, Actin FSâ and APTT SPâ) Clauss Fibrinogen
(C.Fbg) (Thrombinâ) and Thrombin Time (TT) (Thromboclotinâ)
were performed on plasma samples from 10 normal volunteers spiked
with Apixaban to attain 8 concentrations 0–1000 ng mL–1 based on
Anti Xa results.
Results: PT results using Innovinâ CS2100i analyzer (least sensitive
method) revealed a mean prolongation of PT of 0.6 s at 91 ng mL–1
(approximately equivalent to mean peak concentrations during 5 mg
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
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ABSTRACTS
COA37
Anticoagulation reversal and event study collaborative
(ARES) – initial results
COA38
Measuring dabigatran with the dilute Russell viper
venom confirm assay in an anticoagulation clinic population
Baker RI1,2,3, Curnow J4, Brighton T5, Harper P6, Angelatos W2,
James I3, Gallus A7 and on behalf of on behalf of the ARES
Collaborative Investigators of the Australasian Society of
Thrombosis and Haemostasis (ASTH)
1
Western Australian Centre for Thrombosis and Haemostasis,
Murdoch University; 2Perth Blood Institute; 3Institute of
Immunology and Infectious Disease, Murdoch University, Perth;
4
Haematology, Concord Hospital; 5Haematology, Prince of Wales
Hospital, Sydney, Australia; 6Haematology, Palmerston North
Hospital, Palmerston North, New Zealand; 7Haematology,
Flinders Medical Centre, Adelaide, Australia
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Objectives: The Stago Sta-Clot DRVV Confirm is a clot-based dilute
Russell viper venom assay employed to confirm lupus anticoagulant.
We compared DRVVC to the Biophen Hemoclot Thrombin Inhibitor
Assay for its ability to measure dabigatran in samples from anticoagulation clinic patients. The protocol was sponsored by the USAF Office
of the Surgeon General and monitored by the local IRB.
Methods: Consented subjects were 18 or older with creatinine clearances (CCR) > 30 mL/min. All were taking 150 mg oral dabigatran
BID for at least 1 month. A subject who had one kidney but adequate
CCR was taking 75 mg BID for at least 1 month. Subjects were not
excluded for other medications or health issues. We enrolled 102 subjects: 64 males, mean age 77; 38 females, mean age 76. The mean
CHA2DS2-VASc score was 3.2, mean BMI was 29.8. A monthly 3.2%
sodium citrate specimen was collected from each subject
within 5 days of the same date of the first month for a total of 6
monthly specimens, yielding 418 data points after attrition. We did
not correlate specimen collection time with medication time. We collected specimens from 44 subjects who were not on dabigatran to
establish reference intervals (RIs). Specimens were centrifuged immediately after collection to produce platelet poor plasma, aliquotted,
and stored at 70°C. Specimens were rapidly thawed and mixed
immediately prior to analysis. An HTI and a DRVVC was performed
on each using a STAR-Evolution coagulometer (Stago).
Results: The DRVVC RI was 35.9–42.4 s (mean 34.7 2 SD). Normal HTI dabigatran concentrations were zero. Test sample DRVVC
range was 42.2–193.9 s, mean 97.6; test sample HTI range was 0.0–
770 ng mL–1, mean 184.1. Linear regression comparing DRVVC to
HTI generated an r = 0.86. DRVVC failed linearity at levels compared
to HTI values below 30 and above 550 ng mL–1.
Conclusion: The DRVVC, a readily available assay, could be employed
to measure dabigatran within the range of 30–550 ng mL–1.
Disclosure of Interest: None declared.
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Objectives: The ARES Collaborative is a large multicentre international observational prospective study of consecutive patients who
present with hemorrhage, urgent surgery or thromboembolism who
are taking the new oral anticoagulants (NOAC - dabigatran, rivaroxaban, apixaban) or warfarin therapy. The clinical circumstances and
severity of the event is recorded and the hemostatic strategies observed
for urgent anticoagulant reversal or treatment of thromboembolism.
Methods: Data from the first 130 patients from the initial active 5 centers were analyzed. Outcome measures include detailed description of
the presenting population, coagulation and specific assays for NOAC,
type and efficacy assessment of reversal agent used for major hemorrhage and management of thromboembolism.
Results: ARES patients were elderly (mean age 74.1 13.2 years) and
almost all (96.3%) had a creatinine clearance of > 30 mL/min. The
main indications for OAC were atrial fibrillation (64%), venous
thromboembolism (26%) and mechanical heart valves (10%). 26% of
patients were on NOACS (Dabigatran 15%, Rivaroxaban 11%). The
median duration on OAC was 18 months (range 0.1–477 months).
Presenting events include hemorrhage (65%), urgent reversal for surgery (20%) or thromboembolism (15%). Life threatening hemorrhage
occurred in 31% of patients with the gastrointestinal tract the major
site. 27% of patients were on concurrent anti-platelet agents. For
patients on a NOAC, over half received specific hemostatic agent
including Prothrombinex-VF, FEIBA or rFVIIa compared to 92% of
patients of warfarin (Prothrombinex-VF). Length of stay was prolonged (mean 12 1.4 days) and 30 day mortality was 13%.
Conclusion: Initial analysis of ARES data provides important observational data concerning current anticoagulant practice in patients on
the new oral anticoagulants when compared to those on warfarin.
ARES is now recruiting 2000 patients in 20 sites to provide comprehensive data that will improve anticoagulation practice.
Disclosure of Interest: R. Baker has grant/research support from: for
clinical trials from Biogen Idec, Boehringer Ingelheim, Bayer, Baxter
Healthcare, Pfizer, Daiichi Sankyo, Astellas and CSL Behring, has
participated in clinical advisory boards for Amgen, Biogen Idec, Baxter Healthcare, Boehringer Ingelheim, Bayer, and Pfizer, and has
received conference travel support from Novo Nordisk, Baxter
Healthcare and Alexion pharmaceuticals., J. Curnow has grant/
research support from: Bayer, Consultant for: Pfizer/BMS,Boehringer
Ingelheim, Speakers Bureau: Bayer, Boehringer Ingelheim, T. Brighton Consultant for: Bayer and Boehringer Ingelheim, P. Harper Shareholder of: INR Online Ltd, W. Angelatos: none declared, I. James:
none declared, A. Gallus Consultant for: Bayer,Boehringer Ingelheim,
BMS, Daiichi Sankyo & Pfizer.
McGlasson DL1, Fritsma GA2, Ezzell EE3 and Anderson NA4
Hematology/Hemostasis Research, 59th Clinical Research
Division, Lackland; 2Hematology/Hemostasis Research,
University of Alabama Birmingham, Birmingham AL;
3
Hematology/Hemostasis Research, Travis AFB, Travis AFB;
4
Cardiology, SAMMC, Ft. Sam Houston, TX, USA
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COA39
Measuring new oral anticoagulants in thrombin
generation on Ceveronâ alpha
Binder N1, Wagner L1 and Olteanu A2
1
Technoclone GmbH, Vienna, Austria; 2SCJU, Sibiu, Romania
Objectives: The increasing use of the new oral anticoagulants creates
the need of their measurement in clinical routine. A modified thrombin
time-based assay can be used for the measurement of thrombin inhibitors and the direct Xa inhibitors can be measured with a chromogenic
anti-Xa assay. However, these assays only measure the initiation phase
of the coagulation cascade. In this study we asked the question if the
thrombin generation assay, which measures the entire thrombin generation process, could be used to better discriminate the inhibitory profile of the NOACs in patients.
Methods: For this proof of concept study, platelet-poor plasma spiked
with apixaban, rivaroxaban, dabigatran, arixtra or LMWH as well as
warfarin plasma with different INR values was tested in the TGA. As
initiators of thrombin generation two triggers differing in phospholipid concentrations were used. Analysis was performed on the coagulation analyzer Ceveronâ alpha.
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© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
ABSTRACTS
Results: All anticoagulants inhibited thrombin generation in a concentration-dependent manner after activation with both triggers, influencing all TGA parameters. Peak thrombin decreased from 521 nm in
normal plasma to 122 nm at 500 ng mL–1 dabigatran, and to 94 nm at
433 ng mL–1 rivaroxaban. The reduction at 1.76 IU mL–1 LMW was
to 6.9 nm and at INR 3.5 Peak thrombin value was 54 nm. In addition, dabigatran prolonged the Lag Time from 2.5 min to 11.5 min at
500 ng mL–1, the prolongation with rivaroxaban at 450 ng mL–1 was
to 5.7 min. For the direct Xa inhibitors peak thrombin is the most sensitive parameter and for the direct thrombin inhibitor dabigatran it is
the lag time and Peak thrombin and Area under the curve are inhibited
to the same extend, similar to LMWH and warfarin. At high dabigatran concentrations the inhibition is close to 100%. For direct Xa
Inhibitors AUC gives a plateau at 50% inhibition.
Conclusion: Thrombin generation inhibition by NOACs at clinically
relevant plasma levels could serve as a fine-tuned indicator for hemostatic balance in patients using the NOACs.
Disclosure of Interest: N. Binder is an employee of: Technoclone
GmbH, L. Wagner is an employee of: Technoclone GmbH, A. Olteanu: None declared.
Vis Consultant for: Bayer Pharma AG, B. Ploeger is an employee of: is
an employee of Bayer Pharma AG.
COA41
Switching from dabigatran or rivaroxaban to edoxaban:
a biomarker assessment in healthy subjects
Parasrampuria DA1, Weilert D2, Maa J-F3, Dishy V1, Kochan J1,
Shi M1 and Brown KS1
1
Daiichi Sankyo Pharma Development, Edison; 2Quintiles,
Overland Park; 3Daiichi Sankyo, Inc., Parsippany, USA
Objectives: This 3-way crossover study evaluated the anticoagulant
effects, safety, and tolerability of edoxaban when dosed alone or upon
switching from dabigatran or rivaroxaban.
Methods: Healthy subjects (N = 24) were randomized to edoxaban
60 mg QD X 4 days (Treatment A); dabigatran 150 mg BID X 3 days,
then edoxaban single dose 60 mg in the morning of day 4 (Treatment
B); or rivaroxaban 20 mg QD X 3 days, then edoxaban single dose
60 mg in the morning of day 4 (Treatment C). The primary anticoagulant assessment for switching from rivaroxaban was prothrombin time
(PT) and for switching from dabigatran was activated partial thromboplastin time (aPTT). Anti-FXa, bleeding time, and thrombin generation were also assessed. Safety assessments including adverse events
(AEs) were monitored throughout the study.
Results: PT values for switching from rivaroxaban to edoxaban were
similar to edoxaban alone (Figure A); peak (mean SD) values were
21.75 2.46 s (Treatment A) and 21.83 2.88 s (Treatment C).
aPTT values for switching from dabigatran to edoxaban were higher
than edoxaban alone (Figure B) but similar to dabigatran alone. Peak
aPTT values were 35.87 3.15 s (Treatment A – Day 4) and
50.83 8.92 s (Treatment B – Day 4) vs. 46.83 5.08 s (Treatment B
– Day 3). The higher aPTT values upon switching from dabigatran represent residual effects of dabigatran and are within 1.5x the reported
range for median aPTT values for the majority of subjects at 12 h after
cessation of dabigatran 150 mg BID therapy. Bleeding times at 4 h post
dose were comparable for both Treatment A (277.20 119.05 s) and
Treatment B (298.70 166.48 s). Anti-FXa and thrombin generation
had results consistent with the primary biomarkers and pharmacology
of each drug. All AEs were mild or moderate in intensity.
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COA40
Characterization of the population pharmacokinetics
of Bayer’s rFVIII-FS in phase I/II shows that clearance
is affected by lean body weight and von Willebrand
factor
Garmann D1, Shah A2, McLeay S3, Vis P4 and Ploeger B5
Quantitative Pharmacology, Bayer Pharma AG, Wuppertal,
Germany; 2Bayer HealthCare Pharmaceuticals, Whippany, USA;
3
Model Answers Pty Ltd, Brisbane, Australia; 4LAP&P
Consultants BV, Leiden, Netherlands; 5Quantitative
Pharmacology, Bayer Pharma AG, Berlin, Germany
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Objectives: Subjects with hemophilia A are currently treated by intravenous administration of factor VIII (FVIII) on demand or as a prophylactic therapy administered 2–4 times a week. This study aims to
characterize the variability in the pharmacokinetics (PK) of rFVIII-FS
and to identify covariates which could explain the variability in the PK.
Methods: About 200 male subjects from several phase I/II studies with
~3000 PK samples measured using the one-stage assay were included in
a mixed effects or population PK analysis. The covariate effects were
evaluated using a forward inclusion/backward deletion procedure.
Models were qualified using visual predictive checks and the uncertainty
in the parameter estimates were derived from a bootstrap analysis.
Results: Approximately 80% of the subjects were white in the age
range of 4–61 years. The best PK model was a 2-compartment model
with zero-order input and first-order elimination defined by the clearance (CL), volume of distribution of the central (Vc) and peripheral
(Vp) volumes of distribution and the intercompartmental CL (CLp).
In the covariate analysis, age, weight, body mass index (BMI), lean
body weight (LBW), and von Willebrand factor (VWF) were evaluated
for their effect on CL: The covariate analysis revealed that CL and Vc
increased with LBW and CL decreased with increasing VWF.
Although age was also found to have a significant effect on Vc during
covariate analysis, it was highly correlated with LBW; therefore, only
LBW was considered as a covariate. Model qualification showed that
the model can be used for simulations (e.g. to determine the percentage
of patients who maintain FVIII levels above a certain threshold for
different dosing regimens).
Conclusion: The PK of FVIII after administration of rFVIII-FS could
be best described with a 2-compartment model with zero-order input.
Both CL and Vc were found to increase with lean body weight,
whereas increasing von Willebrand factor decreased the clearance.
Disclosure of Interest: D. Garmann is an employee of: is an employee
of Bayer Pharma AG, A. Shah is an employee of: is an employee of
Bayer HealthCare, S. McLeay Consultant for: Bayer Pharma AG, P.
A
B
Conclusion: These data suggest that switching from either dabigatran
or rivaroxaban to edoxaban at the next scheduled dosing time should
maintain adequate anticoagulation.
Disclosure of Interest: D. Parasrampuria is an employee of: Daiichi
Sankyo, D. Weilert: none declared, J.-F. Maa is an employee of: Daiichi Sankyo, V. Dishy is an employee of: Daiichi Sankyo, J. Kochan is
an employee of: Daiichi Sankyo, M. Shi is an employee of: Daiichi
Sankyo, K. Brown is an employee of: Daiichi Sankyo.
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
12/06/2014
24
ABSTRACTS
COA42
Inflammatory and thrombotic response to arthroplasty
in patients undergoing total hip/knee arthroplasty
Results: Experiments using 1:1250 final TP dilution are shown but
experiments using different TP dilutions were tested yielding a similar
effect. The dFiix-PT in PPP was 67–73 s. The dFiix-PT was sensitive
to warfarin, dabigatran and rivaroxaban in a linear manner. For a
warfarin range of INR 2-3 (shaded area) the dFiix-PT was 150–250 s.
For dabigatran the dFiix-PT was 110–210 s at levels corresponding to
peak concentrations obtained with dabigatran etixilate (DE) 150 mg
bid (76–409 ng mL–1) and 80–150 s for levels corresponding to the
trough concentration with DE 110 mg bid (28–155 ng mL–1). The
dFiix-PT for rivaroxaban was 95–125 s for a level corresponding to
peak concentrations obtained with 20 mg qd (160–360 ng mL–1) and
70–90 s for levels corresponding to trough concentrations after administration of 20 mg qd (28–155 ng mL–1).
The test could be easily automated using standard coagulation equipment.
Co
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Objectives: Patients undergoing total hip or knee arthroplasty (THA/
TKA) are at a high risk for thrombotic events. Pre-existing pathophysiologic factors and surgical intervention contributes to this risk. This
study investigated the inflammatory and thrombotic response to
arthroplasty procedures in patients undergoing THA or TKA.
Methods: Seventy five plasmas collected from patients undergoing
THA and TKA procedures were collected pre-operatively and day 1
post-operatively. Commercially obtained plasmas from 48 healthy,
non-smoking, non-medicated donors served as controls (George King
Bio-Medical Inc.) All samples were analyzed for full length tissue factor (TF), von Willebrand factor (vWF), microparticles (MP), microparticle-tissue factor complex (MP-TF), plasminogen-activator
inhibitor 1 (PAI-1), C-reactive protein (CRP), and total tissue factor
pathway inhibitor (TFPI) were measured using ELISA methods.
Functional antithrombin levels (ATIII), protein C, and total thrombin
generation (TGA) after TF activation were also measured. IL-2, IL-4,
IL-6, IL-8, IL-10, VEGF, IFNc, TNFa, IL1a, IL1b, MCP1, and EGF
were measured using a multiplex biochip array method (Randox.) Wilcoxon tests were employed to compare pre and post-op levels. MannWhitney U tests were used to compare pre-op levels with controls.
Results: Compared to controls, pre-operative levels of TF, MP, TGA,
vWF, PAI-1, CRP, IL-4, IL-6, IL-8, IL-10, VEGF, IFNc, TNFa,
IL1a, IL1b, MCP1, and EGF were elevated (P < 0.05). TFPI was
decreased pre-operatively compared to controls. On day 1, PAI-1,
MP-TF, CRP, IL-6, IL-10, VEGF, and MCP1 levels were increased
compared to pre-op levels. TGA, ATIII, vWF, and protein C levels
were decreased on day 1 compared to pre-op levels (P < 0.05).
Conclusion: Arthroplasty patients exhibit a pre-existing inflammatory
and hypercoagulable state which is further augmented by surgical
interventions. Profiling of some of these mediators may be useful in
the prognostic management of TH/TKA.
Disclosure of Interest: None declared.
R
Syed D1, Banos A2, Hopkinson W2, Bacher P3, Rees H2,
Hoppensteadt D1, Abro S1 and Fareed J1
1
Pathology; 2Orthopaedic Surgery and Rehabilitaion, Loyola
University Medical Center, Maywood; 3Abbott Laboratories,
Green Oaks, USA
COA43
Dilute FIIX prothrombin time as a single test to assess
the anticoagulant effect of warfarin, dabigatran and
rivaroxaban
Letertre LR1, Gudmundsdottir BR1 and Onundarson PT1,2
1
Laboratory Hematology and Coagulation Disorders, Landspitali
National University Hospital; 2Faculty of Medicine, School of
Health Sciences, University of Iceland, Reykjavik, Iceland
Objectives: A simple and fast measurement of oral anticoagulants
(OACs) is needed to ensure efficacy and safety, in preparation for
invasive procedures, during emergencies and to confirm their effective
reversal. Currently, different tests are used to measure different OACs.
We aimed to develop a single test to assess warfarin and direct (D)
OACs and simultaneously reducing any confounding effect of coagulation factors (F) other than FII and FX.
Methods: Citrated test samples were obtained from patients on warfarin. Rivaroxaban or dabigatran calibrator or control plasmas were
mixed with normal pooled PPP to derive increasing concentrations of the
DOAC in vitro. Test plasmas were mixed with factor II and X double immunodepleted plasma in the ratio of 1:2.3 to correct for any deficiency in
factors other than FII or FX, especially FVII. Diluted thromboplastin
(TP) and CaCl2 were added and the clotting time was measured.
Conclusion: The dilute Fiix-PT can be used to assess the anticoagulant
effect of warfarin, dabigatran and rivaroxaban.
Disclosure of Interest: L. Letertre: none declared, B. Gudmundsdottir
Shareholder of: Fiix Diagnostics Inc., a start up company involved in
a patent application for Fiix prothrombin time, P. Onundarson Shareholder of: Fiix Diagnostics Inc., a start up company involved in a patent application for Fiix prothrombin time.
COA44
Assessment of rivaroxaban and edoxaban on automated analyzers using a liquid anti-XA assay for apixaban
Cao Z1, Kung C1, Bottenus R1 and Triscott M2
R&D, Instrumentation Laboratory, Orangeburg; 2Instrumentation Laboratory, Bedford, USA
1
Objectives: Rivaroxaban (XARELTOâ), apixaban (ELIQUISâ) and
edoxaban (LIXIANAâ) are direct factor Xa inhibitors, which
although do not require regular monitoring, their assessment in certain
situations is helpful.
Methods: We previously reported on the performance of an automated
chromogenic assay, HemosIL Liquid Anti-Xa assay (Instrumentation
Laboratory, Bedford, MA, USA) to measure apixaban in citrated
human plasma samples on the ACL TOP family of analyzers. In this
research study, the Apixaban Assay was used to measure rivaroxaban
and edoxaban. Two calibrator levels (0 and 500 ng mL–1 apixaban)
were used to create a 5-point apixaban calibration curve (Curve A),
12/06/2014
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
ABSTRACTS
COA46
Upper extremity deep vein thrombosis: a longitudinal
follow-up
and edoxaban values in spiked human plasmas were assessed using the
stored calibration curve. A regression plot was performed on the
recovered values vs. their known edoxaban content, and the edoxaban
concentration (EDO CAL) equivalent to 500 ng mL–1 apixaban was
determined. The edoxaban sample values were then re-calculated using
Curve B (Curve A with the 500 ng mL–1 calibrator point set to EDO
CAL) or a new curve made from edoxaban calibrators (Curve C). A
similar study was done for rivaroxaban.
Results:
DXaI
Parameter
Sample
value
(Curve A
vs. known
content)
Edoxaban
Slope
Intercept
R
Slope
Intercept
R
0.8134
4.1
1.00
1.174
4.8
1.00
Rivaroxaban
Sample
value
(Curve C
vs. from
Curve B)
1.03
1.5
1.00
0.978
11.4
0.999
25
Desancho MT and Shillingford K
New York Presbyterian Hospital, New York, USA
DXaI (ng mL–1)
equivalent to
500 ng mL–1
apixaban
615
426
iza
COA45
Mean normal prothrombin time determination for use
in the pt INR calculation: methods of verification and
picking the ‘right’ number
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Conclusion: The Apixaban Assay effectively assesses edoxaban content
in citrated human plasma factoring in the molecular weight difference
between apixaban and edoxaban; however, an adjustment factor is
needed to assess rivaroxaban content.
Disclosure of Interest: None declared.
Objectives: Management of upper extremity deep upper thrombosis
(UEDVT) is challenging and depends on the risk factors, clinical presentation and risk of bleeding. Our objectives were to evaluate risk
factors, location of thrombosis, management and outcomes of patients
with UEDVT referred to a hematology clinic in a tertiary center.
Methods: Retrospective analysis of 25 consecutive patients with UEDVT seen from Jan 2006 to Dec 2013. We collected demographics, risk
factors for, and location of VT, type and duration of anticoagulation,
use of thrombolysis or thrombectomy and clinical outcomes.
Results: There were 20 females and 5 males, mean age was 40 years
(range 16–86); 21 were Whites, 2 Hispanic, 1 Black, and 1 Indian. 10
patients had multiple VTs; 9 involved the subclavian vein, 2 internal
jugular, 2 axillary, and 1 cephalic. One patient had no specific site of
VT. 3 patients had a prior history of VT, 6 had catheter-related thrombosis, 3 unprovoked, 2 each had pacemakers (PM), thoracic outlet
syndrome (TOS), or effort thrombosis, 1 was pregnant with TOS, 1
was postpartum, 2 underwent assisted reproductive techniques (ART),
2 were on hormonal contraceptives (HC), 2 had HC and TOS, 2 were
either on HC and catheter-related or HC and effort-related. 5 patients
had thrombophilia. 13 patients received Vitamin K Antagonists
(VKA) ranging from 3 months to > 6 months, 6 LMWH, 3 underwent
thrombolysis and thrombectomy, 1 was bridged from LMWH to
VKA, 1 remained on LMWH, and another on rivaroxaban; 2 patients
had catheter removal only, 1 was on rivaroxaban and had surgery for
TOS. 2 patients had recurrent UEDVT, 2 PE, 13 had residual VT, 8
had resolution of VT, 1 had progression of VT and 3 were lost to follow-up. There were no major bleeding episodes. All 22 patients who
were followed remained alive.
Conclusion: The main risk factor for UEDVT in our series was the
presence of a CVC and the subclavian vein was most frequently
affected. The vast majority of patients responded favorably to anticoagulant therapy.
Disclosure of Interest: None declared.
ut
or
Pruthi R, Tange JI, Fylling K and Chen D
Hematology, Mayo Clinic, Rochester, USA
Co
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Objectives: The international normalized ratio (INR) calculation is
based on the patient prothrombin time (PT), the mean normal prothrombin time (MNPT), and the International Sensitivity Index (ISI).
After our recent efforts at standardizing local ISI (Semin Thromb Hemost 2014;40:115), we focused on optimizing MNPT determination.
Based on CLSI guidelines (H54-A), MNPT is determined by testing at
least 20 normal donors (ND). An alternate method uses the plasma
samples from the local ISI assignment (Clin Chem Lab Med
2010;48:1079). Herein, we compared the two methods of determining
MNPT and their potential impact on INR.
Methods: We used one lot of R2G reagent and the IL TOP instrument.
The MNPT was assigned using 24 ND plasmas (Lab MNPT). A second MNPT was derived using the antilog of the y-intercept when plotting the log of assigned INR plasma samples on the x-axis and log of
their PTs on the y-axis (Calc MNPT). Next, PTs and INR were
obtained on 100 warfarin patient samples using the same ISI but differently determined MNPTs. INR results were categorized as sub therapeutic (< 2.0), therapeutic (2.0–3.0), and supra therapeutic (> 3.0)
and compared using the Lab MNPT as the reference.
Results: The Lab MNPT was 10.5s and Calc MNPT 11.4s. 20 samples
(100%) were subtherapeutic by both methods. of the 58 pt considered
therapeutic using the Lab MNPT, 5 (9%) would be considered subtherapeutic using the Calc MNPT. of the 22 pt considered to be supra
therapeutic by the Lab MNPT, 9 (41%) were considered therapeutic
by Calc MNPT.
Conclusion: Calc MNPT is different than the Lab MNPT. The disparity results in variable INR results, and it is unclear which INR is the
‘right’ INR. Similar to ISI assignment, a reliable and widely applicable
method is also needed for MNPT determination. The Calc MNPT
may be an alternative method which requires further evaluation.
Disclosure of Interest: None declared.
COA47
Tissue factor pathway inhibitor (TFPI) in patients with
sepsis receiving prophylactic enoxaparin
Abdelgadir AM1,2 and on behalf of Hadeel A. Al Otair, Syed M.
Khurshid, Abdul Aziz A. Alzeer, Abdul Kareem Al Momen,
Mashael Al Shaikh, Farja Al Gahtani, Zuhair Al Assiri, Hossam A.H.
Abdelrazik
1
Basic Medical Sciences, College of Medicine, King Saud
University for Health Sciences; 2King Khalid University Hos,
Riyadh, Saudi Arabia
Objectives: To characterize the contribution of the fluctuations in the
measured levels of TFPI in septic patients, to get a more accurate view
of the dynamics of TFPI in an approach not been tried before.
Methods: We studied 51 consecutive patients with sepsis who were
admitted to the intensive care unit (ICU) at King Khalid University
Hospital, Riyadh. Blood samples were collected at baseline (0 time,
arrival to the A & E Department and before anticoagulation) and at 4,
12 and 24 h after receiving Enoxaparin.
The following assays were undertaken using commercial kits:
Coagulation screening tests: Activated partial thromboplastin time
(APTT), prothrombin time (PT), and thrombin time (TT). Coagulation inhibitors total and free: TFPI and protein S, protein C; antithrombin (AT).
Results: On arrival to the A & E and before received any treatment,
the first blood samples showed marked disturbance of the clotting sys-
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
12/06/2014
26
ABSTRACTS
COA49
Impact of local prothrombin time reagent ISI assignment on proficiency testing results: a multi-instrument
and laboratory experience
tem in the form of significant prolongation of the PT and APTT and
this significant prolongation of both the PT and APTT remained in
the three subsequent samples. There was significant reduction in the
levels of AT, protein C and total and free PS, below control values,
throughout. In contrast, we noted with much interest that the plasma
levels of total and free TFPI were markedly elevated throughout the
study period. The mean levels of total TFPI, on arrival and increased
further slightly after the Enoxaparin therapy.
Conclusion: Sepsis triggers marked release of TFPI inhibitor from
endothelial cells and the administration of Enoxaparin enhances the
release of TFPI further. These results go some way to explain why the
therapeutic use of rTFPI fails to correct the coagulopathy associated
with sepsis.
Disclosure of Interest: None declared.
Fylling KA1, Tange JI1, Grill DE2, Ybabez RJ1, Wiese CR1, Rossini
LA1, Baumann NA1, Block DR1, Karon BS1, Donato LJ1, Pruthi RK1
and Chen D1
1
Laboratory Medicine and Pathology; 2Biomedical Statistics and
Informatics, Mayo Clinic, Rochester, USA
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Gentian D1, Pengo V1, Antonucci E2, Poli D3, Tripodi A4, Magrini
N5, Marongiu F6 and Palareti G7
1
Department Cardiac Thoracic and Vascular Sciences, University
of Padova, Padova; 2Department of Experimental and Clinical
Medicine, University of Florence; 3Department of Heart and
Vessel, AOU Careggi, Florence; 4Angelo Bianchi Bonomi
Hemophilia and Thrombosis Center, University of Milan, Milano;
5
Drug Evaluation Unit, Regional Agency for Health and Social
Care of Emilia-Romagna, Bologna; 6Department of Biomedical
Sciences, University of Cagliari, Cagliari; 7Unit of Angiology and
Blood Coagulation, University Hospital Policlinic S. OrsolaMalpighi, Bologna, Italy
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COA48
The start-register (survey on anticoagulated patients
register): baseline data of included patients, with focus
on those treated for non-valvular atrial fibrillation
Co
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Objectives: START-Register – Survey on anTicoagulated pAtients
RegisTer – is an independent, inception-cohort, collaborative database
aimed to prospectively record the clinical history of adult patients
starting an anticoagulant treatment for any reason and whatever the
drug used. The registry is designed solely for observational purposes
and to execute collaborative clinical studies. In this article we present
the START-Register and give a cross section of baseline data, in particular focusing on non valvular atrial fibrillation (NVAF).
Methods: Participating members prospectively insert recorded as electronic file on the web-site of the registry consecutive patients. Prospective data required are: demographic and clinical characteristics of
patients, associated risk factors for stroke and bleeding, laboratory
routine data, clinical indication for treatment, therapeutic range
expected (in cases of treatment with vitamin K antagonists -VKAs).
The follow-up is conducted to record: quality of treatment (for
patients on VKAs), bleeding complications, thrombotic events, and
the onset of any type of associated disease
Results: At this time 5252 patients have been enrolled; 97.6% of
patients were on VKAs treatment because of recent availability of DOACs in Italy. The median age was 74 years [interquartile range (IQR)
64–80]; males 53.7%. The present analysis is focused on the 3209
(61.1%) NVAF patients. Mean CHADS2 score was 2.1 1.1, median
age was 76 years (IQR 70–81); 168 patients (5.3%) had severe renal
failure (CrCl < 30 mL/min). Moderate renal failure (CrCl 30–59 mL/
min) was found in 1265 patients (39.5%).
Conclusion: In conclusion, the analysis of START-Register data shows
that two-third of patients who started a chronic anticoagulant treatment had a NVAF, one-third of them had > 80 years with high prevalence of renal insufficiency
Disclosure of Interest: None declared.
Objectives: Given the lack of international sensitivity index (ISI)
assignments for all reagent/instrument combinations, we recently
implemented an approach to ISI assignment/verification across different labs in our institution (Semin Thromb Hemost. 2014;40:115–20).
Since implementation of this process, we have undergone several interlab comparisons (ILC) and external proficiency testing (EPT) exercises. The goal of this study is to analyze the results to assess if our
goal of consistent INR results across different labs has been accomplished.
Methods: The 6 participating labs use RecombiPlasTin 2G (R2G)
reagent on the IL-TOP 700 (the reference lab), Stago-STA-R Evolutionâ, STA Compactâ, and STA Satelliteâ instruments. Pooled
patient samples of varying INRs (0.9–5.0) were tested in each lab.
Results were compared to the reference lab and the within group maximum difference caluculated. ILC rejection rules are as follows: INR
absolute difference > 0.2 (low and medium INR pools) or > 10%
(high INR pool) and min-max difference > 0.3 (low and medium
INR pools) or > 15% (high INR pool). Three College of American
Pathology (CAP) EPTs were conducted in 2013 with a total of 15 samples. Their results were analyzed.
Results: of the 9 ILC exercises, only one high sample (INR = 3.7–4.3)
demonstrated results outside acceptable criteria. Root cause analysis
revealed a compromised sample integrity. The results of a repeat run
using a newly prepared high INR pool were all acceptable. All 15 reference lab CAP EPT samples were within acceptable limits. When
compared to the reference lab’s peer group, all 15 CAP EPT samples
analyzed in the other 5 labs were also within acceptable limits.
Conclusion: It is important to follow up implementation of an ISI verification/assignment process with ILC and/or EPT studies. Our experience demonstrates excellent agreement of INR results across our 6
different labs, indicating that our goal of harmonizing INR results
across our institution has been achieved.
Disclosure of Interest: None declared.
COA50
Comparison of local and systemic inflammatory,
fibrinolytic and hemostatic parameters in patients with
varicose veins
Jezovnik M1, Syed D2, Hoppensteadt D2, Poredos P1, Tetik S2 and
Fareed J2
1
Vascular Disease, University medical centre Ljubljana, Ljubljana,
Slovenia; 2Pathology, Loyola University Medical Center,
Maywood, USA
Objectives: Varicose veins of the lower limbs are a manifestation of
chronic venous disease of the leg and valvular incompetence. This
study was performed to compare local and systemic inflammatory,
fibrinolytic and hemostatic parameters in patients with varicose veins.
Methods: Blood samples were drawn from both varicose and cubital
veins of 37 patients with superficial varicose veins at University clinic
(Ljubljana, Slovenia). Biochip array analysis included C-reactive protein (CRP), D dimer and tumor necrosis factor receptor 1 (TNFR1)
(Randox UK). Microparticle levels (MP) were measured using an
ELISA method (Hyphen) and anti-thrombin III (ATIII) were measured using a chromogenic method (Hyphen). Means, standard error,
12/06/2014
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
ABSTRACTS
27
DIC02
Disseminated intravascular coagulation scoring system
in cardiac surgery patients with postoperative severe
sepsis
quartile analysis, confidence intervals, paired, two-tailed t-tests and
correlations compared varicose vein samples with cubital vein samples.
Results: No difference in CRP levels was found between varicose and
cubital vein samples (varicose; 2.117 0.3633 mg/L, cubital; 2.041 0.3691 mg/L) and a strong positive correlation was observed (pearson
r = 0.9862). D dimer was observed to be elevated in varicose vein samples (varicose; 104.3 9.196 ng mL–1, cubital; 89.54 8.344 ng mL–
1
, P = 0.0387) and a positive correlation in d-dimer levels between
varicose and cubital vein samples was evident (pearson r = 0.6977).
There was no difference in TNFR1 levels (varicose; 0.5250 0.02225,
cubital; 0.4997 0.02481) but a positive correlation between varicose
and cubital vein samples existed (pearson r = 0.8104).
Conclusion: An increase in D-dimer levels in varicose vein samples suggests increased endogenous fibrin formation at the varicose vein site.
Correlations between varicose and cubital vein samples in terms of levels of CRP and TNFR1 suggest similar amounts of release of these
inflammatory markers at the two sites. Differences in levels of hemostatic mediators between varicose and cubital veins exist within
patients and should be further explored.
Disclosure of Interest: None declared.
Rogalskaya E1, Samsonova N1, Climovich L1, Yaroustovsky M2
and Krotenko N2
1
Clinical Laboratory Diagnostics Division; 2Department of
Detoxication and Endoscopy, Bakoulev Scientific Center for
Cardiovascular Surgery of the Russian Academy of Medical
Sciences, Moscow, Russian Federation
or
or
iza
Cruz MA1, Nguyen T2, Gushiken F1, Dong J-F1, Thiagarajan P3
and Dasgupta S3
1
Medicine/Cardiovascular Research; 2Pediatric; 3Pathology,
Baylor College of Medicine, Houston, USA
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DIC01
Isolated A2 domain of von Willebrand factor that binds
to fibrin inhibits microvascular thrombosis and reduces
mortality of mice with endotoxemia-induced disseminated intravascular coagulation
CD
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Disseminated Intravascular
Coagulation
Objectives: Etiology and pathogenesis of cardiovascular disease are
strongly linked to the functioning of hemostasis system. Open-heart
surgery with cardiopulmonary bypass is an additional pathogenic factor in the development of complications such as SIRS and DIC.
Objective: To determine the clinical diagnostic value of severity criteria
for disseminated intravascular coagulation syndrome in accordance
with the scoring system of Japanese Association for Acute Medicine
(JAAM DIC) in cardiac surgery patients.
Methods: The study group included 38 patients, 57 (48–62) years old,
with severe sepsis. Clinical signs of severe sepsis (SIRS + infection
site + failure of two or more organs) together with endotoxin activity
assay ≥ 0.6 and procalcitonin levels ≥ 2 ng mL–1 served as an inclusion criteria for the group. Ventilator-associated pneumonia confirmed
by clinical assessment and X-ray imaging was the source of infection.
Levels of hemostasis system markers were measured using analyzer
ACL TOP 700, (Instrumentation Laboratory). JAAM DIC was scored
at the time of diagnosis of severe sepsis (day 1) and day 3. Scores for
disease severity and organ dysfunction were also evaluated. All data
are expressed by median and interquartile range – Me (P25–P75). SPSS
for Windows version 17.0 was used for all statistical analysis. The level
of significance was set at P < 0.05.
Results: It was found that septic patients had evidence of DIC syndrome in the immediate postoperative period. 63% of patients had
overt DIC and 37% - non-overt DIC. The severity of the estimated
APACHE II and SOFA in these groups did not differ – 28 (21; 32) vs.
28 (21; 30) and 13 (12; 14) vs. 12 (9; 14) respectively, but the analysis
of the 28-day survival rate showed that mortality was higher in the
overt DIC group – 50% vs. 29%.
Conclusion: Our results demonstrated that overt DIC in cardiac surgical patients with sepsis is an independent predictor of mortality. The
JAAM DIC scoring system helps to predict decreased survival rate.
Disclosure of Interest: None declared.
Co
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Objectives: The coagulation system can be activated by a wide variety
of clinical conditions, causing disseminated intravascular coagulation
(DIC). This complex disorder is characterized by fibrin deposition and
fibrin-rich microthrombi in different organs. Von Willebrand factor
(VWF) mediates the incorporation of platelets to the nascent fibrin in
the growing thrombus as demonstrated by histologic studies from
patients that succumbed to DIC. Because the molecular mechanism by
which VWF interacts with fibrin remains unclear, this study was
designed to identify potential contact sites for fibrin within the structure of VWF.
Methods: We employed purified VWF, fibrinogen, fibrin degradation
products, and recombinant wild type and mutant VWF-fragments to
investigate the VWF-fibrin binding. This study also used parallel-flow
chamber adhesion assays, fibrin polymerization assays, and a mouse
model of endotoxemia-induced DIC.
Results: We demonstrate an additional cryptic binding site for fibrin
located within the A2 domain of VWF, a site that is exposed upon a
conformational change. Interestingly, the purified recombinant A2
domain, which constitutively expresses this site, bound to fibrin,
blocked flow-dependent platelet adhesion to fibrin under high shear
stress, impaired fibrin polymerization, and inhibited platelet-fibrin clot
formation in vitro. Furthermore, infusion of the purified A2 domain to
mice 1.5 h after the endotoxin insult prevented fibrin-rich microthrombi, attenuated levels of inflammatory mediators, and reduced
mortality rates at 96 h from 60% to 0% in a mouse model of endotoxemia-induced DIC.
Conclusion: In addition to the C domains, the interaction between
VWF and fibrin is also mediated by the A2 domain of VWF, and this
binding can be prevented with the purified A2 domain. This isolated
domain, which contains a fibrin-binding site, represents a potential
agent to treat DIC in different clinical conditions.
Disclosure of Interest: None declared.
DIC03
Tracking progression of LPS induced disseminated
intravascular coagulopathy using a native whole blood
thrombin generation assay
Baumgartner CK1, Mattson JG1, Shi Q2 and Montgomery RR1
1
Blood Research Institute, Blood Center of Wisconsin; 2Medical
College of Wisconsin, Milwaukee, USA
Objectives: Disseminated intravascular coagulopathy (DIC) results
from dramatically increased coagulation due to a variety of diseases,
including sepsis. Due to massive coagulation, clotting factors and
platelets are depleted leaving patients with increased risk for severe
bleeding after minor injury while simultaneously suffering from
thrombosis. High dose lipopolysaccharide (LPS) challenge resembles a
murine model for sepsis. In previous studies plasma levels of thrombin/anti-thrombin complexes (TAT) have been reported as an evaluation of thrombin generation. With the exception of one clinical study
using plasma, data of actual thrombin generation during DIC has not
been reported yet.
Methods: Because red blood cells and platelets have been shown to
impact thrombin generation we utilized a native whole blood thrombin
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
12/06/2014
28
ABSTRACTS
DIC05
Up-regulation of a novel inflammatory mediator, endocan, in patients with sepsis associated coagulopathy
generation assay (nWB-TGA) in which clotting was initiated by recalcification only. Mice were observed for up to 16 h post LPS injection.
Results: In accordance with previous reports, TAT levels in plasma
increased over time. We detected 3.6 0.7 ng mL–1 (mean SEM) of
TAT complexes at baseline, 10 3 and 15 5 ng mL–1 at 6 and 16 h
after LPS challenge, respectively. The nWB-TGA revealed a gradual
progression of DIC with significantly reduced thrombin generation over
time. Comparing baseline and 16 h post-challenge we found that lag
and peak times increased from 8 0.6 to 15 2 min and from 14 1
to 24 2 min respectively. Peak thrombin and endogenous thrombin
potential decreased from 131 7 to 56 8 nmol L–1 and from
1107 32 to 782 33 nmol L–1 thrombin respectively, and thrombin
generation rate was reduced from 25 3 to 6 1 nmol L min–1.
Increased coagulation and progressive DIC was confirmed by assessing
plasma levels of VWF, fibrinogen, D-Dimer and by platelet counts.
Conclusion: Thus, nWB-TGA provides a tool to track the progression
of DIC in in vivo sepsis models and potentially patients by real time
monitoring thrombin generation in whole blood samples.
Disclosure of Interest: C. Baumgartner has grant/research support
from: Novo Nordisk, J. Mattson: none declared, Q. Shi: none
declared, R. Montgomery: none declared.
Fareed J1, Syed D1, Hoppensteadt D1, Mosier M2, Low C1,
George M1 and VanThiel D3
1
Pathology; 2Surgery, Loyola University medical Center,
Maywood; 3Rush University Hospital, Chicago, USA
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Objectives: The diagnosis of DIC relies on the combination of clinical
presentation and laboratory evidence of increased thrombin generation and fibrinolysis. We examined the laboratory characteristics of
the quantitative plasma SFMC assay and its utility for DIC diagnosis.
Methods: SFMC (Diagnostica Stago) laboratory performance (precision, linearity, lower limit of detection) was characterized, extending
the upper limit with an additional 1:50 plasma dilution. Stored plasma
from 141 clinically-suspected DIC patients and 93 healthy controls
were analyzed for the operating characteristics for DIC diagnosis. We
reviewed the patients’ medical records for clinical characteristics of
DIC and assayed other plasma analytes that could be useful for diagnosing DIC, including the platelet count, PT, APTT, fibrinogen, DDimer, thrombin and reptilase times, factors V & VIII, antithrombin
and plasminogen activities, and thrombin-antithrombin complex.
Patients were assigned a DIC diagnosis based on (i) their clinical characteristics and the above assay results (not including SFMC), and (ii)
ISTH overt and non-overt DIC scores (108/141). Optimal SFMC cutoffs for DIC were estimated by ROC curve analysis.
Results: SFMC precision (< 10% CV) and linearity (including an
extended upper range) were acceptable, with a normal range < 8 lg
mL–1. SFMC levels ranged from 2- > 2000 and 8–506 lg mL–1 in
patients with and without clinical DIC, respectively; two patients with
clinical DIC had < 8 lg mL–1. Using a < 8 lg mL–1 cutoff, the SFMC
sensitivity/specificity for DIC assigned by clinical determination and
by ISTH overt and non-overt DIC scores were 90%/69%, 90%/63%
and 81%/67%, respectively. Positive and negative predictive values
were 44%/96%, 37%/96%, 51%/89% respectively.
Conclusion: The quantitative SFMC assay has acceptable laboratory
performance characteristics and utility, including a high negative predictive value, for the diagnosis of DIC.
Disclosure of Interest: None declared.
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Hematology, Mayo Clinic, Rochester, USA
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DIC04
Utility of soluble fibrin monomer complex (SFMC) in
the laboratory evaluation of disseminated intravascular
coagulation (DIC)
Objectives: Sepsis associated coagulopathy (SAC) is a complex syndrome where hemostatic dysregulation and inflammatory responses
along with endothelial dysfunction contribute to the negative outcomes. Endocan is a human endothelial specific proteoglycan, originally identified as endothelial cell-specific molecule 1 (ESM-1).
Endocan is implicated as a major mediator in the regulation of cell
adhesion, inflammation, and proliferation. Since widespread endothelial dysfunction is commonly seen in SAC, we hypothesized that this
proteoglycan is up-regulated in this syndrome. The aim of this study
was to compare the circulating levels of endocan in normal, healthy
individuals and in patients with suspected SAC.
Methods: Plasma samples from thirteen patients with SAC were collected at the time of initial diagnosis and frozen at -80 C. Plasma from
nineteen normal, healthy individuals served as control group. Endocan
levels were measured using an ELISA method (Lunginnov Lille,
France). These samples were also profiled for various inflammatory
cytokines using a biochip array (Randox UK). In addition, microparticle-tissue factor (MP-TF) levels were measured using an ELISA
method (Hyphen France).
Results: In comparison to normals (1.71 0.22 ng mL–1, range: 1.34–
2.21 ng mL–1), the SAC patients showed a significant increase in the
endocan levels (5.16 2.54 ng mL–1, range: 2.79–11.4 ng mL–1)
(P < 0.0001). The inflammatory cytokine showed wide variations with
a significant increase in TNFa (13.5 2.05 ng mL–1, range: 0.87–
216.48 ng mL–1) vs. normals (1.73 0.15 ng mL–1, range: 0–6.55 ng
mL–1) and MP-TF (1.38 0.16 ng mL–1, range: 0–10.6 ng mL–1) vs.
normals (0.46 0.08 ng mL–1, range: 0–1.99 ng mL–1).
Conclusion: Endocan levels in patients with SAC may be a useful marker to predict the pathogenesis of this syndrome. The cumulative
increase in endocan along with TNFa and MP-TF is suggestive of the
interplay of the activation of coagulation and inflammation with endothelial dysfunction in SAC.
Disclosure of Interest: None declared.
DIC06
Immunity-coagulation interface in patients with Stevens–Johnson syndrome/toxic epidermal necrolysis
Iqbal OM1, Syed D1, Malais J2, Mosier M3, Abro S1,
Hoppensteadt D1, Lin A2, Mata C2, Fareed J1 and Bouchard C2
1
Pathology; 2Ophthalmology; 3Surgery, Loyola University
Medical Center, Maywood, USA
Objectives: Stevens-Johnson Syndrome/Toxic Epidermal Necrolysis
(SJS/TEN) are specific drug hypersensitivity reactions initiated by
cytotoxic T-lymphocytes.
Methods: Following an Institutional Review Board approved protocol,
blood samples were obtained from 8 biopsy-confirmed and 8 biopsy
unconfirmed SJS/TEN patients. Commercially available plasmas from
48 healthy volunteers served as controls. Cytokine levels were measured using the Cytokine High Sensitivity Array biochip from Randox
Laboratories Limited. Mann–Whitney U-tests were conducted to compare biopsy-confirmed patients, unconfirmed patients, and normal
using statistical software (GraphPad Prism.) A P-value < 0.05 was
considered significant.
Results: Compared to controls, plasma levels of IL-4, IL-6, IL-8, IL10, VEGF, IFNc, TNFa, IL1a, IL1b, MCP1, and EGF were significantly increased in biopsy-confirmed and unconfirmed patients. IL-10
and TNFa levels were significantly lower in biopsy-confirmed patients
12/06/2014
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
ABSTRACTS
compared to unconfirmed patients (P = 0.0281 and 0.0104, respectively.) A marked increase in the TAT complexes (6.3 5.9 lg mL–1),
F1.2 (430.4 202.4 pM), platelet microparticles (13.1 9.3 nmol L–
1
) and protein C levels (90.5 63.4%) with a corresponding decrease
in PAI-1 (53.3 18.8 ng mL–1) and antithrombin levels
(80.7 42.4%) were also observed.
Conclusion: An interplay between immune mechanisms and coagulation is evident by an increased expression of cytokines, TAT complexes, F1.2 and platelet microparticles and corresponding decrease of
protein C, antithrombin, and PAI-1 which may progress to sepsisassociated coagulopathy and overt disseminated intravascular coagulation.
Disclosure of Interest: None declared.
Exogenous Hemostatic Factors
EHF01
Abstract withdrawn.
EHF02
Evidence that extravascular human factor IX influences
pharmacokinetics in hemophilic B dogs
Vanderslice N1, Xu W1, Gui T2, Hu G2, Masiello N3, Merricks EP4,
Monahan PE2, Nichols TC4 and Velander WH1
1
Chemical and Biomolecular Engineering, University of Nebraska,
Lincoln; 2Department of Pediatrics, University of North Carolina
at Chapel Hill, Chapel Hill; 3Revobiologics, Framingham;
4
Department of Pathology and Laboratory Medicine, University
of North Carolina at Chapel Hill, Chapel Hill, USA
DIC07
The intensity of the intravascular microcoagulation at
the patients with atherothrombosis and thrombophilia
1
1
2
3
Objectives: Multiple gene transgenesis was used to produce a biologically active, recombinant human Factor IX (r-FIX) protein with no
residual propeptide. This protein was studied in hemophilic B dogs to
assess safety and efficaciousness.
Methods: Human furin was co-expressed with human FIX in the milk
of transgenic pigs using the milk promoter from murine Whey Acidic
Protein. Purified plasma-derived FIX (pd-FIX) and r-FIX were intravenously administered at 50 IU/kg to hemophilic B dogs which were
monitored for 14–15 days. These studies in four hemophilic B dogs
were:
Study A (Dog O06): a single infusion of r-FIX;: Study B (Dog O66):
two successive infusions of r-FIX where the second infusion occurred
at 72 h; Study C (Dog O05): an infusion of r-FIX followed by a second
infusion of pd-FIX at 48 h; Study D (Dog O25): an infusion of pdFIX followed by a second infusion of r-FIX at 48 h. For all studies,
plasma antigen, single stage clotting activity, and whole blood clotting
times (WBCT) were assessed. Mean residence times (MRT) were calculated from the time course values of above assays.
Results: The baseline WBCT in all hemophilic B dogs studied was typically > 50 min. In studies A, B, and C, the average WBCT for the first
infused on of r-FIX was 14.5 min for 2 days. Similarly, the WBCT of
dog D after the first infusion of pd-FIX was 11.2 min for 2 days.
These values are comparable to the normal value of 8–12 min for
WBCT in normal dogs. Studies C and D showed an increase in MRT
when preceded by an infusion of the other species: the MRT of pdFIX increased 30% when preceded by r-FIX but the MRT of the rFIX increased by >700% when preceded by pd-FIX. Taken together,
studies C and D showed evidence of differential extravascular partitioning between r-FIX and pd-FIX.
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Shelest EA , Popova L , Matveeva M , Patrushev L , Shuganov
A2, Gneusheva T2 and Bokarev I1
1
Laboratory of Thrombosis, Haemostasis and Vascular Wall
Pathology Problems, Hospital Thepapy no. 1; 2I.M. Sechenov
First Moscow State Medical University (Msmu); 3Shemyakin and
Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russian
Federation
Objectives: Atherothrombosis (AT) is a leading cause of death worldwide. Thrombophilia is the well-known cause of arterial and vein
thrombosis. The influence of thrombophilia on the intensity of the
intravascular microcoagulation in patients with AT is unknown. The
aim of our study was to determine the intensity of the intravascular
microcoagulation in patients with atherothrombosis and thrombophilia.
Methods: Investigated 37 patients with atherothrombosis (23 with
thrombophilia and 14 – without).The study included patients who
have had a myocardial infarction more than 6 months ago with the
known anatomy of the coronary arteries. In control group was 53
healthy people (39 with thrombophilia and 14 – without). The intensity
of intravascular microcoagulation was measured: platelet factor 4
(PF4) by ELISA, D-dimer by ELISA, euglobulinlysis time (ELT).Persons with thrombophilia had Leiden mutation, prothrombin gene
mutation 20210A, mutation in the gene of methylenetetrahydrofolate
reductase (MTHFR) and plasminogen activator inhibitor-1 (PAI-1)
that was found by polymerase chain reaction (PCR).
Results: The level of PF4, D- dimer and ELT was significantly higher
in AT group compared with control group (P < 0,05). During analyzing the level of parameters of intravascular coagulation in (AT)
patients with thrombophilia and without, levels of PF4, D-dimer and
ELT were significantly higher in thrombophilia subgroup (PF4: 127.1
vs. 103.7 U mL–1 P < 0.001; D-dimer: 96.9 vs. 72.5 ng mL–1,
P < 0.001; ELT: 850 vs. 620 s, P = 0.027). In subgroup with thrombophilia levels of these parameters were significantly higher in patients
with two or more thrombophilia than with one (P < 0.01).
Conclusion: The intensity of intravascular microcoagulation increases
in patients with atherothrombosis. Patients with thrombophilia have
much more increased parameters of intravascular microcoagulation
especially with two or more thrombophilia.
Disclosure of Interest: None declared.
29
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
12/06/2014
30
ABSTRACTS
Conclusion: These four intravenous r-FIX studies indicate that r-FIX
was safe and efficacious in hemophilic B dogs.
Disclosure of Interest: N. Vanderslice: none declared, W. Xu: none
declared, T. Gui: none declared, G. Hu: none declared, N. Masiello:
none declared, E. Merricks: none declared, P. Monahan: none
declared, T. Nichols: none declared, W. Velander Shareholder of:
Progenetics LLC.
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Objectives: First study (hemophilia A/B with inhibitor): The comparison of two drugs effects to stop bleeding episodes in hemophilia with
inhibitor, Comparison of two drugs effects on FVII:C level
Seecond study (congenital factor VII deficiency): The comparison of
two drugs effects on FVII:C level,Comparison of two drugs effects on
prophylactic treatment of bleeding episodes.
Methods: 2 multicenter randomized double blind clinical trials were
conducted. In the first study we randomized 66 male patients into two
groups for treatment of one hemorrhage. Group A and B received
AryosevenTM and Novosevenâ respectively. Factor VII dosage was
90–120 lg/kg every 2 h till 3 doses. Primary outcome was self-reported
joint pain and joint movement with Kavakli scoring system. In second
study, we randomized 66 male and female patients into two groups for
prophylactic therapy.Group A and B received and Novosevenâ
respectively. rFVIIa dosage was 30 lg/kg, once per week for 4 weeks.
Primary outcome was FVII: C level (IU/dL) 20 min after injection.
Results: In the first study, group A = 31, group B = 35 patients. Median Kavakli score for post-injection pain and joint movement was similar in two groups, at 7.00 (6–8). Response to treatment was positive in
96.8% of group A, and 91.4% of group B. Reported side effects were
minor and occurred in similar frequency in both groups. In second
study. There was no difference in median increment of FVIIa activity
level 20 min after rFVIIa injection between two groups in any of the
4 weeks. The severity of bleeding was also similar between two groups,
with a similar reduction in median bleeding frequency, Reported side
effects were minor and occurred in similar frequency in both groups.
Conclusion: We studied efficacy and safety of AryoSevenTM in comparison with Novosevenâ in hemophilia with inhibitor and congenital factor VII deficiency, AryoSevenTM was similar to Novosevenâ in clinical
efficacy as well as post-injection FVIIa activity (FVII:C). The frequency of side-effects was also similar.
Disclosure of Interest: P. Eshghi has grant/research support from: This
work was supported financially by Aryogen Pharmaceutical Company
without any permission and role in design;data collection and analyzing the results which was performed by scientists of medical universities and research centers, K. Kamyar has grant/research support from:
This work was supported financially by Aryogen Pharmaceutical
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Eshghi P1, Kamyar K2, Abolghasemi H3, Karimi M4, Faranoush
M5, Toogeh G6, Hoorfar H7, Dehdezi BK8 and Khoeiny B9
1
Pediatric Congenital Hematologic Disorders Research Center,
Shahid Beheshti University of Medical Sciences, Tehran, Iran;
2
Tehran Clinic Hospital; 3Pediatric Hematology Oncology,
Shahid Beheshti University of Medical Sciences, Tehran, Iran,
Tehran; 4Pediatric Hematology Oncology, Shahid Beheshti
University of Medical Sciences, Tehran, Iran, shiraz; 5Pediatric
Hematology Oncology, High Institute of Research, Iranian Blood
Transfusion Organization, Tehran, Iran; 6Adult Hematology
Oncology, Tehran University of Medical Sciences, Tehran, Iran;
7
General Practitioner, Isfahan University of Medical Sciences,
Isfahan, Iran; 8Pediatric Hematology Oncology, Ahwaz University
of Medical Sciences, Ahwaz, Iran; 9General Practitioner, Karaj,
Iran, Tehran, Iran
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EHF03
Comparison of efficacy and safety between aryogen
recombinant activated factor VII (AryosevenTM) and
Novosevenâ in the main on-label (approved) indications of recombinant activated factor VII in Iran
Company without any permission and role in design;data collection
and analyzing the results which was performed by scientists of medical
universities and research centers, H. Abolghasemi has grant/research
support from: This work was supported financially by Aryogen Pharmaceutical Company without any permission and role in design;data
collection and analyzing the results which was performed by scientists
of medical universities and research centers, M. Karimi has grant/
research support from: This work was supported financially by Aryogen Pharmaceutical Company without any permission and role in
design;data collection and analyzing the results which was performed
by scientists of medical universities and research centers, Consultant
for: performed by scientists of medical universities and research centers, M. Faranoush has grant/research support from: This work was
supported financially by Aryogen Pharmaceutical Company without
any permission and role in design;data collection and analyzing the
results which was performed by scientists of medical universities and
research centers, Consultant for: performed by scientists of medical
universities and research centers, G. Toogeh has grant/research support from: This work was supported financially by Aryogen Pharmaceutical Company without any permission and role in design;data
collection and analyzing the results which was performed by scientists
of medical universities and research centers, Consultant for: performed
by scientists of medical universities and research centers, H. Hoorfar
has grant/research support from: This work was supported financially
by Aryogen Pharmaceutical Company without any permission and
role in design;data collection and analyzing the results which was performed by scientists of medical universities and research centers, Consultant for: performed by scientists of medical universities and
research centers, B. Keikhaei Dehdezi has grant/research support
from: This work was supported financially by Aryogen Pharmaceutical
Company without any permission and role in design;data collection
and analyzing the results which was performed by scientists of medical
universities and research centers, B. Khoeiny is an employee of: Aryogen and only hepled for the writing of the paper.
EHF04
Impairment of plasma clotting by crystalloid solutions
is related to ionic strength and presence of chloride ions
Ogweno G
Medical Physiology, Kenyatta University, Nairobi, Kenya
Objectives: To determine the effects of ionic vs. non-ionic crystalloid
on routine plasma clotting tests.
Methods: After IRB approval, 17 consenting healthy human subjects
provided 10 mL blood sample.Separated plasma aliquots hemodiluted
4:1 with crystalloid solutions. PT and aPTT Coagulation testing was
done.
Results:
Solution
Isotonic (300 mOsm/L)
Mannitol
Dextrose 5%
Nagluconate
NaCl
Choline chloride
Hypertonic
Mannitol (1080 mOsm/L)
Nagluconate (1800 mOsm/L)
NaCl (1800 mOsm/L)
Choline chloride (1800 mOsm/L)
PT (ref range
13–15 s) SE
aPTT (ref range
27–37 s) SE
14.39
14.34
14.82
14.73
15.01
0.26
0.29
0.27
0.27
0.28*
35.11
35.20
30.82
31.85
36.08
14.77
17.62
18.44
20.31
0.27
0.33*
0.38*
0.44**
35.24 3.41
30.31 3.69
33.58 4.15
78.42†3.75†
3.53
3.21
3.07
3.3
4.12
*Significant above ref range.
**Highly significant.
†
Unrecordably high.
12/06/2014
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
ABSTRACTS
Conclusion: Hypertonic ionic crystalloids significantly prolonged clotting times, strongly influenced by presence of chloride ions.
Disclosure of Interest: None declared.
EHF05
Assessment of protein aggregates and sub-visible particles in different recombinant FVIII preparations
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Objectives: The development of neutralizing antibodies against FVIII
is the major complication in current hemophilia A care. The initial
trigger of these antibodies has not been clarified. Studies with other
biotherapeutics indicated, that certain protein aggregates and sub-visible particles can initiate unwanted immune responses. Therefore, we
assessed the presence of such aggregates and particles in different
recombinant FVIII (rFVIII) preparations.
Methods: Five commercially available rFVIII products (A, B, C, D, E)
were included. At least three different lots of each product were freshly
reconstituted and tested for soluble protein aggregates and sub-visible
particles. Soluble protein aggregates were detected by size exclusion
chromatography, sub-visible particles were analyzed by a novel flow
cytometry based method and by Micro-Flow Imaging. The flow
cytometry based method used a combination of size calibration beads,
counting beads and a number of different fluorescent probes for distinct structural elements, thereby enabling the assessment of amount,
size and nature of sub-visible particles.
Results: Soluble protein aggregates were found in all rFVIII products
in concentrations ranging from 0.7% (product A, full-length rFVIII)
up to 8.4% (product C, BDD-rFVIII). The concentration of sub-visible particles ranged from 3789 particles/lg (product A) to 395,771 particles/lg (product C). The structural properties of sub-visible particles
were similar in all freshly reconstituted products
Conclusion: We conclude that there is a considerable variation in the
quality of rFVIII products for critical attributes such as protein aggregates and sub-visible particles. FVIII products lacking most of the B
domain seem prone towards aggregation which may be due to the lack
of most of the glycosylation sites contained in the B domain of FVIII.
Whether aggregates in rFVIII products can trigger unwanted immune
responses remains to be investigated.
Disclosure of Interest: M. Malisauskas is an employee of: Baxter Innovations GmbH, C. Lubich is an employee of: Baxter Innovations
GmbH, T. Prenninger is an employee of: Baxter Innovations GmbH,
P. Matthiessen is an employee of: Baxter Innovations GmbH, P. Turecek is an employee of: Baxter Innovations GmbH, F. Scheiflinger is an
employee of: Baxter Innovations GmbH, B. Reipert is an employee of:
Baxter Innovations GmbH.
(NovoNordisk) in several one stage clotting and chromogenic assays.
One stage and chromogenic FVIII:C were measured in 6 blinded samples: FVIII deficient plasma spiked to 20, 60 or 90 IU/dL with N8-GP
or recombinant FVIII (Advate, Baxter).
Methods: One stage FVIII:C assays were performed with Siemens
FVIII-deficient plasma with the following aPTT reagents: Actin FS
(Siemens), APTT-SP (Instrumentation laboratory, IL), DG-APTT
Synth (Diagnostic Grifols), Pathromtin (Siemens), SynthAFax (IL) or
SynthASil (IL). Chromogenic FVIII:C assays were performed with
kits from Siemens, Coamatic (Chromogenix), Coatest SP4 (Chromogenix) in both centers and Biophen (Hyphen Biomed) in one centre.
Samples were tested on 3 days using Sysmex CS5100 or Siemens BCS
XP against SSC reference plasma.
Results: The average FVIII:C was used to calculate recovery as a percentage of the target value. 100 25% was deemed a suitable recovery. The one-stage FVIII:C assays using Actin FS, DG-APTT Synth
and Pathromtin and Siemens chromogenic assay were within acceptable limits for N8-GP. APTT-SP significantly underestimated the
activity (< 50% of target). N8-GP results with SynthAFax and SynthASil were within target in one centre but reduced (70% of target) in
the second. Coamatic and Coatest FVIII:C were acceptable in one centre but overestimated in the second (130% and 140% of target). Biophen overestimated the FVIII:C level (130% of target).
Conclusion: N8-GP can be accurately measured in a one-stage assay
using Actin FS, DG-APTT Synth and Pathromtin, whilst SynthAFax
and SynthASil tend to underestimate and APTT-SP significantly
underestimates FVIII:C. Measurement of N8-GP by chromogenic
assay was generally acceptable but with a tendency to overestimate in
some cases.
Disclosure of Interest: A. Bowyer has grant/research support from:
Novo Nordisk, A. Hillarp has grant/research support from: Novo
Nordisk, M. Persson has grant/research support from: Novo Nordisk,
M. Ezban is an employee of: Novo Nordisk, P. Persson is an employee
of: Novo Nordisk, S. Kitchen has grant/research support from: Novo
Nordisk.
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Malisauskas M, Lubich C, Prenninger T, Matthiessen P, Turecek P,
Scheiflinger F and Reipert BM
Baxter Innovations GmbH, Vienna, Austria
31
EHF06
The laboratory measurement of a glycopegylated
recombinant FVIII, N8-GP: a two centre study
Bowyer A1, Hillarp A2, Persson M3, Ezban M4, Persson P5 and
Kitchen S1
1
Coagulation, Sheffield Teaching Hospitals, Sheffield, UK;
2
Clinical Chemistry and Transfusion Medicine, Region Halland,
Halmstad; 3Clinical Chemistry, Skane University Hospital,
Malmo, Sweden; 4Haemophilia; 5Clinical Pharmacology, Novo
Nordisk, Copenhagen, Denmark
Objectives: The plasma elimination half-life of FVIII may be increased
by glycopegylation. Difficulties in the measurement of the activity of
modified FVIII products have been reported. Two European centers
assessed the measurement of FVIII:C of glycopegylated FVIII N8-GP
EHF07
Abstract withdrawn.
EHF08
Clinician reported ease of use for a novel fibrin sealant
patch for hemostasis: results from three randomized
controlled trials
Corral M1, Ferko N2, Hollmann S2, Jamous N3, Batiller J1, Shen JX1
and Riebman J1
1
Ethicon Inc., Somerville, USA; 2Cornerstone Research Group
Inc., Burlington, Canada; 3Ethicon Inc., Berkshire, UK
Objectives: To evaluate clinician reported ease of use (EOU) for a
novel fibrin sealant patch (EVARREST) across different surgical
bleeding populations using an Ease of Use Questionnaire (EUQ).
Methods: The EUQ was administered in 3 randomized controlled studies of EVARREST vs. standard of care (SOC) (e.g. Surgicel) including
mild-moderate soft-tissue bleeding (400-07-02), severe soft-tissue
bleeding (400-08-02), and hepatic parenchymal bleeding (400-10-001).
The EUQ, administered for 100 subjects, was an endpoint for all EVARREST groups, but only once for Surgicel (400-07-02). The EUQ is
a 19-item instrument evaluating clinician’s perceptions and preferences
on 5 subscales (Table 1). An ANOVA model evaluated differences
between EVARREST and Surgicel (400-07-02), as well as EVARREST composite scores (3 studies) and Surgicel.
Results: Mean subscale scores for Confidence in Efficacy, Global Confidence, and Global Satisfaction were significantly higher for EVARREST vs. Surgicel in mild-moderate soft tissue bleeding (P < 0.05).
Across trials, EVARREST EUQ results remained consistently high
despite more problematic bleeding. The EVARREST composite scores
were > 4.4 out of 5 for every subscale, with EOU having the highest
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
12/06/2014
32
ABSTRACTS
is an employee of: Novo Nordisk A/S, M. Ezban is an employee of:
Novo Nordisk A/S, V. Jimenez-Yuste: none declared.
score. The EVARREST composite scores were significantly higher
than Surgicel scores for Confidence in Efficacy, Global Confidence,
and Global Satisfaction (P < 0.05).
Mean EUQ scores
EUQ subscale
Ease of use
Satisfaction
Confidence in
efficacy
Global
confidence
Global
satisfaction
ANOVA test (P -value)
Trial 400-07-02
EVARREST vs.
SOC
EVARREST
composite
(3
studies)
EVARREST vs.
SOC
(400-07-002)
EVARREST
composite vs.
SOC
EHF10
Safety and efficacy profile of a biosimilar recombinant
factor VIIA in patients with factor VII deficiency
4.82 vs. 4.67
4.71 vs. 4.31
4.70 vs. 3.51
4.64
4.53
4.44
0.38
0.05
0.0009
0.85
0.23
<0.0001
Faranoush M and on behalf of Biosimilar VIIa Study Group
Oncology, Tehran University of Medical Sciences, Tehran, Iran
4.70 vs. 4.13
4.57
0.03
0.044
4.85 vs. 4.06
4.56
0.02
0.014
CD
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Fernandez-Bello I1, Stenmo C2, Butta N1, Lind V2, Ezban M2 and
Jimenez-Yuste V1
1
University Hospital La Paz-IdiPaz, Madrid, Spain; 2Novo Nordisk
A/S, Bagsvaerd, Denmark
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EHF09
Assessment of in vivo administered recombinant activated factor VII by thrombelastography using two different coagulation activators
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Conclusion: Physicians consistently reported high EOU for EVARREST across various surgery and bleeding population types. Future
studies should evaluate EOU for SOC across the range of bleeding
severities, as these data are not currently available.
Disclosure of Interest: M. Corral is an employee of: Ethicon Inc., N.
Ferko Consultant for: Ethicon Inc., S. Hollmann Consultant for: Ethicon Inc., N. Jamous is an employee of: Ethicon Inc., J. Batiller is an
employee of: Ethicon Inc., J. Shen is an employee of: Ethicon Inc., J.
Riebman is an employee of: Ethicon Inc.
Co
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Objectives: Global assays, such as e.g. thrombelastography (TEG), are
currently being assessed as possible methods for monitoring treatment
of hemophilia patients. In particular treatment response to by-passing
agents represents a challenge as no established validated assays exist.
The aim of this clinical study was to assess the TEG profiles for up to
24 h after administration of a single dose of rFVIIa (NovoSevenâ,
Novo Nordisk A/S) to patients with hemophilia using two different
methods of activation (kaolin and tissue factor [TF]), and to compare
the TEG parameters with the individual pharmacokinetic (PK) profiles.
Methods: Whole blood from 6 patients (age ≥ 18 year old) with severe
hemophilia (2 with inhibitors) was collected in 3.2% citrate tubes
before dosing, and 10 min, 1, 3, 6, 9, and 24 h after dosing of 270 lg/
kg rFVIIa. TEG was triggered by intrinsic pathway activation with kaolin and by extrinsic pathway activation with TF (0.1 pM) (Innovinâ;
final dilution 1:50,000) plus tPA (Actilyseâ, final concentration
1.5 nmol L–1). FVIIa activity levels and TEG parameters (r-time,
alpha-angle, MA and maximum thrombus generation [MTG; the peak
of the first derivative curve]) were obtained at each time point.
Results: The TEG profiles generated after activation with Kaolin were
comparable to when activated with TF-tPA, supporting that both
methods can be used when monitoring rFVIIa administration. The
MTG and alpha-angle showed a better correlation to FVIIa activity
than r-time and MA. The TEG parameters appeared to be more sensitive as they showed a prolonged effect compared to FVIIa activity,
which declines more rapidly following administration.
Conclusion: Kaolin is preferable to TF-tPA for initiation of hemostasis
when monitoring coagulation with TEG after NovoSevenâ treatment
in vivo, as it provides a simple and reliable method that can readily be
used.
Disclosure of Interest: I. Fernandez-Bello: none declared, C. Stenmo is
an employee of: Novo Nordisk A/S, N. Butta: none declared, V. Lind
Objectives: This study was designed to compare the efficacy of NovoSevenâ with that of a biosimilar recombinant factor VIIa concentrate
(AryoSevenTM).
Methods: In a multicenter double-blind randomized clinical trial conducted in comprehensive hemophilia care centers in Iran, patients
(> 2 years) with congenital factor VII deficiency and > 1 bleeding episode per month were treated with AryoSevenTM or NovoSevenâ
(30 lg/kg, intravenously, once per week for 4 weeks). Exclusion criteria included other coagulopathies, rFVIIa prophylactic therapy during
the previous month, platelet count < 50,000, presence of a neutralizing
anti-factor VII antibody and history of severe atherosclerosis. The primary outcome was increased plasma FVII clotting activity (FVII:C)
20 min post- injection; FVII:C below 30 IU/dL was considered a treatment failure. Secondary outcome was self-reported bleeding frequency.
Results: The mean age was 21.8 (range: 2–65 years) and 33 patients
(50%) were male. Median (IQR) plasma FVII:C in AryoSevenTM and
NovoSevenâ groups were 1.6 (1.1–14.0) IU/dL and 5.0 (1.1–25.5) IU/
dL before injection. All patients had FVII:C above 30 IU/dL 20 min
post-injection. The FVIIa:C level 20 min post-injection was not significantly different between two groups during the treatment period. A
similar reduction in bleeding severity and frequency compared to the
last month prior to treatment was observed. Reported side effects were
minor and occurred in similar frequency in both groups.
Conclusion: AryoSevenTM is similar to NovoSevenâ in increasing postinjection FVII clotting activity (FVII:C), as well as in clinical efficacy.
The frequency of side-effects was also similar to the branded product.
Although the study was underpowered to detect differences in rare
complications between the two compounds.
Disclosure of Interest: None declared.
EHF11
The laboratory measurement of a glycopegylated
recombinant FIX, N9-GP: a two centre study
Bowyer A1, Hillarp A2, Persson M3, Ezban M4, Persson P5 and
Kitchen S1
1
Coagulation, Sheffield Teaching Hospitals, Sheffield, UK;
2
Clinical Chemistry and Transfusion Medicine, Region Halland,
Halmstad; 3Clinical Chemistry, Skane University Hospital,
Malmo, Sweden; 4Haemophilia; 5Clinical Pharmacology, Novo
Nordisk, Copenhagen, Denmark
Objectives: FIX:C half-life may be increased by glycopegylation of the
FIX molecule during manufacture. Difficulties in the measurement of
modified FIX products with some FIX:C assays have previously been
reported. The aim of this two European centre study was to assess the
measurement of glycopegylated FIX concentrate N9-GP (Novo Nordisk, Denmark) in several one stage and chromogenic assays of FIX:C.
Methods: One stage and chromogenic FIX:C were measured in 12
blinded samples; FIX-deficient plasma spiked to 3, 20, 60 or 90 IU/dL
with N9-GP, plasma-derived FIX (Mononine, CSL Behring) or
recombinant FIX (BeneFIX, Pfizer).
One stage FIX:C assays were performed with FIX-deficient plasma
(Precision Biologic) in conjunction with the following aPTT reagents;
Actin FS (Siemens), APTT-SP (Instrumentation laboratory, IL), DGAPTT Synth (Diagnostic Grifols), Pathromtin (Siemens), SynthAFax
12/06/2014
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
ABSTRACTS
EHF13
The procoagulant activity on nephrotic syndrome
induced by adriamycin in rats
(IL) or SynthASil (IL). Chromogenic FIX:C assays were performed
with kits from Rossix in both centers and Biophen (Hyphen Biomed)
in one centre. Samples were tested on 3 days using Sysmex CS5100 or
Siemens BCS XP against SSC reference plasma.
Results: The average FIX:C was used to calculate a percentage of the
target value. 100 25% was deemed a suitable recovery.
The one-stage FIX:C using DG-APTT Synth and SynthAFax and
both chromogenic FIX:C kits produced N9-GP results within the
acceptable range. Actin FS and SynthASil significantly underestimated
the activity of N9-GP (< 60% of target value). APTT-SP and Pathromtin grossly overestimated the activity of N9-GP (> 300% of target value). The FIX:C of BeneFIX was underestimated by the Biophen
kit tested in a single centre.
Conclusion: In this study, N9-GP could only be accurately measured in
a one-stage assay using DG-APTT Synth and SynthAFax or a chromogenic FIX assay with either kit.
Disclosure of Interest: A. Bowyer has grant/research support from:
Novo Nordisk, A. Hillarp has grant/research support from: Novo
Nordisk, M. Persson has grant/research support from: Novo Nordisk,
M. Ezban is an employee of: Novo Nordisk, P. Persson is an employee
of: Novo Nordisk, S. Kitchen has grant/research support from: Novo
Nordisk.
He M-X1, He S-L2, Zhang Y3, Zhu J-L1 and Hua H-Y1
1
Henan Academy of Medical and Pharmaceutical Sciences,
Zhengzhou University, Zhengzhou; 2Central South University,
Changsha; 3The Sixth Hospital of Zhengzhou, Zhengzhou, China
Objectives: Adriamycin nephropathy (AN) in rats has been used to
studied the progression of nephrotic syndrome (NS), however, the rat
species, batch and dose of adriamycin severely affected the model, and
the blood coagulation system has not been studied in the AN. The aim
of the study was to establish a stable AN rats model and to analyze
plasma coagulation parameters during the experiment.
Methods: The male SD rats were randomly divided into the control
group (n = 10) and the AN group (n = 22). Adriamycin (4.0 and
3.5 mg/kg) was tail intravenously administered to NS rats twice at a 7day interval, and the AN group was given high protein diets from the
first adriamycin injection for 2 weeks, after which, the rats were fed
the normal diets as the control group for 5 weeks. At 0, 3, 5 and
7 weeks, 24 h urinary proteinuria (24huPro) and serum were collected
to observe the levels of albumin (ALB), triglyceride (TG), cholesterol
(CHO) and creatinine (CRE) were detected to confirm if the AN model
had been successfully established, plasm prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT) and recalcification time (RT) were detected to analyze plasma coagulation.
Results: 2 rats died during the experiment because of severely diarrhea,
1 rat was eliminated because of the 24huPro was lower than 30 mg.
Compared with the controls, significant decrease of serum albumin
with marked increase in TG and CHO was observed at week 3 and
continued throughout the experiment in AN group, and CRE
increased significantly at week 5 later. Compared to the control group,
the PT of AN group was significantly shorter at week 3, and APTT,
TT and RT also showed the same tendency at week 3 or later.
Conclusion: These observations indicate that the rats fed high protein
diets induced reproducible, steady and chronic AN. The blood coagulation system was activated during the development of AN, this may
explain the hypercoagulable state in the progression of nephrotic syndrome.
Disclosure of Interest: None declared.
CD
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EHF12
Non-pathogenic antibodies against human recombinant proteins – what is their relevance?
Co
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€hl B, Scheiflinger F and
Horling FM, Hofbauer CJ, Allacher P, Abbu
Reipert BM
Baxter BioScience, Vienna, Austria
Objectives: Today it is well established that healthy individuals produce non-pathogenic antibodies against a range of autoantigens. The
origin and biological significance of these autoantibodies is still a matter of debate. Recently, we showed that healthy individuals express
low-titer antibodies which bind to human recombinant C (FVIII).
Here, we extend this study and ask if healthy individuals express antibodies that bind to a variety of human recombinant proteins, including FVIII, FIX, VWF and Furin.
Methods: Specific antibodies were detected using ELISA assays established in compliance with regulatory guidelines. Plasma samples
obtained from cohorts of 480–600 healthy individuals were screened
for the presence of antibodies against each protein followed by the
determination of antibody titers and the confirmation of antibody
specificities.
Results: Antibodies against human recombinant proteins are present
in a number of healthy individuals, with a prevalence ranging from
1.25% to 19%. Most antibodies had titers below 1:80 (1:20, 1:40), but
some samples contained antibodies with titers up to 1:640. Importantly, the presence of antibodies against one protein did not predict
the presence of antibodies against another protein in the same sample.
Longitudinal studies indicated that antibodies against human proteins
in healthy individuals could persist for the whole observation period
(1–3 years).
Conclusion: Our results provide evidence for the presence of non-pathogenic antibodies against a range of human recombinant proteins in
healthy individuals which resemble non-pathogenic self-reactive autoantibodies. The physiological relevance of these antibodies and the
regulatory pathways which give rise to their generation are not clear.
A better understanding of the nature of these antibodies should facilitate the differentiation between pathogenic and non-pathogenic antibodies which develop in some patients following treatment with
biotherapeutics.
Disclosure of Interest: F. Horling is an employee of: Baxter Innovations GmbH, C. Hofbauer is an employee of: Baxter Innovations
GmbH, P. Allacher is an employee of: Baxter Innovations GmbH, B.
Abb€
uhl is an employee of: Baxter Innovations GmbH, F. Scheiflinger
is an employee of: Baxter Innovations GmbH, B. Reipert is an
employee of: Baxter Innovations GmbH.
33
Factor VIII and IX
FEN01
In vivo selection of genetically manipulated platelets
corrects murine hemophilic phenotype and induces
immune tolerance even using a low multiplicity of
infection for transduction
Schroeder JA, Chen Y, Fang J, Wilcox DA and Shi Q
Medical College of Wisconsin, Blood Research Institute,
Children’s Research Institute, Milwaukee, USA
Objectives: Our previous studies have demonstrated that lentivirusmediated platelet-specific (2bF8) gene therapy can restore hemostasis
in hemophilia A (HA) mice with or without inhibitors. In this study,
we aimed to enhance platelet-FVIII (Plt-F8) expression while minimizing potential toxicities.
Methods: A novel lentiviral vector (LV), which harbors dual genes, the
2bF8 gene and a drug-resistance gene, the MGMTP140K cassette, was
constructed. Plt-F8 expression in HA mice was introduced by bone
marrow (BM) transduction and syngeneic transplantation. After BM
reconstitution, the recipients were treated with BG/BCNU monthly
for 3 or 4 times. Animals were analyzed by PCR, qPCR, FVIII:C
assays, and inhibitor assays. Phenotypic correction was assessed by tail
clipping tests and ROTEM analysis.
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
12/06/2014
34
ABSTRACTS
FEN03
Risk of inhibitor development in previously treated
patients with positive inhibitor history or low titer of
inhibitor at baseline: results of a survey of hemophilia
experts
Results: When an MOI (multiplicity of infection) of 1 was used for
transduction, Plt-F8 expression in recipients was only 0.22 0.15 mU/
108 platelets before the drug treatment, but remarkably increased to
4.33 5.48 mU/108 platelets (n = 16) after BG/BCNU treatments,
which is 2.89-fold higher than the data obtained from our regular
2bF8LV with an MOI of 10. 2bF8 proviral DNA was barely detectable
(0.01 0.02 copies/cell) before chemoselection, but it increased to
0.42 0.15 copies/cell after BG/BCNU treatments. Fifteen of 16 treated animals survived tail clipping. Blood loss and whole blood clotting
time were normalized in the treated recipients. When an MOI of 10 was
used, Plt-F8 expression was enhanced from 1.63 0.36 mU/108 platelets to 14.18 5.39 mU/108 platelets after 3 BG/BCNU treatments.
Notably, no anti-FVIII antibodies were detected in the treated animals
even after exogenous rhFVIII challenge.
Conclusion: We have established a powerful in vivo selective system
that allows us to enhance the therapeutic efficacy of 2F8 gene therapy
and induce immune tolerance in hemophilia A mice.
Disclosure of Interest: None declared.header>
Marcucci M1,2, Fisher K3, Kenet G4, Young G5, Walker I1,
Thabane L1 and Iorio A1
1
Clinical Epidemiology & Biostatistics, McMaster University,
Hamilton, Canada; 2Department of Clinical Sciences &
Community Health, University of Milan, Milan, Italy; 3van
Creveld Klinik, Utrecht, The Netherlands; 4Tel Hashomer
Hospital, Tel Aviv University, Tel Hashomer, Israel; 5Hemophilia
Center, Children Hospital, Los Angeles, USA
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Reilley MJ1, Blair A2, Matthai WH3, Vega R4, Gimotty PA5 and
Fogarty P6
1
Department of Medicine, University of Pennsylvania Health
System; 2Department of Biostatistics and Epidemiology,
Perelman School of Medicine, University of Pennsylvania;
3
Cardiovascular Division, Penn Presbyterian Medical Center;
4
Division of Hematology/Oncology, Perelman School of
Medicine, University of Pennsylvania; 5Department of
Biostatistics and Epidemiology, University of Pennsylvania;
6
Division of Hematology/Oncology, University of Pennsylvania
Health System, Philadelphia, USA
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FEN02
Revascularization strategies and in-hospital outcomes
in acute coronary syndromes (ACS) complicated by
hemophilia A (HA) or B (HB)
Co
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Objectives: Information is limited regarding in-hospital management
of ACS, which typically requires invasive procedures and/or exposure
to antithrombotic agents in HA and HB. We sought to identify clinical
characteristics and in-hospital outcomes among ACS patients with
hemophilia, compared to matched noncoagulopathic ACS controls.
Methods: Case discharges from the Nationwide Inpatient Sample (NIS),
Healthcare Cost and Utilization Project (HCUP), Agency for Healthcare
Research and Quality (1998–2011) had ICD-9 codes for HA or HB, and
ACS. Control discharges were matched to cases by year of discharge and
hospital. Discharges in both groups were assessed for cardiovascular risk
factors, type of ACS, use of coronary artery bypass grafting (CABG),
percutaneous coronary intervention (PCI), bare-metal stent (BMS) and/
or drug-eluting stent (DES), bleeding, and death.
Results: 237 cases and 148,848 matched controls were identified. The
mean age of cases and controls was 66 and 64 years, respectively.
Among cases, HIV/HCV were more common and obesity/hyperlipidemia less common. ST-elevation myocardial infarction (STEMI)
occurred less frequently among HA cases (18.6%; [CI: 12.8–24.4]) than
controls (24.4%; [CI: 24.2–24.6]). HA and HB cases were more likely
to be managed medically compared with controls (55.8% [CI: 48.4–
63.2] and 50.8% [CI: 38.6–62.9] vs. 32.0% [CI: 31.8–32.3]). When stented, cases more often received BMS than controls (77.2% [CI: 66.3–
88.1] vs. 53.7% [CI: 53.3–54.1]). Among PCI, bleeding was more
common among HA (12.0% [CI: 3.0–21.0]) than controls (2.8% [CI:
2.7–2.9]). The death rate appeared to be comparable among the groups.
Conclusion: ACS-HA/HB cases were more often treated non-invasively
compared with controls, suggesting an avoidance of PCI/CABG in
this population, although the clinical impact of these findings is uncertain. Standardized approaches for management of ACS in the congenital bleeding disorder population are needed.
Disclosure of Interest: None declared.
Objectives: Good quality evidence on the risk of inhibitors in patients
with low titer inhibitors or a past history of inhibitors lacks although
these patients are frequently encountered in clinical practice. Aim of
this study was to elicit experts’ opinion on the topic.
Methods: We prepared a survey including 2 clinical scenarios, the first
describing a severe hemophilia A patient with borderline inhibitor titer
(≤ 2 BU), the second a severe hemophilia A patient with personal history of inhibitors (disappeared spontaneously or after immunotolerance). For each clinical scenario, the same 5 questions were presented
(2 multiple choice and 3 open response) asking for responders’ opinion
about the risk of high titer inhibitors as compared to a severe hemophilia A patient with negative titers, and for their confidence in switching to a different molecule. The invitation to the survey was sent via
email to 100 hemophilia expert treaters from North and South America, Europe, Africa and Asia. Reminders to invitees were sent until 50
responses were received.
Results: The figure shows the distribution of responses to the 2 multiple
choice questions for each scenario. For the first case, the median elicited relative risk was 6, range 1.5–10; the median absolute risk 15%/
year, range 0.25–60. For the second case, the median relative risk was
6, range 1.25–3; the median absolute risk 3.25%/year, range 1–50. Most
of the responders would monitor the two clinical cases for 50 EDs and/
or 6 months after a hypothetical switch. 2% and 4% of the responders,
for the first and second scenario respectively, believed that monitoring
for inhibitor was not needed after switching; 19% and 15% believed
that monitoring was needed but they could not say for how long.
Conclusion: The risk of inhibitor development in patients with low titer
or past history of inhibitors has been considered increased by most of
responders. Empirical studies to objectively assess the risk of inhibitor
development in these patients are needed.
Disclosure of Interest: None declared.
12/06/2014
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
ABSTRACTS
FEN04
Investigation of the biological fate of 40 kDa PEG
following single dose administration of glycopeglylated
factor IX (N9-GP) to fix knockout mice
APTT reagents which may influence the Bethesda assay as well. Here
we investigate the influence of pegylated FIX in heat modified and normal FIX Bethesda assays in samples with residual PEGylated FIX
activity.
Methods: Samples spiked with different levels of PEGylated FIX and
polyclonal FIX antibodies (inhibitors) were analyzed in a normal -,
heat modified and a heat/cold modified FIX Bethesda assay using a
variety of APTT reagents.
Results: In samples with PEGylated FIX product the normal FIX
Bethesda assay was greatly influenced by the different APTT reagents
making an assessment of early inhibitors difficult. Pre-treatment of
samples with heat (known to deactivate FIX), removed the interference by PEGylated FIX when assayed with different APTT reagents.
This suggests that the PEG moiety of N9-GP does not interfere with
the FIX Bethesda assay when the attached FIX is deactivated. Moreover, the heat inactivation method and in particular the heat/cold
modified FIX Bethesda assay were able to detect low level inhibitors in
the presence of residual PEGylated FIX activity.
Conclusion: Introduction of a heat pre-treatment step removed the variability observed with the use of different APTT reagents in the FIX
Bethesda assay when analysing samples with left over PEGylated FIX.
Moreover, the introduction of the heat/cold FIX Bethesda increased
the drug tolerance when assessing inhibitor samples with residual FIX
activity.
Disclosure of Interest: A. Millner is an employee of: Novo Nordisk, H.
Boesen is an employee of: Novo Nordisk.
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Bjoernsdottir I1, Sternebring O1, Watson E1, Christensen J1,
Kornoe HT2, Kristensen JB2 and Bagger MA1
1
Development DMPK; 2Isotope Chemistry, NOVO NORDISK
A/S, Maaloev, Denmark
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FEN06
Post translational modifications and pharmacokinetics
of recombinant factor IX (RFIX) derived from HEK293
and CHO, and plasma-derived (PD) FIX
€hm E, Seyfried B, Dockal M, Hasslacher M, Ho
€llriegl W,
Bo
Kaliwoda M, Turecek PL, Muchitsch EM and Scheiflinger F
Baxter Innovations GmbH, Vienna, Austria
Co
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Objectives: N9-GP is a recombinant, glycoPEGylated derivative of
human coagulation factor IX (FIX) currently being developed for prophylaxis and on-demand treatment of hemophilia B. The activation
peptide of N9-GP contains a 40 kDa branched PEG. The objectives
for this poster, are to describe the synthesis and stability of the [3H]-radiolabelled N9-GP tracer as well as discussing the provisionally studies
conducted in order to assess the biological fate of [3H]PEG.
Methods: [3H]-radiolabelled 40 kDa PEGylated rFIX (N9-GP) was
synthesized in four steps and subsequently tested with respect to radiochemical stability and biological activity. The methods used in order
to assess the biological fate of the [3H] PEG moiety included single
dose i.v. studies with [3H]labelled N9-GP in Hemophilia B mice (FIX
knockout mice) and subsequent investigations of excretion, distribution and assessment of the pharmacokinetic parameters in plasma.
The various sample types were collected up to 12 weeks post dose in
all studies. The nature of components circulating in plasma over time
was assessed using gel electrophoreses and HPLC.
Results: The [3H]N9-GP tracer was found to be stable, biologically
active and therefore fit for investigation of the biological fate of PEG.
[3H]PEG related radioactivity was found to be excreted in both urine
and feces (40% excreted in urine and 50% recovered in feces, total estimated recovery was 103%). Terminal plasma half-life (t½) of radioactivity was 22 days. The tissue distribution revealed that radioactivity
was widely distributed, mainly in the highly vascularised tissues and
gradually eliminated. The lowest levels of radioactivity were found in
the CNS (brain and spinal cord).
Conclusion: The [3H]N9-GP tracer was found to be fit for investigation
of the biological fate of PEG. [3H]N9-GP and PEG related components were eliminated from plasma with a terminal t½ of 22 days,
widely distributed in the vascularised tissues and excreted both via kidneys and liver.
Disclosure of Interest: I. Bjoernsdottir Shareholder of: Novo Nordisk
A/S, is an employee of: Novo Nordisk A/S, O. Sternebring Shareholder of: Novo Nordisk A/S, is an employee of: Novo Nordisk A/S,
E. Watson Shareholder of: Novo Nordisk A/S, is an employee of:
Novo Nordisk A/S, J. Christensen is an employee of: Novo Nordisk
A/S, H. Kornoe Shareholder of: Novo Nordisk A/S, is an employee
of: Novo Nordisk A/S, J. Kristensen Shareholder of: Novo Nordisk
A/S, is an employee of: Novo Nordisk A/S, M. Bagger Shareholder of:
Novo Nordisk A/S, is an employee of: Novo Nordisk A/S.
35
FEN05
APTT reagents do not interfere with the heat/cold
modified FIX Bethesda assay for detection of inhibitors
in samples with pegylated FIX
Millner A and Boesen HT
DMPK, Cell and Antibody Analysis, Novo Nordisk, Maaloev,
Denmark
Objectives: Two new challenges occur when analysing samples for
early inhibitor formation after prophylactic administration with a long
half-life PEGylated FIX product such as nonacog beta pegol (N9-GP).
The first challenge is measuring early inhibitors in samples with residual FIX activity. This may be circumvented by introducing the heat/
cold modified FIX Bethesda assay (EAHAD 2014). The second challenge is the divergent FIX activity results obtained with different
Objectives: Human FIX undergoes extensive post-translational modification for its proper function. These modifications include g-carboxylation of Glu in the gla-domain, phosphorylation of serine, sulfation of
tyrosine, and N-and O-linked glycosylation. Some of these are colocated in the activation peptide, and their role in the lifecycle of the
protein is not fully understood. We explored whether differences in
post-translational modifications influence the pharmacokinetics of
rFIX produced in different mammalian cell lines.
Methods: rFIX was produced in four cell lines (HEK293, CHO, BHK,
and SkHep) and purified. In addition to conventional purification
yielding total rFIX, HEK293rFIX was subjected to a protocol for
enrichment of rFIX with a significantly higher degree of phosphorylation and sulfation. N-glycans and protein phosphorylation and sulfation were characterized; total rFIX and highly phosphorylated and
sulfated rFIX from HEK293 were compared with CHOrFIX and
pdFIX in a pharmacokinetic study in FIX-ko mice.
Results: Pharmacokinetic analysis revealed that HEK293rFIX had a
lower in vivo recovery (IVR) than CHOrFIX. pdFIX had the highest
IVR, comparable to literature. Highly phosphorylated and sulfated
rFIX showed a higher IVR than total HEK293rFIX. Its similar
degree of N-glycan sialylation, while substantially different to
pdFIX indicated that higher rFIX phosphorylation and sulfation
improved IVR. The high degree of sialylation in CHOrFIX most
probably compensated the low degree of phosphorylation and
sulfation.
Conclusion: Phosphorylation and sulfation of rFIX influenced its IVR.
HEK293rFIX showed a lower IVR than CHOrFIX and pdFIX, indicating no significant advantage of using these cells to produce a ‘more
human-like’ rFIX.
Disclosure of Interest: E. B€
ohm is an employee of: Baxter Innovations
GmbH, B. Seyfried is an employee of: Baxter Innovations GmbH, M.
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
12/06/2014
36
ABSTRACTS
Dockal is an employee of: Baxter Innovations GmbH, M. Hasslacher
is an employee of: Baxter Innovations GmbH, W. H€
ollriegl is an
employee of: Baxter Innovations GmbH, M. Kaliwoda is an employee
of: Baxter Innovations GmbH, P. L. Turecek is an employee of: Baxter
Innovations GmbH, E. M. Muchitsch is an employee of: Baxter Innovations GmbH, F. Scheiflinger is an employee of: Baxter Innovations
GmbH.
Coagulation in the one-stage assay is initiated by addition of negatively charged materials such as kaolin, micronized silica or ellagic acid
to activate factors XII and XI in plasma samples, followed by a calcium-dependent phase that triggers enzymatic reactions resulting in
fibrin polymerization. Generally, ellagic acid based aPTT reagents
result in faster clot times than silica or kaolin. A positive correlation
between the apparent rFIXFc activity and the strength of the aPTT
reagent has been observed. The objective of this study is to investigate
the enzymatic mechanism of the aPTT assay and its potential correlation with FIX assay discrepancies.
Methods: A FXIa-specific fluorogenic substrate was used to monitor
FXIa generation during the contact activation period with different
aPTT reagents in the presence of 1 IU mL–1 FIX products. A thrombin generation assay was performed during the calcium-dependent
phase.
Results: Ellagic acid based aPTT reagents generated higher amounts of
FXIa at the end of the contact activation phase, prior to addition of
calcium. Stronger aPTT reagents resulted in a higher rate and magnitude of thrombin production. The differences in thrombin generation
profiles by different FIX products were minimized with additional
phospholipids.
Conclusion: The amount of FXIa available in the aPTT assay at the
end of the contact phase, and the amount and type of phospholipids
present in the calcium-dependent phase correlated well with the
reagent dependent discrepancies observed for rFIXFc. These observations may contribute to the underlying mechanism of rFIXFc assay
discrepancy with different aPTT reagents.
Disclosure of Interest: Y. Buyue Shareholder of: Biogen Idec, is an
employee of: Biogen Idec, J. Sommer Shareholder of: Biogen Idec, is
an employee of: Biogen Idec.
FEN07
Highly discrepant inhibitor titres in the Bethesda assay
as a function of FVIII source: plasma vs. isolated FVIII
Co
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CD
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Objectives: Performing the Bethesda assay using different factor VIII
(FVIII) concentrates to guide clinical treatment has been suggested. In
this study, we tested inhibitor reactivity against various plasmatic or
recombinant FVIII concentrates.
Methods: Normal plasma, plasma-derived FVIII/von Willebrand factor [pdFVIII/VWF] complex, recombinant FVIII [rFVIII], B-domain
deleted [BDD]-FVIII, and isolated pdFVIII were used as FVIII
sources. Inhibitor IgG was purified from a pool of plasmas with inhibitors. To determine the titre, serial dilutions of inhibitor IgG were
mixed with an equal volume of 2 IU mL–1 VWF (no FVIII present),
and the mixtures were incubated with an equal volume of 1 IU mL–1
FVIII from the different sources. Conversely, the inhibitor IgG was
also added to previously mixed VWF + FVIII. Residual FVIII:C was
determined after incubation at 37°C (up to 2 h) using the Coamatic
FVIII kit and the one-stage assay.
Results: At t = 2 h, the inhibitor titres for normal plasma and
pdFVIII/VWF concentrates were comparable. In contrast, the inhibitor titres for all isolated FVIII concentrates, even after previous binding to VWF, were significantly higher than the titres for plasma or
pdFVIII/VWF (range 1.4–1.9 fold). At t = 0 h, titres for plasma or
pdFVIII/VWF complex were unquantifiable, but titres were detectable
for isolated FVIII concentrates (0.6–1.6 Bethesda Units).
Conclusion: The inhibitor titres from a pool of hemophilic inhibitors
are variable for different FVIII concentrates. pdFVIII/VWF complex
results are similar to normal plasma, whereas isolated FVIII concentrates show higher Bethesda titres, even after previous binding to
VWF. This suggests that VWF protection against inhibitor activity is
higher with native pdFVIII/VWF complex than with the corresponding complex formed from the isolated proteins. These data strongly
support the recommendation to perform Bethesda Assay titration
using different FVIII concentrates to guide the treatment of inhibitor
patients.
Disclosure of Interest: M. I. Bravo is an employee of: Grifols, B. Da
Rocha-Souto is an employee of: Grifols, S. Grancha is an employee
of: Grifols, J. I. Jorquera is an employee of: Grifols.
R
Bravo MI, Da Rocha-Souto B, Grancha S and Jorquera JI
Research & Development, Instituto Grifols S.A., Parets del
Valles, Spain
FEN08
Effect of APTT reagents on factor XI activation and
thrombin generation in one-stage clotting assays and
its correlation with factor IX product activity determination
Buyue Y and Sommer JM
Biogen Idec Hemophilia, Cambridge, USA
Objectives: The one-stage clotting assays based on aPTT are the most
commonly used assays for clinical monitoring and potency assignment
for FIX products for Hemophilia B replacement therapy. Activity discrepancies have been reported when a recombinant FIX Fc fusion protein (rFIXFc) is assayed with different aPTT reagents.
FEN09
Identification of binding partners for b domain of FVIII
by yeast two hybrid screen
Pachlinger R, Baldin-Stoyanova A, Knofl F, Ullrich N, Scheiflinger F
and Dockal M
Baxter Innovations GmbH, Vienna, Austria
Objectives: Coagulation FVIII is a large, complex glycoprotein that is
an essential component of the intrinsic tenase complex. The mature
protein is synthesized as a 2332aa single chain with the following
domain structure: A1-A2-B-A3-C1-C2. The B domain is cleaved intracellularly at positions 1313 and 1648 and extracellularly by thrombin
during activation at site 741. We explored the function of the FVIII B
domain by applying a yeast two hybrid (Y2H) screen to identify new
intra- and extracellular binding partners.
Methods: The A Matchmaker Gold system was used to screen a
human liver cDNA library against the B domain to identify interacting
partners. Three baits were used for the screen consisting of full length
B domain (aa741–aa1648) or truncated forms, aa741–aa1313 and
aa1313–aa1648, respectively. Each of these fragments was used in a
screen and positive clones were isolated on medium containing AureobasidinA and lacking histidine, leucine, tryptophan, and adenine. Bait
and pray swapping was used to verify positive interactions. A quantitative lacZ assay was used to determine the interaction strength.
Results: Nine different B domain interacting partners were identified
by Y2H technology, three of which are secreted while six are located
intracellularly. Quantification of the interaction strength by lacZ assay
revealed an interaction between the B domain and an intracellular and
an endomembrane system protein, while all other identified binding
partners barely activated the lacZ reporter gene.
Conclusion: The Y2H screen elucidated possible new interacting partners
that are located extra- or intracellularly and may therefore modulate the
function of FVIII in the bloodstream or take part in the maturation of
the protein. Verification of the interaction by alternative technologies
such as coimmunoprecipitation may provide further insights.
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© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
ABSTRACTS
Disclosure of Interest: R. Pachlinger is an employee of Baxter Innovations GmbH, A. Baldin-Stoyanova is an employee of Baxter Innovations GmbH, F. Knofl is an employee of Baxter Innovations GmbH,
N. Ullrich is an employee of Baxter Innovations GmbH, F. Scheiflinger is an employee of Baxter Innovations GmbH, M. Dockal is an
employee of Baxter Innovations GmbH.
tion based on ELISA (‘true content’) using N9-GP spiked samples and
patient samples originating from clinical studies of N9-GP.
Methods: Plasma samples from the N9-GP Phase 3 trial, paradigmTM 2
were used. In total 193 samples from 13 patients were used in which
the ELISA values were known for 64. All the samples were part of the
PK profiles. Based on ELISA values, 20 samples were prepared by
adding N9-GP to hemophilia B plasma corresponding to the same
range determined in the clinical study (0.2–9.0 lg mL–1). FIX concentration in the spiked samples was measured using the same assays as
used for the postinfusion patient samples. The APTT reagent for the
clot assay was SynthAFax (IL) and the chromogenic FIX kit from
Hyphen. ELISA was performed using own reagents
Results: The comparison of the FIX activity determined in spiked and
postinfusion samples showed a clear regression on true content
(ELISA/spiked value) both for the one-stage assay and the chromogenic assay (see figures). Data analysis showed that the slopes and
intercepts for the fitted regression lines are very similar for ELISA and
spiked value. As the intercepts were not significantly different from 0,
regression lines with 0 intercepts could also be fitted
FEN10
Sensitivity and variability of the thrombin generation
assay using recombinant factor VIII (turoctocog alfa)
and factor IX, and their glycopegylated derivatives with
either tissue factor or factor XIA to trigger the reaction
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Waters E, Hilden I, Sørensen B, Ezban M and Holm P
Haemophilia Biology, Novo Nordisk A/S, M
aløv, Denmark
Objectives: The thrombin generation assay (TGA) is being assessed as
a possible method for monitoring hemophilia treatment. The aim of
this study was to assess the TGA using turoctocog alfa, factor IX
(FIX) and their glycopegylated derivatives, N8-GP and N9-GP, with
respect to sensitivity and variability, using the calibrated automated
thrombogram.
Methods: The four compounds were tested in hemophilia A or B
plasma at concentrations from 0.13% to 130% (where 100% = 1 IU
mL–1). Samples were tested in triplicate in 12 distinct experimental
runs. Thrombin generation was triggered by either 1 pmol L–1 tissue
factor (TF) or 8.3 mU mL–1 FXIa.
Results: With the TF trigger, 0.4% turoctocog alfa, 0.4% FIX, 0.4%
N9-GP, and 1.2% N8-GP generated thrombin levels statistically different than what was generated in hemophilia A or B plasma alone.
Sensitivity was improved with FXIa as the trigger, and 0.13% of any
of the four compounds was distinguishable from hemophilia plasma
alone. FXIa generated less variability compared to TF, with relative
standard deviations ≤ 20% for most tested concentrations. Turoctocog alfa and N8-GP had similar activity in this assay, whether TF or
FXIa was used to initiate thrombin generation. FIX and N9-GP had
similar activity with the FXIa trigger, but with TF, FIX had more
activity than N9-GP.
Conclusion: This systematic approach indicates that the sensitivity,
and especially the variability, of the TGA with FXIa as the trigger
were improved compared to TF as the trigger. Additionally, it has
been shown previously that N9-GP is activated by FXIa at the same
rate as FIX, but by TF:FVIIa at an impaired rate compared to FIX;
therefore, it was not surprising that the two compounds looked nearly
identical with FXIa as the trigger, but not with the TF trigger. These
differences highlight the importance of the contact pathway, particularly with FIX activation. A standardized, sensitive assay will be
needed to correlate in vitro and in vivo data; perhaps a FXIa-triggered
assay will provide this.
Disclosure of Interest: E. Waters is an employee of Novo Nordisk A/S,
I. Hilden is an employee of Novo Nordisk A/S, B. Sørensen is an
employee of Novo Nordisk A/S, M. Ezban is an employee of Novo
Nordisk A/S, P. Holm is an employee of Novo Nordisk A/S.
37
FEN11
Comparison between spiked and postinfusion samples
of N9-GP evaluated by ELISA, one-stage clot assay,
and chromogenic assay
Conclusion: The linear relation between the clotting/chromogenic
assay and the ELISA for postinfusion samples and spiked samples
indicates a similar assay performance of N9-GP.Thus, for N9-GP
spiked samples can be used to evaluate and optimize assay conditions
and thereby save valuable patient samples.
Disclosure of Interest: M. Sørensen is an employee of Novo Nordisk
A/S, S. Andersen is an employee of Novo Nordisk A/S, M. Ezban is
an employee of Novo Nordisk.
Sørensen MH, Andersen S and Ezban M
Novo Nordisk, Maaloev, Denmark
Objectives: The aim of this study was to evaluate if spiked samples
reflect the amount of N9-GP in clinical samples, as patient postinfusion
samples are limited. In the current study we investigated the relation
between chromogenic and one-stage assay data with FIX concentra© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
12/06/2014
38
ABSTRACTS
FEN12
Feasibility of high through-put rapid turnaround mutational analysis for patients with hemophilia using dried
blood spots
philia A. None of these 77 PTPs developed an inhibitor while
treated exclusively with Octanate. A prospective clinical trial has
been initiated in 2000 to assess the immunogenicity in previously
untreated patients (PUPs). This included 50 PUPs with severe
hemophilia A for an observational period of 100 exposure days
with Octanate.
Methods: Patients with severe hemophilia A without previous exposure
to FVIII or FVIII-containing products were enrolled. Efficacy and tolerability were assessed by a 4-point verbal rating scale. Inhibitor assay,
according to modified Bethesda method, was tested prior to treatment,
every 3–4 exposure days (ED 1-20), and afterwards every 10 EDs (ED
21–100) or every 3 months.
Results: Three of 50 (6%) subjects developed clinically relevant inhibitor titers during the study. Another two displayed inhibitors that disappeared spontaneously without change of dose or dosing interval. All
inhibitors developed under on-demand treatment and before ED 50.
From the 50 subjects, 44 exceeded 50 EDs today. Octanate was welltolerated and, the hemostatic efficacy in prophylaxis and treatment of
bleeding episodes was generally rated as ‘excellent.’ No complication
was reported for any surgical treatment.
Conclusion: Despite frequent inhibitor testing and predominant ondemand treatment, the data indicate a low overall inhibitor rate for
Octanate in patients who exceeded 50 exposure days (5/44) of which
only 3 (6.8%) were clinically relevant.
Disclosure of Interest: None declared.
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FEN13
Latest results from the PUP-GCP clinical trial: a low
inhibitor rate in previously untreated patients with
severe hemophilia A treated with octanate
Klukowska A1, Jansen M2, Komrska V3, Laguna P4, Vdovin V5 and
Knaub S6
1
Department of Paediatrics, Haematology and Oncology,
Warsaw Medical University, Warasw, Poland; 2Octapharma
Produktionsgesellschaft mbH, Vienna, Austria; 3Department of
Pediatric Haematology and Oncology, University Hospital Motol,
Prague, Czech Republic; 4Department of Paediatrics,
Haematology and Oncology, Warsaw Medical University,
Warsaw, Poland; 5Izmaylovo Children’s Hospital Haematological
Centre, Moscow, Russian Federation; 6CR&D, Octapharma AG,
Lachen, Switzerland
Objectives: Octanate is a double virus inactivated, human plasmaderived factor VIII (FVIII) concentrate, naturally stabilized with
VWF. Five prospective GCP studies with Octanate were conducted in 77 previously treated patients (PTPs) with severe hemo-
CD
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FEN14
In vitro characterization of the first plasma-derived
factor V concentrate in development
da
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Objectives: For individuals diagnosed with hemophilia, mutational
analysis ideally would be available to better predict disease manifestations such as inhibitor development and to provide family planning
to female carriers. However, many patients do not undergo testing
due to high cost and lack of insurance reimbursement. Additionally,
the time to receive sequencing results can be several weeks. We are
developing a sequencing assay which can identify genetic mutations
in the factor 8 and 9 genes using dried blood spots on filter paper
with the goal of significantly lowering the turnaround time and cost
of testing.
Methods: This is a single-center, prospective, pilot study. Thus far, we
have isolated DNA from blood spots on filter paper for sample preparation. Samples are prepared for next generation sequencing utilizing
the Nextera rapid capture and enrichment workflow followed by
sequencing on a Miseq.
Results: Using a previously designed targeted panel, we have successfully sequenced and analyzed patients with known F8 and F9 variants
within 1 week of sample receipt. Currently we are able to detect
sequence variants and insertions and deletion events up to 40 base
pairs. Further method development is under way to detect the known
factor 8 inversions from the sequencing data, providing a rapid single
molecular test for both factor 8 andf 9 patients.
Conclusion: DNA can successfully be extracted and analyzed for mutations in factor 8 and 9 genes from known hemophilia patients. Once
we have finalized the process for identifying inversions, we will compare results from 24 individuals known to be affected with hemophilia
(16 hemophilia A, 8 hemophilia B) to 24 banked control DNA samples
to provide proof of principle. Subsequently, we plan to expand the
project to a larger clinical trial.
Disclosure of Interest: S. Carpenter Grant/Research support from:
Pfizer, Baxter, Consultant for: Grifols, Pfizer, E. Farrow: None
declared, N. Miller: None declared, L. Amos: None declared, M. Gibson: None declared, S. Streeter: None declared, B. Yoo: None
declared, S. Herd: None declared, S. Soden: None declared, S. Kingsmore: None declared.
R
Carpenter SL1, Farrow EG2, Miller N2, Amos L3, Gibson M2,
Streeter S1, Yoo B2, Herd S2, Soden S2 and Kingsmore S2
1
Pediatric Hematology/Oncology; 2Pediatric Genomic Medicine;
3
Pediatrics, Children’s Mercy Hospital, Kansas City, MO, USA
Lawrie AS1, Berbenni C2, Nardini I3, Mackie I1, Machin S1 and
Peyvandi F1,2
1
Haemostasis Research Unit, Department of Haematology,
University College London, London, UK; 2Angelo Bianchi Bonomi
Hemophilia and Thrombosis Centre, Department of
Pathophysiology and Transplantation, Fondazione IRCCS Ca’
Granda Ospedale Maggiore Policlinico Milano, Universit
a degli
Studi di Milano, Milan; 3Kedrion S.p.A - Loc. Ai Conti 5505,
Castelvecchio, Pascoli Barga Lucca, Italy
Objectives: Congenital factor V (FV) deficiency is a rare bleeding disorder and as such no therapeutic concentrate is currently available.
The only existing treatment available is fresh frozen plasma (FFP),
which has potential safety and hypervolemia problems, since the FV
level can realistically only be elevated to approximately 20% in
severely affected patients (i.e. FV < 1%). Kedrion S.p.A. is developing
a plasma-derived therapeutic FV concentrate with solvent-detergent
and nanofiltration steps in the manufacturing process.
Methods: In order to study hemostatic efficacy, FV procoagulant
activity (FV:C) and antigen (FV:Ag) levels of this new product were
assayed. FV:C relative to the WHO International Standard for FV
activity (03/116) using a range of thromboplastin reagents and FV
depleted plasmas in parallel-line bioassays. FV:Ag was measured using
Zymutest Factor V (Hyphen BioMed). On 3 consecutive days, concentrate was thawed at 37 °C and then left for 30 min at ambient temperature to equilibrate. The analyser (Sysmex CS-5100) was calibrated
using four thromboplastin/deficient plasma combinations. The FV
concentrate was then prediluted in three sources of deficient plasma
prior to assay. This process was repeated 2 h after thawing the FV
concentrate.
Results: The four reagent combinations over the 3 days gave a mean
relative potency at 30 min of: FV:C = 14.8 IU mL–1 (CV = 9.2%) and
at 2 h: FV:C = 14.6 IU mL–1 (CV = 10.4%), although some reagent
combinations gave poor assay parallelism, possibly due to residual
FV:Ag in one deficient plasma. Using the antigen kit reference preparation (the WHO standard is only calibrated for activity), mean FV:
12/06/2014
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
ABSTRACTS
Ag = 16.1 U mL–1 (CV = 9.3%) was obtained giving a FV: C/FV:Ag
ratio of 92%.
Conclusion: This novel and first therapeutic plasma derived FV concentrate potentially offers a major advance for treatment of patients
with FV and FV+ FVIII deficiency, however care must be taken in
selecting assay reagents for this product.
Disclosure of Interest: A. Lawrie Grant/Research support from: Kedrion S.p.A.: The UCL and Milan centers performed this study independently from Kedrion S.p.A. who funded the work by unrestricted
research grants and did not have sight of the data until the study was
completed, or take part in data analysis, C. Berbenni Grant/Research
support from: Kedrion S.p.A.: The UCL and Milan centers performed
this study independently from Kedrion S.p.A. who funded the work
by unrestricted research grants and did not have sight of the data until
the study was completed, or take part in data analysis, I. Nardini is an
employee of Kedrion S.p.A., I. Mackie Grant/Research support from:
Kedrion S.p.A.: The UCL and Milan centers performed this study
independently from Kedrion S.p.A. who funded the work by unrestricted research grants and did not have sight of the data until the
study was completed, or take part in data analysis, S. Machin: None
declared, F. Peyvandi Grant/Research support from: Kedrion S.p.A:
The UCL and Milan centers performed this study independently from
Kedrion S.p.A. who funded the work by unrestricted research grants
and did not have sight of the data until the study was completed, or
take part in data analysis.
FEN16
Investigation of hemostasis and fibrinolysis in FVIIIsensitive thromboelastometry
Palige M, Knappe S, Scheiflinger F and Dockal M
Baxter Innovations GmbH, Vienna, Austria
or
CD
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Objectives: Rotational thromboelastometry (ROTEMâ) is widely used
to monitor coagulation in whole blood. Here, we assessed and standardized a method to investigate coagulation and fibrinolysis in
FVIII-inhibited whole blood as a potential tool for testing bypassing
agents such as tissue factor pathway inhibitor (TFPI) antagonists for
hemophilia therapy.
Methods: As a model for hemophilia, blood from a healthy donor was
drawn into a tube containing citrate and corn trypsin inhibitor (CTI)
and preincubated with a goat anti-human FVIII antibody to block
FVIII activity. Minimizing shear stress resulted in highly consistent
ROTEMâ analysis. Coagulation was initiated by adding calcium chloride and 44 fmol L–1 of tissue factor (TF). Analysis of 66 blood samples collected on different days yielded a coefficient of variation (CV)
of 14% for normal whole blood and 13% for FVIII-inhibited blood
for the clotting time (CT). Fibrinolysis in FVIII-inhibited whole blood
was studied by adding 90 ng mL–1 tissue plasminogen activator (tPA)
and adjusting TF to 0.2 pmol L–1 to generate parameters within a
measurement for 6000 s.
Results: We demonstrated the sensitivity of the assay to FVIII and
TFPI by adding a TFPI inhibitory antibody to normal and FVIIIinhibited blood. Coagulation and fibrinolysis were determined by calculating the magnitude of the ROTEMâ tracings using the integral of
the curves (AUC). The overall coagulation potential was calculated
from samples without tPA, whereas fibrinolysis-induced FVIII-inhibited blood reflected its overall hemostasis potential. The overall fibrinolysis potential was calculated as the difference between these two
areas as a parameter for fibrinolysis in FVIII-inhibited human blood.
Within several measurements (N = 9), the values for the calculated
areas were reproducible (CV% < 20).
Conclusion: In summary, we established robust ROTEMâ protocols to
determine the potential of bypassing agents including TFPI antagonists to improve hemostasis and inhibit fibrinolysis.
Disclosure of Interest: M. Palige is an employee of Baxter Innovations
GmbH, S. Knappe is an employee of Baxter Innovations GmbH, F.
Scheiflinger is an employee of Baxter Innovations GmbH, M. Dockal
is an employee of Baxter Innovations GmbH.
or
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Turecek PL, Schrenk G, Hoellriegl W, Schiviz A, Rottensteiner H,
Schwarz HP, Muchitsch E-M and Scheiflinger F
Baxter Innovations GmbH, Vienna, Austria
Hoellriegl is an employee of Baxter Innovations GmbH, A. Schiviz is
an employee of Baxter Innovations GmbH, H. Rottensteiner is an
employee of Baxter Innovations GmbH, H. P. Schwarz is an employee
of Baxter Innovations GmbH, E.-M. Muchitsch is an employee of
Baxter Innovations GmbH, F. Scheiflinger is an employee of Baxter
Innovations GmbH
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FEN15
Comparison of RIXUBIS with another recombinant factor IX product for function, safety, and efficacy with
focus on factor IXA content
39
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Objectives: RIXUBIS is a new recombinant factor IX product produced from CHO cells using the protein-free manufacturing technology. The presented studies evaluate the function, safety, and efficacy
of RIXUBIS in comparison to commercially available rFIX with
regard to differences in activated FIX (FIXa) content.
Methods: The FIXa concentration of RIXUBIS was 0.01 IU mL–1
and approximately 10-fold less than the comparator rFIX. RIXUBIS
samples with an increased FIXa content were prepared and analyzed
in vitro by one-stage clotting assay, non-activated partial thromboplastin time assay and thrombin generation assay. The thrombogenic
potential of RIXUBIS was assessed in vivo using a modified Wessler
Test in rabbits at 750 IU kg–1 (10-fold human clinical dose). Efficacy
of the rFIX products was studied in hemophilia B (FIX ko) mice at a
dose of 75 IU kg–1 of both products and analyzed in a carotid occlusion model and using thrombelastography as endpoint.
Results: In all functional in vitro assays, spiking of FIX with FIXa
caused an increase in the measured FIX activity, with thrombin generation assay being affected most. No thrombogenic potential was
observed with RIXUBIS (individual scores of 0), whereas the mean
score for the commercially available rFIX was 0.5. After increasing the
FIXa concentration of RIXUBIS to that of the comparator rFIX a
mean score of 0.42 (individual scores 0–0.5) was determined. Efficacy
was comparable for both products.
Conclusion: FIXa interferes with potency assignment of FIX. The
results of the thrombogenicity model suggested that the differences in
preclinical thrombogenicity were caused by the higher FIXa content of
commercially available rFIX. The efficacy studies demonstrated that
despite its lower FIXa content, RIXUBIS was as efficacious as a commercially available rFIX indicating that FIXa does not contribute to
the efficacy of a FIX product. The results indicate that rFIX products
should preferentially contain a low FIXa content.
Disclosure of Interest: P. Turecek is an employee of Baxter Innovations
GmbH, G. Schrenk is an employee of Baxter Innovations GmbH, W.
FEN17
A generic version of recombinant factor VIIA is comparable to the branded product (NovoSeven)
Sadeghi N1, Kahn D1, Jeske W2, Hoppensteadt D1, Wahi R1 and
Fareed J1
1
Pathology; 2Thoracic and Cardiovascular Surgery, Loyola
University Medical Center, Maywood, USA
Objectives: A recombinant version of human factor VIIa (NovoSeven)
is commercially available for the management of hemophilic patients
with inhibitors. Recently several generic versions of NovoSeven have
been developed. Aryoseven (Aryogen, Tehran, Iran) represents a generic recombinant FVIIa preparation that is currently available in Iran
and other Asian countries. This study assessed the biosimilarity of
branded and generic human FVIIa preparations.
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
12/06/2014
40
ABSTRACTS
Methods: Four batches of NovoSeven were obtained from various vendors. Nine batches of Aryoseven were obtained from various vendors.
Each of these products was diluted in saline to obtain concentrations
ranging from 0.001 to 1.0 mg mL–1. The molecular profiles of the
products were measured using SELDI-TOF mass spectrometry. In
addition, SDS gel electrophoresis studies were carried out. Factor VII
(FVII)-related antigen, FVIIa activity, and thrombin generation studies were carried out in various systems. In addition, corrective actions
of the products were studied in FVIIa-deficient plasma and orally anticoagulated plasma.
Results: Both NovoSeven and Aryoseven exhibited a single peak at
50 kDa in the SELDI-TOF mass spectrometry analysis. Neither
preparation showed any additional peaks in any of the batches studied. In the SDS gel electrophoresis analysis each preparation exhibited a band at 50 kDa with a doublet at 27 and 28 kDa. The
FVIIa-related activity and FVII antigen levels were comparable in
both groups of products. In the thrombin generation studies, both
products exhibited comparable activities. Both groups of product
produced comparable corrective actions in FVIIa-deficient and oral
anticoagulated plasma.
Conclusion: The generic version of factor VIIa, namely, Aryoseven,
exhibits an identical molecular and biochemical profile in comparison
to the brand NovoSeven. These studies indicate that the brand and
generic products are similar.
Disclosure of Interest: None declared.
Kahn D, Sadeghi N, Hoppensteadt D, Fareed J and Fareed J
Pathology, Loyola University Medical Center, Maywood, USA
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Objectives: RIXUBIS is a new recombinant factor IX (FIX) product
produced from CHO cells using the protein-free manufacturing technology. Potency assignment is performed with a one-stage clotting
assay (OSCA) based on the activated partial thromboplastin time
assay (APTT) with DAPTTIN as reagent. The test is calibrated
against the 4th WHO IS for FIX concentrates. RIXUBIS and
another rFIX product were analyzed for FIX activity using a panel
of APTT reagents from various manufacturers and with chromogenic assays. Results were compared with the labeled potency. The
impacts of type and source of the APTT reagent were investigated.
Methods: OSCA was performed on the BCS/XP coagulation analyzer
from Siemens. FIX activities were calculated relative to a secondary
in-house standard, traceable to the WHO standard for FIX concentrates. In addition, the chromogenic activity was analyzed using test
kits Biophen Factor IX (Aniara/Hyphen Biomed, Coachrom) and
Rox Factor IX (Rossix, Haemachrom Diagnostica).
Results: The FIX activity was dependent on the type of APTT reagent
used, and discrepancies up to 40% were found with both recombinant
products. The dependency on the specific reagent was similar for both
rFIX concentrates. For RIXUBIS, good agreement between the labeled
potency and the FIX potency obtained with two different FIX chromogenic assays was found. The comparator product resulted in lower
chromogenic activities compared to the label. When RIXUBIS and the
comparator rFIX product were spiked in vitro into plasma from
hemophilia B patients the resulting FIX activity was also dependent on
the type of APTT reagent used for the one-stage clotting assay.
Conclusion: Potency of rFIX products is dependent on the APTT
reagent used for OSCA. Both RIXUBIS and the comparator rFIX product are affected in a similar way. For RIXUBIS the labeled one-stage
clotting potency is in good agreement with the chromogenic activity,
while for the other rFIX product lower chromogenic values were found.
Disclosure of Interest: H. Gritsch is an employee of Baxter Innovations
GmbH, S. Romeder-Finger is an employee of Baxter Innovations
Objectives: Recombinant factor VIIa (rFVIIa) is used for the control
of bleeding in hemophilia patients with inhibitors. Besides the branded
FVIIa (NovoSeven, Novo Nordisk, Copenhagen, Denmark), a generic
version of FVIIa (AryoSeven) has been developed by Aryogen (Tehran, Iran). We used surface enhanced laser desorption ionization
(SELDI) mass spectrometry to compare the composition of these
agents. Following this we supplemented rFVIIa with prothrombin
complex concentrate (PCC) and activated with tissue factor to determine the conversion of prothrombin to thrombin by these agents.
Methods: The FVIIa, AryoSevenTM and NovoSevenâ, and the PCC,
Profilnineâ, were obtained from various vendors. The proteomic profile of each of these agents was obtained in the MW range of 3–
150 kDa. Following this the Profilnineâ and rFVIIa mixtures were
activated with thromboplastin (RecombiPlasTinâ) and activation profiles were determined over 30 min. Three different time points were
evaluated to monitor the effect in terms of thrombin generation.
Results: Both rFVIIa preparations exhibited a distinct and homogenous peak at 50 kDa. When supplemented into Profilnineâ at a fixed
concentration, no activation was observed. However, in the presence
of tissue factor the initial rate of the reaction was increased, as measured by the degradation of the prothrombin peak (72 kDa) and the
generation of peaks at 50, 36, and 12.6 kDa. This 50-kDa peak represented prothrombin, whereas the 36-kDa peak represented thrombin.
The peak intensities at 50 kDa for Aryoseven was 0.701 0.233 U
and for Novoseven was 0.746 0.199 U (P > 0.05). The generation
of thrombin with both agents was time dependent and comparable.
Conclusion: Both the AryoSeven and NovoSeven exhibited comparable molecular composition. Both rFVIIa also produced comparable
activation of Profilnine in terms of the activation of prothrombin and
thrombin generation. Thus, AryoSeven represents a biosimilarly
equivalent product to NovoSeven.
Disclosure of Interest: None declared.
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Gritsch H, Romeder-Finger S, Scheiflinger F and Turecek PL
Baxter Innovations GmbH, Vienna, Austria
FEN19
Molecular profile and prothrombin complex activation
studies on a branded factor VIIA and a biosimilar product
iza
FEN18
Comparison of RIXUBIS with another recombinant FIX
product for behavior in chromogenic and one-stage
clotting assays using different APTT reagents
GmbH, F. Scheiflinger is an employee of Baxter Innovations GmbH,
P. Turecek is an employee of Baxter Innovations GmbH.
FEN20
Variability in the thromboplastin-mediated thrombin
generation in commercially available prothrombin
complex concentrates
Fareed J1, Sadeghi N1, Jeske W2, Hoppensteadt D1 and Wahi R1
1
Pathology; 2Thoracic and Cardiovascular Surgery, Loyola
University Medical Center, Maywood, USA
Objectives: Commercially available prothrombin complex concentrates
(PCCs) are usually standardized in terms of factor IX (FIX) units.
Besides FII, FVII, FIX, and FX, variable amounts of proteins C, S,
and Z are also present in these concentrates. Additives such as heparin
and anti-thrombin are also present in some of these PCCs. The relative
hemostatic profile of these PCCs is dependent on their composition
and is not proportional to their FIX levels. The purpose of this study is
to compare the variability in thrombin generation in various matrices.
Methods: Beriplex, Cofact, Feiba, Konyne, Octaplex, Preconativ, Profilnine, and Prothromplex were obtained from various vendors. These
agents were supplemented to saline, 5% albumin, NHP, and plateletrich plasma (PRP) in a concentration of 1 U mL–1. Thrombin generation studies were carried out using a kinetic fluorimetric method using
thromboplastin (Technoclone Vienna, Austria). Peak thrombin generation was measured as nmol L–1. Thrombin generation kinetics was
evaluated using AUC and other kinetic parameters.
12/06/2014
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
ABSTRACTS
FEN22
Impact of corn trypsin inhibitor (CTI) on the thrombin
generation assay of plasma samples collected from a
phase 3 multicenter clinical trial in severe hemophilia
A subjects
Results: All different PCCs produce matrix dependent thrombin generation profiles. In saline, the amount of thrombin generated varied from
0.6 to 1097 nmol L–1. Whereas in albumin, this range was 24 to
750 nmol L–1. In NHP, prothromplex and octaplex failed to generate
any thrombin. The range for other PCCs was 332–1246 nmol L–1 in
NHP. In PRP, all agents generated higher amounts of thrombin ranging
from 100 to 1638 nmol L–1. The kinetics for the thrombin generation
initiation, AUC, and other kinetic parameters varied in all matrices.
Conclusion: These results clearly suggest that compositional variations
in commercially available PCCs may impact on their interactions with
the solution matrix and cellular components and the overall hemostatic effects. Such additives as heparin and anti-thrombin, which are
found in some of these concentrates, may also influence their ability to
generate thrombin and other proteases.
Disclosure of Interest: None declared.
Buyue Y, Jiang H, Brennan A and Sommer JM
Biogen Idec Hemophilia, Cambridge, USA
Objectives: A thrombin generation assay (TGA) was used as an exploratory biomarker in a phase 3 study of recombinant factor VIII fusion
protein (rFVIIIFc). Samples were collected into custom tubes containing corn trypsin inhibitor (CTI), a specific FXIIa inhibitor, to attenuate potential contact activation. The necessity of this practice has been
the focus of debate. This study is aimed at investigating the effect of
CTI in TGA sample collection.
Methods: Thrombin generation tests (triggered with 1:6000 dilution of
Innovin and 4 lmol L–1 synthetic phospholipids) were performed on
(1) paired blood samples collected in house under strictly controlled
experimental conditions into a vacutainer containing 11 mmol L–1
Na-citrate with or without 50 lg mL–1 CTI and (2) paired blood samples (109 pairs) collected with or without CTI pre- and post-infusion
of rFVIII or rFVIIIFc from 16 hemophilia subjects during a phase 3
clinical trial.
Results: (1) In-house collected samples (n = 3) showed no significant
differences with or without CTI in all TGA parameters. (2) Clinical
samples collected without CTI showed significantly shortened lag time
and time to peak and increased ETP (endogenous thrombin potential),
peak thrombin, and slope (P < 0.01) relative to samples containing
CTI. (3) For individual subjects, the fold increase without CTI varied
significantly. The intersubject variance was significantly higher without
CTI than that with CTI. (4) For most subjects, a better correlation
with one-stage clotting activity was achieved with CTI present during
blood collection.
Conclusion: For samples collected carefully following a standard procedure, CTI did not affect TGA results. However, in a multicenter
clinical trial where consistent sample processing may not be possible,
contact activation during blood collection and handling significantly
affected the final results. Therefore, blood collection in specialized
tubes containing CTI to prevent contact activation in vitro could be
beneficial in multicenter clinical studies.
Disclosure of Interest: Y. Buyue Shareholder of: Biogen Idec, is an
employee of Biogen Idec, H. Jiang Shareholder of: Biogen Idec, is an
employee of Biogen Idec, A. Brennan Shareholder of: Biogen Idec, is
an employee of Biogen Idec, J. Sommer Shareholder of: Biogen Idec,
is an employee of Biogen Idec.
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FEN21
Observational Immune Tolerance Induction Research
Program (ObsITI)―a multifaceted approach to explore
immune tolerance induction
Kreuz W1, Escuriola-Ettingshausen C1, Gutowski-Eckel Z1,
Berntorp E2, Astermark J2, Oldenburg J3, Pavlova A3, Lacroix€nigs C5, N
Desmazes S4, Ko
egrier C6 and Dargaud Y6
1
€rfelden,
HZRM Haemophilia Centre Rhine-Main, Frankfurt-Mo
€ Centre for Thrombosis and Haemostasis,
Germany; 2Malmo
€ University Hospital, Malmo
€, Sweden; 3Institute of
Malmo
Experimental Haematology and Transfusion Medicine, University
of Bonn, Bonn, Germany; 4Centre de Recherches des Cordeliers,
INSERM UMRS 872, Paris, France; 5Johann Wolfgang-Goethe
University Hospital, Department of Paediatrics, Molecular
Haemostasis, Frankfurt, Germany; 6Haematology Department,
Edouard Herriot Hospital, Lyon, France
41
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Objectives: ObsITI is an international, open-label, uncontrolled multicenter observational program initiated in December 2005. The study
admits HA patients of any age and with any severity, with a confirmed
inhibitor titer ≥ 0.6 BU, and reduced FVIII incremental in vivo recovery and/or reduced FVIII half-life. Patients with risk factors historically associated with a poor ITI prognosis as well as good prognosis
are included. Patients are treated preferably according to the Bonn
protocol.
Methods: The aim of the program is to evaluate patient and therapy
related variables on ITI course, outcome and morbidity in HA
patients. ObsITI satellite studies additionally look at other factors
related to tolerization: The ObsITI thrombin generation sub-study has
been initiated to evaluate the correlation between the clinical bleeding
phenotype and their thrombin generation capacity before and during
ITI, in order to predict the individual bleeding risk. Host genetic factors such as the F8 gene defect or polymorphism of the immune
response genes, HLA class II alleles might act as predictors of the ITI
outcome. TGA, the collection of data on inhibitor antibodies that recognize specific FVIII epitopes and correlation of thess data with ITI
outcome and duration is performed. Additionally, the modifications of
the immune system and the inflammatory status of the patient during the
course are investigated. The results obtained from the substudies will be
correlated with the individual success rates of ITI, allowing the investigators to further personalize the ITI treatment of patients with inhibitors.
Results: As of February 2014, a total of 256 patients from 22 countries
have been screened for ObsITI. In 149 patients ITI has been documented, and 94 patients completed the study.
Conclusion: ObsITI is a large ongoing study on ITI with the potential
to extend our knowledge on ITI and to tailor ITI treatment to each
individual patient in order to help HA patients with inhibitors to
achieve treatment success.
Disclosure of Interest: None declared.
FEN23
Large external quality assessment survey on thrombin
generation with CAT: further evidence for the usefulness of normalization with external reference plasma
Perrin J1, Depasse F2, Lecompte T3 and on behalf of Frenchspeaking CAT Club
1
H
emotologie biologique, Chu Nancy, Nancy; 2Diagnostica
^pitaux
Stago, Asni
eres, France; 3Service d’h
ematologie, Ho
Universitaires de Gen
eve HUG, Gen
eve, Switzerland
Objectives: For the last decade, CAT has been widely used to determine thrombin generation in order to get a better insight into the coagulation in vitro phenotype. Interlaboratory exercises (e.g., ECAT),
however. have documented a worrisome poor reproducibility, but
Dargaud et al. have demonstrated the potential usefulness of the normalization with an appropriate external reference plasma. This multicentric study of the French-speaking CAT Club aimed at providing
further evidence for the usefulness of such a normalization.
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
12/06/2014
42
ABSTRACTS
0.01 IU mL–1. The rate of break-through bleeds and consumption of
FVIII concentrate will be analyzed for both treatment phases.
Results: As of mid February 2014, enrollment is nearly complete and
dosing recommendations for the personalized prophylaxis Treatment
Phase II have been provided for 36 subjects. By summer 2014 interim
data on a significant number of patients will be available to evaluate
the validity of this treatment approach.
Conclusion: The results of this study may improve the practice of preventing bleeds by extending the treatment interval in hemophilia A
patients.
Disclosure of Interest: None declared.
Methods: 34 laboratories agreed to participate (with a total of 38
instruments). All reagents with different batches were from Stago,
France. Lyophilized aliquots of a reference plasma were provided
along with three plasmas: P1 = normal, P2 = hypo-, or P3 = hypercoagulable. Tests had to be performed over 3 days in a row. Plates
and software were those locally used. Interlaboratory CVs were calculated for each plasma before and after normalization.
Results: Mean endogenous thrombin potential expressed as percentage of reference plasma were P1: 98% and 94%; P2: 10% and 36%;
P3: 156% and 130%; with 1 and 5 pmol L–1 TF, respectively. A
good discrimination between the three plasmas was achieved in all
laboratories but there was no overlap after normalization only. The
difference between plasmas was greater with FT 1 pmol L–1 compared to FT 5 pmol L–1, albeit at the expense of a greater variability. CVs were improved with normalization and often dropped to
< 10%. Some parameters had a greater variability despite normalization. Difficulties were obvious with P2 (very hypocoagulable) and
some laboratories even being unable to get a useable increase in fluorescence.
FEN25
Anti-inhibitor complex concentrate prophylaxis in
hemophiliacs with inhibitors
R
Zulfikar OB1, Koc B2 and Ozdemir N3
1
Pediatric Haematology and Oncology, Istanbul University
Cerrahpasa Medical Faculty and Oncology Institute; Haemophilia
Society of Turkey; 2Pediatric Haematology and Oncology,
Istanbul University Cerrahpasa Medical Faculty and Oncology
Institute; 3Pediatric Haematology and Oncology, Istanbul
University, Cerrahpasa Medical Faculty, Istanbul, Turkey
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FEN24
Personalizing prophylactic treatment in adult patients
with severe hemophilia A
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Klamroth R1, Lissitchkov T2, Walter O3, Knaub S4, Bichler J4 and
Tiede A5
1
Center of Angiology, Vivantes Klinikum im Friedrichshain,
Berlin, Germany; 2Specialized Hospital for Active Treatment
‘Joan Pavel’, Sofia, Bulgaria; 3Haematology; 4CR&D, Octapharma
AG, Lachen, Switzerland; 5Haematology, Haemostasis, Oncology
and Stem Cell Transplantation, Hannover Medical School,
Hannover, Hannover, Germany
Objectives: People with hemophilia and inhibitors are at high risk for
severe and uncontrolled bleeding. This life-threatening bleeding episodes can cause severe morbidity and mortality. In these patients antiinhibitor complex concentrate (aPCC) as a prophylaxis has been
shown to reduce the frequency of bleeding. The goal of this study is to
show our aPCC prophylaxis experience in hemophiliacs with inhibitors.
Methods: We enrolled hemophiliacs with inhibitors who are at aPCC
prophylaxis. We recorded their age, highest inhibitor titers and the
severe/moderate bleeding episodes before and after treatment at the
end of the one calendar year.
Results: Five of the 24 hemophiliacs with inhibitors were treated with
aPCC as a prophylaxis. aPCC was given 50–70 U kg dose–1 twice or
three times a week.
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Conclusion: We confirm with the largest study to date that normalization of CAT results with suitable reference plasma is useful in ‘real life’
practice and reaches an acceptable level of variability. In case of frank
hypocoagulability, the small remaining thrombin generation might be
of clinical importance, and further improvements are required to get a
reliable result.
Disclosure of Interest: None declared.
Objectives: Prophylactic therapy in hemophilia A patients is aimed to
maintain sufficient FVIII plasma concentrations between infusions of
FVIII concentrate to prevent bleeding. This prospective, multicenter
study investigates the efficacy of individually PK-tailored prophylaxis
with human-cl rhFVIII, a recombinant FVIII expressed in a human
cell line, in previously treated adult patients with severe hemophilia A.
Methods: The study is being performed in accordance with the Declaration of Helsinki and approved by the Ethics Committees of each participating institution. Informed consent is obtained from the subject
prior to any trial-related activity. Approximately 65 subjects will be
enrolled from various study centers across Europe. Following initial
PK evaluation, subjects enter the Treatment Phase I during which they
will be treated prophylactically every other day or 3 times per week
with a dose of 30–40 IU kg body weight–1 for up to 3 months. For the
ensuing 6-month Treatment Phase II, the dose and dosing interval will
be individually determined for each patient based on their PK data.
The goal is to determine the longest dosing interval that can be
achieved with a dose of not more than 60–80 IU kg–1 capable of hypothetically maintaining FVIII:C plasma concentrations of at least
Patient
No.
Age
(years)
Highest
inhibitor
(BU)
1 YE
2C
B
3 MS
4 AT
5 VE
9
20
20
39
23
25
7.9
13.8
37,5
23.3
Bleeding
(before
aPCC prophylaxis)
Bleeding
(1 year
after aPCCprophylaxis)
2/months
2/months
1–2/months
4/months
3/months
4/years
2/years
1/years
1/months
2/months
In four of five patients, aPCC prophylaxis was successful. The success
was excellent in three of them and average in one. In one patient,
aPCC prophylaxis did not work and we stopped the prophylaxis after
2 months.
Conclusion: In our patients, clinically relevant reductions in bleeding
frequency were achieved. aPCC could be an effective prophylactic
treatment choice for patients with inhibitors.
Disclosure of Interest: None declared.
12/06/2014
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
ABSTRACTS
FEN26
The coherence of menorrhagia and rare bleeding disorders
mands, answered inappropriately and disoriented. Motor was 5/5 in
the left upper extremity and 4/5 in all other extremities with decreased
reflexes. She had an ataxic gait. There was no meningeal sign.
Methods: Cranial MRI revealed microhemorrhages in both thalami
and right deep periventricular region. Work-up for metabolic, infectious and neoplastic causes were negative. On further evaluation, the
inferior sagittal, internal cerebral vein and straight sinus were not visualized on cranial MRV, indicative of CVT. CBC, PT, aPTT, D-dimer,
duplex scan, ANA and EEG were unremarkable.
Results: Anticoagulation in CVT has been controversial for it may
promote hemorrhagic transformation of venous infarcts while arresting the thrombotic process. Our patient was treated with Enoxaparin 0.8 mL SQ BID. Follow-up cranial CT scan showed interval
progression of the bilateral thalamic lesion with hemorrhages
extending to the lateral ventricles. She deteriorated then expired on
the 49th hospital day. Autopsy report revealed CVT involving the
superficial and deep veins with bilateral acute hemorrhagic infarcts,
bilateral intraparenchyma hemorrhage, medial and temporal herniation with necrosis, cerebral edema, anoxic-ischemic injury and atherosclerosis.
Conclusion: Anticoagulation in CVT, while indicated, should be used
with caution in a patient with concomitant cerebral hemorrhage to
avoid life-threatening complications.
Disclosure of Interest: None Declared.
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Halimeh S, Rott H, Kappert G and Siebert M
Gerinnungszentrum Rhein Ruhr, Duisburg, Germany
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FEN28
Analysis of calreticulin mutations in Chinese patients
with essential thrombocythemia: clinical implications
in diagnosis, prognosis and treatment
Fu R, Xuan M, Sun T, Bai J, Cao Z, Zhang L, Yang R and Zhang L
State Key Laboratory of Experimental Hematology, Institute of
Hematology & Hospital of Blood Disease, Chinese Academy of
Medical Sciences & Peking Union Medical College, Tianjin,
China
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Objectives: Menorrhagia or severe menstrual bleeding (HMB) is the
most common symptom of women with bleeding disorders. HMB is
defined as bleeding that lasts for more than 7 days or as the loss of
more than 80 mL of blood per menstrual cycle (1).
The menstrual blood loss can be quantified by the use of a pictorial
blood assessement chart (PBAC). With this study we compared the
PBAC-Score of controls to patients with and without the proof of a
hemorrhagic disorder.
Methods: In 198 women with menorrhagia and in 106 controls menstrual blood loss was quantified using PBAC and results were compared. We analyzed samples conducting blood count and all
coagulation factor activities. The women with menorrhagia did not
recieve any treatment for menorrhagia while we collected the PBACScore. The control group were not affected family members, friends
and colleagues of our patients.
Results: In 152 of 198 women with menorrhagia (76.7%) a bleeding
disorder could be detected. of these 75% a Von Willebrand Disease
could be detected The remaining 25% had other rare bleeding disorders (e. g. hypofibrinogenemia, FVII deficiency and other mild factor
deficiencies). In 2.8% of the control group a rare bleeding disorder
(VWD) was found.
Median PBAC score of the control group was 59.5 (range 4 – 100).
Patients median PBAC score were 260 (range 31–4212)
Conclusion: In our study a PBAC-Score above 100 was suspicious of
having a bleeding disorder.
We found a high correlation of a PBAC > 100 and an inherited bleeding disorder (P < 0.001). We keep on collecting the PBAC-Score from
women with menorrhagia and women with normal menses to raise
more awareness on menorrhagia and the coherence with rare bleeding
disorders.
Literature: 1 ACOG. Management of anuvulatory bleeding. Amcerican College of Obstetricians and Gynecologists 2000: Washington,
DC; 1-12
Disclosure of Interest: None Declared.
43
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FEN27
Acute deep cerebral venous thrombosis associated
with cerebral hemorrhage in an elderly female: a case
report
Acosta KR1, Cano J2, Rosales M2, Cuanang J2, Ang J2
and Dejan RJ2
1
Medicine; 2Neuroscience, St. Luke’s Medical Center, Quezon
City, Philippines
Objectives: We report a case of a 63 year-old right-handed, hypertensive and diabetic female with generalized body weakness and progressive change in sensorium as the initial presentation of acute deep
cerebral venous thrombosis (CVT), a rare case with an estimated incidence of ~2–4/million people/year. She was previously admitted for a
subgaleal hematoma in the left frontal area due to fall, then re-admitted
for a 1-week history of dizziness, generalized weakness, urinary incontinence, decrease in verbal output and progressive change in sensorium.
On examination, she was drowsy but easily arousable, followed com-
Objectives: Discovery of calreticulin (CALR) mutations fills the molecular diagnostic gap in essential thrombocythemia (ET). We studied
profiles of CALR mutations in Chinese patients with ET to provide
additional details on the utility of CALR mutations in the diagnosis,
prognosis and treatment of ET.
Methods: Mutations in MPL exon 10, CALR exon 9 and JAK2
V617F were investigated in 436 Chinese patients diagnosed with ET.
Their clinical records were carefully reassessed.
Results: Compared with Caucasian patients, Chinese patients had a
lower frequency of MPL mutations (1.4%) but comparable JAK2
V617F (53.9%) and CALR mutations (22.7%). Two patients harbored
both JAK2 V617F and CALR mutations; one of whom had a novel
point mutation in CALR exon 9. CALR-mutated patients displayed a
lower frequency of endogenous erythroid colonies but higher degree of
reticulin deposition (P < 0.05) than V617F+ patients did. Triple-negative patients showed intermediate hematological features between the
two subgroups above (Figure 1). JAK2 V617F but not CALR mutation was a risk factor for thrombosis (hazard ratio = 1.838;
P = 0.009), and CALR-mutated patients showed better thrombosisfree survival (P = 0.024). Screening CALR mutations in paired samples before and after treatment revealed that CALR-mutated clones
were not completely eliminated by cytoreductive agents. Antiplatelet
and cytoreductive agents decreased the risk of thrombosis in V617F+
patients (P = 0.034), but not in CALR-mutated cases (P = 1.000).
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
12/06/2014
44
ABSTRACTS
A
B
C
D
tions in the genes encoding ITGA2B or ITGB3 that result in qualitative
or quantitative abnormalities in the platelet membrane proteins leading to bleeding disorders. A precise molecular diagnosis in patients
with suspected GT allows an increased understanding of the disease
pathogenisis, and is important for screening of at risk family members
and establishing a prenatal diagnosis.
Disclosure of Interest: None Declared.
FEN30
Impact of ADAMT13 deficiency on the pathogenesis
and treatment of thrombotic thrombocytopenic purpura (TTP): a systematic review
Abegunde S
Clinical Pathology, National Orthopaedic Hospital Lagos Nigeria,
Lagos, Nigeria
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Conclusion: We confirm the value of CALR mutations in the diagnosis
and prognosis of ET in Chinese patients and provide new evidence for
making treatment strategies based on molecular markers.
Disclosure of Interest: None Declared.
Objectives: ADAMTS 13 is the 13th member of the ADAMTS (a disintegrin and metalloprotease with thrombospondin repeats} family of
metallopproteases. It is a VWF cleaving protease located on chromosome 9q34 and the principal regulator of VWF size. The regulation of
VWF multimer size is important in preventing spontaneous microvascular platelet clumping, a central pathophysiologic finding in TTP.
Studies have identified severe deficiency of ADAMT13 in majority of
TTP patients. This review is aimed at determining the impact of
ADAMT13 deficiency on the diagnosis and treatment of TTP.
Methods: The PUBMED, EMBASE,MEDLINE and the Cochrane
Database of systematic reviews were searched for relevant English-language reports of clinical trials, studies, abstracts, and case reports..
The reference lists of the identified articles were reviewed for additional relevant publications. Congress Web sites were also consulted.
Results: Showed two forms of severe ADAMTS13 deficiency have
been identified. However severe ADAMTS13 deficiency (< 10% activity) is not sufficiently sensitive or specific for the diagnosis of TTP.
Patients with severe acquired ADAMTS13 deficiency define the characteristics features of TTP and their response to treatment. This
acquired form is caused by circulating autoantibodies which inhibits
ADAMTS13 activity or increase ADAMTS13 clearance, and the antiADAMTS 13 autoantibodies are mostly IgG. Constitutional ADAMTS 13 deficiency leading to hereditary TTP is due to homozygous or
compound heterozygous ADAMTS13 gene mutation. Severe ADAMTS 13 deficiency is also associated with increased risk of relapse.
Conclusion: The focus of future challenges should include improvement of the assays for reliable diagnosis of TTP, delineation of modifiers affecting the severity of the disease, and development of new
therapeutic measures for more effective treatments. With heightened
interest in the investigation of this disease, the prospect of meeting
these challenges is encouraging.
Disclosure of Interest: None Declared.
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Borhany M1, Haghighi AR2, Sayer J3, Fatima N4 and Shamsi T5
1
Haematology, Haemostasis & Thrombosis, National Institute of
Blood Disease & Bone Marrow Transplant (Nibd), KARACHI,
Pakistan; 2Department of Genetics, Harvard Medical School,
Boston, Department of Medicine and the Howard Hughes
Medical Institute, Brigham and Women’s Hospital, Boston, MA,
USA, Harvard Medical School,the Howard Hughes Medical
Institute, Boston, USA; 3Institute of Genetic Medicine, Newcastle
University, UK; 4Department of Haematology, Haemostasis &
Thrombosis, National Institute of Blood Disease & Bone Marrow
Transplantation; 5Department of Haematology, Haemostasis &
Thrombosis., Karachi, Pakistan
or
FEN29
Molecular genetic analysis of glanzmann thrombasthenia in Pakistani cohort reveals novel mutations
Objectives: Glanzmann thrombasthenia (GT) is a rare autosomal
recessive bleeding disorder characterized by quantitative and/or
qualitative defects of the platelet glycoprotein (GP) IIb/IIIa complex.
Using a cohort of patients with a clinical phenotype consistent with
GT we undertook a molecular genetic approach to solve these cases.
Methods: Patients of GT were included in the study. They were
screened using complete blood counts, platelet aggregation studies,
flowcytometry and positive family history of bleeding. Screening of
genes ITGA2B and ITGB3 using exon PCR and Sanger sequencing
was peformed and parental samples were used to confirm segregation
of mutations.
Results: A total of 10 GT patients (4 males and 6 females) came to our
attention: their mean age was 6 4.0 years in six families. The most
common bleeding manifestations were epistaxis, bruises, prolong
bleeding after trauma, ear bleeding, gum bleeding, and hematomas.
History of consanguinity was present in 80% of cases and significant
family history of bleeding in 50% of patients. We identified 3 novel
homozygous mutations in ITGA2B (R551Q, Y411X and
c.1427_1428insAGGT) in 6 families with GT and 1 novel homozygous
mutation (C258X) in ITGB3. Segregation analysis confirmed heterozygous carrier status in all parents (who were unaffected). All novel mutations were predicted to be disease casuing using in silico modelling.
Conclusion: GT occurs in high frequency in certain ethnic populations
with an increased incidence of consanguinity. GT is caused by muta-
FEN31
The validity of algorithms for identifying suspected
and confirmed heparin-induced thrombocytopenia
Patel V1, Walton S1, Galanter W2, Lee T1, Schumock G1 and
Nutescu E3
1
Department of Pharmacy Systems, Outcomes and Policy;
2
Department of Medicine, General Internal Medicine;
3
Departments of Pharmacy Practice and Pharmacy Systems,
Outcomes and Policy, University of Illinois at Chicago, Chicago,
USA
Objectives: This study developed and compared the performance of
three algorithms based on claims data for identifying HIT using gold
standard measures of suspected and actual HIT.
12/06/2014
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
ABSTRACTS
Methods: A retrospective cohort study was conducted at an urban academic medical center. A total of 45,096 patients exposed to heparin
during hospitalization were identified between April 1, 2006 and
March 31, 2011. Claims data were used to develop 3 algorithms for
identification of HIT patients: (A) initiation of direct thrombin inhibitor treatment and a diagnostic test ordered more than 4 days after
exposure to heparin; (B) algorithm A combined with any of the International Classification of Disease, 9th version, Clinical Modification
(ICD-9-CM) codes for thrombocytopenia (287.4/287.5/289.84); and
(C) algorithm A combined with the ICD-9-CM code for HIT (289.84).
The data used in this study were collected from medical charts for
patients (n = 39) identified by algorithm A and a random sample
(n = 250) of patients who did not meet the algorithm’s definition. The
performance of the three algorithms was examined by comparing the
confirmed/suspected HIT status determined by the algorithms with an
assessment by clinical experts (gold standard) based on review of the
medical chart.
Results: Both negative predictive value and specificity were high
(> 99%) across the algorithms for confirmed and suspected HIT. The
positive predictive values (PPV) for confirmed HIT were 30.77%,
37.50% and 50% for algorithms A, B and C, respectively (P = 0.547).
However, the PPV was 100% for suspected HIT for all 3 algorithms.
The differences in sensitivity between the algorithms A, B and C for
confirmed (100% vs. 75% vs. 50%; P = 0.024) and suspected (100%
vs. 61.54% vs. 40%; P < 0.001) HIT were statistically significant.
Conclusion: These findings suggest that algorithms based on pharmacy
medication data, diagnostic test orders and diagnostic codes can be
used to identify suspected HIT in claims data.
Disclosure of Interest: None Declared.
Conclusion: Patients with beta-thalassemia are at high risk of thromboembolic events due to hypercoagulable state .
Disclosure of Interest: None Declared.
Factor XI and the Contact System
FNCS01
Characterization and development of factor XIIA inhibitors for assaying tissue factor–triggered coagulation
Kolyadko V and on behalf of Dr. T.A. Vuimo, Dr. S.S. Surov, Ms.
R.A. Ovsepyan, Ms. V.A. Korneeva, Dr. I.I. Vorobiev, Dr. N.N.
Orlova, Dr. L. Minakhin, Dr. K. Kuznedelov, Prof. K. Severinov,
Prof. F.I. Ataullakhanov, Prof. M.A. Panteleev
Laboratory for Molecular Mechanisms of Hemostasis, Center for
Theoretical Problems of Physicochemical Pharmacology Russian
Academy of Sciences, Moscow, Russian Federation
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Objectives: Global coagulation assays (measuring thrombin generation, spatial clot growth, and its elasticity) could be improved for better sensitivity with corn trypsin inhibitor (CTI), but its selectivity has
not been studied to date. We characterize factor XIIa (FXIIa) inhibitors and develop the most appropriate one for assaying of tissue factor
(TF)-triggered coagulation.
Methods: Infestin-4 (Inf4) mutated at the reactive site positions, and
CTI and squash-type trypsin inhibitors LCTI-III and CMTI-III were
produced and assayed. They were compared by inhibitory constants Ki
measured in chromogenic assays, by CT3 concentration that caused a
3-fold APTT elongation, and by elongation of the time to clot of uncitrated whole blood. The parameters (mean SD, n ≥ 3) are presented
in Table 1 (‘NI’ = no inhibition). The potency and selectivity were estimated in normal, FXII-depleted, and hemophilia A plasma samples
by examining the dynamics of spatial clot formation initiated by surface-localized TF (thrombodynamics assay).
Results: Unexpectedly, CTI inhibited FXIa and activated protein C
(aPC). LCTI-III and CMTI-III had low anti-FXIIa activity (Ki ca.
20 nmol L–1, CT3 ≥ 30 lmol L–1), inhibited factors XIa and Xa (Ki
ca. 30 lmol L–1), and decreased the clot growth rate in all tested
plasma by 25% compared with no inhibitor. Inf4 inhibited factor Xa
(2.21 0.19 lmol L–1) and changed the coagulation dynamics in
hemophilia A plasma. Inf4-MutB, Inf4 mutant with P2-P4’ sequence
TRNFVA, inhibited FXIIa and did not alter the coagulation dynamics in all tested plasma, despite its minor activity against thrombin. It
prolonged the blood clotting time and blocked contact activation in
the thrombodynamics assay.
iza
FEN32
Psychiatric disorder as expression of beta-thalassemia
intermedia
45
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Olaya V1,2, Alvarez MF3, Restrepo R3, Gomez G1 and Galvez K1
1
Antioquia, Hospital Pablo Tobon Uribe, Medellin; 2Valle,
Universidad Icesi, Cali; 3Antioquia, Universidad Pontificia
Bolivariana, Medellin, Colombia
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Objectives: Describe a case of beta-thalassemia intermedia with multiple cerebral infarctions
Methods: Case report.
Results: 37 year old woman with medical history of depressive disorder, transient ischemic attack; obstetric history 5 pregnancies, 2 births
and 3 abortions. The patient presented to a regional hospital, fatigue,
flight of ideas, tachylalia and insomnia with 3 days of evolution, in
paraclinical microcytic anemia is founded, with increased red blood
cell distribution width, leucopenia and thrombocytopenia. Refer to
our hospital on suspicion of neuropsychiatric lupus. However, rheumatology discarded systemic lupus erythematosus (SLE) since laboratories was negative. Magnetic Resonance Imaging brain reporting
diffuse cortical atrophy, multiple ischemic events from different times
of evolution and in several vascular territories: Multiple hyperintensities in radiated crowns and semi-oval centers by penetrating branches
of ischemic events with normal Willis polygon
Studies for antiphospholipid syndrome (APS) were then initiated, the
coagulation tests were reported as normal, VDRL, lupus anticoagulant, anticardiolipins and B2 glycoproteins, by the above APS is discarded. Electrophoresis of hemoglobin (Hb) shows Hb A 95.6%, Hb
A2 4.4%, confirming the diagnosis of beta-thalassemia intermedia
with thrombotic events in the central nervous system. Management
begins with hydroxyurea, acetyl salicylic acid and folic acid, whereby
the patient’s condition improves your blood count.
Additionally, the family is studied and diagnosed brother with betathalassemia intermedia with hemoglobin electrophoresis showing
93.2% Hb A, Hb A2 6.1%.
Conclusion: Inf4-MutB, FXIIa inhibitor based on Inf4, is more selective than previously known inhibitors and can be successfully applied
in global coagulation assays.
Disclosure of Interest: V. Kolyadko was an employee of HemaCore
(Moscow, Russia) during the time when this work was performed.
FNCS02
A retrospective review of rare coagulation factor deficiencies in a tertiary hospital (Singapore)
De Guzman MR, Tha MH, Ng GM and Tien SL
Department of Haematology, Singapore General Hospital,
Singapore City, Singapore
Objectives: Singapore is a country of about 5.4 million inhabitants of
diverse ethnicity. This study aims to review the prevalence, clinical pre-
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
12/06/2014
46
ABSTRACTS
sentation, and management of rare coagulation factor deficiencies in
this population.
Methods: A retrospective review of the records of the patients with rare
coagulation factor deficiencies in Singapore General Hospital (SGH)
dated from 1975 to 2011 was performed. The list of the patients was
retrieved from the laboratory database, electronic patient information
system and the case notes. All patients with relevant clinical information and a clotting factor (factors V, VII, X, XI, XIII) level of < 50%
were included.
Results: Summary of the findings as follows
X
3
XI
48
6
FFP
Novoseven
FFP prophylaxis
before procedure
FFP
Factor XIII and Fibrinogen
FF01
Identification of sequence variations in fibrinogen
genes causing hemorrhagic or thrombotic diathesis
Not
available
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XIII
< 1–49% Traumatic
injury
Asymptomatic
Epistaxis
Menorrhagia
Spontaneous L rectus
hematoma
Postoperative
bleeding
Hemoptysis
Hematuria
<1–50% Not available
(managed in pediatric
hospital)
FFP, tranexamic
acid
R
11
< 1–47% Hemoptysis
Menorrhagia
Gluteus
maximus hematoma
Subdural hematoma
Hemarthrosis
Peritoneal hematoma
hematuria
2–38%
Asymptomatic
Bleeding gastric ulcer
Menorrhagia
35–48% Asymptomatic
CD
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Management
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Clinical
presentations
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Factor
level
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Prevalence*
or
Factor
that products exhibiting high activity are not distributed for patient
use.
Methods: We have used 11/236 to calibrate nine FXIa-sensitive assays:
two commercial chromogenic FXIa activity assays (CA); a non-activated partial thromboplastin time (NaPTT); an in-house microplate
fibrin generation assay (FGA); an in-house thrombin generation assay
(TGA); and an assay for FXIa- and kallikrein-like proteolytic activities based on cleavage of substrate SN13a (D-LPR-ANSNH-C3H7
2HCl). NaPTT, FGA, and TGA were tested in either normal or FXIdeficient plasma.
Results: Each method demonstrated a sigmoidal dose-response to serially diluted 11/236. NaPTT was the least sensitive to low FXIa concentrations and the least robust; the in-house TGA was the most sensitive;
and the two CA were the most robust. All methods, except for SN13a
which is less specific for thrombotic impurities, gave comparable
(within 20% difference) potency assignments for IG lots.
Conclusion: Using 11/236 allows assignment of FXIa potency to IG
products that is independent of assay methodology. Also, some assays
may be more suitable than others to assess the thrombotic potential of
IG products.
Disclosure of Interest: Y. Liang is an employee of FDA. This is an
informal communication and it represents authors’ own best judgment. These comments do not bind or obligate the FDA. S. Woodle:
None declared, M. Weinstein: None declared, T. Lee: None declared,
D. Scott: None declared, M. Ovanesov: None declared.
aa
*Per 2 million (estimated according to the geographical population coverage by
SGH), FFP: fresh frozen plasma.
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Conclusion: This is the first review on rare coagulation factor deficiencies in Singapore. Factor XI deficiency was found to be the most prevalent disorder. Various manifestations were noted ranging from
being asymptomatic to potentially life-threatening bleeding. There was
no fatal outcome directly related to the bleeding symptoms among
these patients.
Disclosure of Interest: None declared.
FNCS03
Evaluation of assays for factor XIa to reduce the thrombotic risk of immunoglobulins
Liang Y, Woodle SA, Weinstein M, Lee TK, Scott D and Ovanesov
MV
Office of Blood Research and Review, US FDA, CBER, Rockville,
USA
Objectives: Although coagulation factor XIa (FXIa) impurity is the
primary source of procoagulant activity in thrombosis-implicated
immunoglobulin (IG) product lots, poor consistency among various
procoagulant assay methodologies and questions about whether global
coagulation assays give results equivalent to assays specific for FXIa
limit our ability to identify lots of IG with high thrombogenic activity.
Here we have used the WHO Reference Reagent for FXIa, NIBSC 11/
236, to present assay results of various procoagulant methodologies in
units of FXIa activity and have ranked the sensitivity and robustness
of these methodologies. These studies should help to standardize measurement of the thrombotic risk of IG products and will help to ensure
Ekhlasi-Hundrieser M, Halves C, Detering C and von Depka M
Werlhof-Institute, Hannover, Germany
Objectives: Fibrinogen consists of three pairs of polypeptide chains,
Aa, Bb, and c, which are encoded by paralogous genes FGA, FGB,
and FGG. Inherited fibrinogen disorders include a-, hypo-, and dysfibrinogenemia. Clinical manifestations of fibrinogen defects vary from
being asymptomatic to life-threatening bleeding or thrombophilic
diathesis. Here we present data of patients with various coagulation
disorders caused by missense mutations in fibrinogen genes.
Methods: Genomic DNA was extracted from 35 patients with conspicuous laboratory values (fibrinogen/Clauss, thrombin, and batroxobin
time). Complete fibrinogen gene loci (exons, introns, and flanking
regions) were sequenced using long-range PCRs combined with Sanger/next generation sequencing.
Results: Molecular analysis of the three fibrinogen genes in 35 unrelated patients with fibrinogen defects has led to the identification of
four novel heterozygous missense mutations. Three mutations have
been located in the Bb-chain gene (Cys211Arg, Glu145Gln,
Lys178Asn). One mutation has been detected in the fibrinogen c-chain
gene (Tyr237His). Numerous intronic variants were detected, in addition.
Conclusion: Hypo-/dysfibrinogenemia in patients with hemorrhagic
diathesis is caused by different heterozygous missense mutations in
FGB and FGG genes affecting the functional properties of fibrinogen.
In this analysis, there were no clear hotspots detectable. Sequence variations are highly variable among unrelated patients, although patients
are recruited from a clearly defined area of North Germany. Correlations with the type of mutation may help to understand the function of
the protein and to improve the management of the patients.
Disclosure of Interest: None declared.
12/06/2014
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
ABSTRACTS
FF02
Congenital fibrinogen deficiency in Pakistan and identification of five novel mutations
Methods: This cross-sectional study was conducted in NIBD Karachi
in conjunction with Children Hospital and Chughtai’s Lab Lahore.
Comprehensive data were collected with a detailed questionnaire
about the patient’s demographics, complete history including past
medical and surgical, family history, current status of disease, physical
examination and baseline lab investigations including CBC, liver profile, and viral markers (HBsAg, anti HCV and HIV), and coagulation
profile including PT, APTT, fibrinogen clottable assay, and fibrinogen
antigen. The genetic screening to look for any mutation in index
patients was accomplished by direct sequencing of PCR-amplified gene
segments using genomic DNA. All DNA samples were verified at the
University of Geneva, Switzerland, and University of Bonn, Germany.
Simple descriptive was applied for frequency.
Results: Nine of 10 patients were found to have mutations; 7 (70%) of
10 showed mutations in FGA; two (20%) have shown mutations in
FGB; and no mutation so far has been detected in FGG. FGA gene
mutation is seemingly the most commonly occurring mutation in local
population with two novel homozygous nonsense mutations in the
same gene.
Conclusion: Congenital afibrinogenemia is an inherited bleeding disorder. Its rarity can be questioned now due to its swiftly increasing incidence over the past decade in particular. In this study, two novel
mutations in the FGA gene has been identified along with one missense
mutation in FGA in our population, which has not yet been reported
in the literature. As reported in the literature, FGA is the most commonly occurring mutation in regions of the world where consanguinity
prevails. This study transpires the fact that mutations in FGA frequently exist in local populations followed by FGB mutations with
variation in severity of phenotypic expression.
Disclosure of Interest: None declared.
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Borhany M1, Robusto M2, Fatima N3, Shamsi TS1, Asselta R2,
Duga S2 and Peyvandi F4
1
Haematology,Haemostasis & Thrombosis, National Institute Of
Blood Disease & Bone Marrow Transplant (NIBD), KARACHI,
Pakistan; 2Department of Medical Biotechnologies and
Translational Medicine, University of Milan, Milan, Italy;
3
Research and Development, National Institute Of Blood Disease
& Bone Marrow Transplant (NIBD), Karachi, Pakistan; 4Angelo
Bianchi Bonomi Hemophilia and Thrombosis Center, University
of Milan, Milan, Italy
FF04
Measuring plasma fibrinogen levels below 40 mg dL–1
using the STAâ-Liquid FIB reagent
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Objectives: The aim of the study is to identify the underlying mutations
of afibrinogenemia and possibly to correlate bleeding symptoms with
the identified genotype.
Methods: Subjects with inherited bleeding tendency in the absence of
any acquired cause of hemostatic defect were included in the study.
Afibrinogenemia was diagnosed after analysis of PT, APTT, and measurements of functional and antigen fibrinogen levels. Genomic DNA
was extracted from whole blood by the QiAamp DNA Blood mini kit
(Qiagen).Genomic DNA was PCR amplified by using sense and antisense primers designed on the basis of known sequences of the three
fibrinogen genes and intergenic regions (GenBank accession numbers
M64982, M64983, M10014, U36478, and AF229198). Molecular
analysis was performed by DNA sequencing on both strands, by using
the Big Dye Terminator Cycle Sequencing kit and an ABI-3130-XL
automated DNA sequencer (Applied Biosystems). The Variant Reporter software (Applied Biosystems) was used for mutation detection.
Results: A total of 21 afibrinogenemic patients (10 males and 11
females) came to our attention: their mean age was 11.54 11.2 years
(range 1–40) and the mean bleeding score was 13.3 6.7.The most
common bleeding manifestations were bruises, umbilical cord bleeding, gum bleeding, hematomas, circumcision bleeding, epistaxis, and
menorrhagia. Genetic analysis was completed in nine cases, revealing a
total of six different mutations: four in FGA (p.Arg 129 X, p.Cys8 X,
p.Gln 200X,p. Gln162 X, the last three being novel) and two in FGB
(p.Arg 47 X, p.Thr 407 Lys, both being novel). All afibrinogenemic
patients resulted homozygous for the identified mutation.
Conclusion: We have reported five novel mutations leading to congenital afibrinogenemia in a cohort of 21 patients.
Disclosure of Interest: None declared.
47
FF03
Identification of novel mutations in fibrinogen gene
alpha (FGA) and frequency of mutations in Pakistani
population
Naz A1, Neerman-Arbez M2, Ivaskevicius V3, Khan TN4, Ahmed
S4, de Moerloose P2, Oldenburg J3, Tariq S5, Saqlain N6, Ahmed
N6, Shamsi TS4 and on behalf of Arshi Naz1, Marguerite
Neerman-Arbez2, Vytautas Ivaskevicius3, Tehmina Nafees Khan1,
Shariq Ahmed1, Philippe de Moerloose2, Johannes Oldenburg3,
Shehla Tariq4, Nazish Saqlain5, Nisar Ahmed5, Tahir S Shamsi1
1
Thrombosis and Haemostasis/Genome Lab, National Institute of
Blood Disease & Bone Marrow Transplantation, Karachi,
Pakistan; 2University of Geneva, Geneva, Switzerland; 3Institute
of Experimental Haematology and Transfusion medicine, Bonn,
Germany; 4National Institute of Blood Disease & Bone Marrow
Transplantation, Karachi; 5Chughtai’s Lab; 6Children Hospital
Lahore, Lahore, Pakistan
Objectives: The study focuses to detect the most commonly occurring
mutation of fibrinogen gene in Pakistani patients.
Grimaux M, Carrion C, Chtourou D, Magdelaine A, Nicham F and
Mazet C
R&D, Diagnostica Stago, PARIS, France
Objectives: Fibrinogen plays a major role in wound healing and clotting. A decreased fibrinogen level is observed in acquired diseases like
DIC and fibrinogenolysis but also in heritable fibrinogen deficiency
and in thrombolytic therapy. In these cases, it can be necessary to measure very low plasmatic fibrinogen levels. The aim of our study was to
evaluate the accuracy of a modified STAâ-Liquid Fib methodology
for the determination of low plasmatic fibrinogen activity levels.
Methods: STAâ-Liquid Fib is a new reagent for the quantitative determination of fibrinogen level in plasma based on the Clauss method.
The measuring range of 100–800 mg dL–1 (sample dilution 1:20) can
be extended to 40–1200 mg dL–1 using, respectively, 1:8 or 1:40 dilutions. This study goes further by demonstrating the reliability of measuring fibrinogen levels below 40 mg dL–1.
A range of fibrinogen levels between 10 and 40 mg dL–1 was obtained
by diluting hypofibrinogenemic plasma samples in Owren’s buffer.
Samples were tested using a 1:2 dilution. Evaluation of the total precision was carried out according to CLSI guidelines. Interferences were
investigated towards unfractionated and low molecular heparins
(UFH and LMWH), fibrin degradation products (FDP), hirudin, and
dabigatran etexilate.
Results: Accuracy was shown to be highly satisfactory with a bias
< 10% from 10 to 40 mg dL–1 and reproducible from one lot to
another. Within-run and within-laboratory precision were found to be
≤ 4% for the tested fibrinogen level (30 mg dL–1). Regarding interferences, STAâ-Liquid Fib appears to be insensitive to 30 lg mL–1 of
FDP, 125 ng mL–1 of dabigatran, 2 lg mL–1 of hirudin, and 2 IU
mL–1 of UFH or LMWH.
Conclusion: This study demonstrates the possibility to accurately measure fibrinogen levels down to 10 mg dL–1 by modifying the plasma
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
12/06/2014
48
ABSTRACTS
sample dilution of the STAâ-Liquid Fib methodology. The satisfactory results for the precision study and the method insensitivity to a
number of potentially interfering substances make this modified
method of high interest.
Disclosure of Interest: M. Grimaux is an employee of Diagnostica
Stago, C. Carrion is an employee of Diagnostica Stago, D. Chtourou
is an employee of Diagnostica Stago, A. Magdelaine is an employee of
Diagnostica Stago, F. Nicham is an employee of Diagnostica Stago,
C. Mazet is an employee of Diagnostica Stago.
the difference is even larger. These variations may be caused by clot
detection method and the effect of fibrin degradation products on
thrombin and fibrin polymerization. Given the potential clinical consequences, including plasma transfusion, accurate determination of low
fibrinogen levels is pivotal and the commercial assays need to be
selected and optimized accordingly. The use of integral methods like
FIBTEM requires clinical validation.
Disclosure of Interest: None declared.
Fibrinolysis
FF05
Clinically relevant discrepancies between fibrinogen
analysis methods in patients undergoing thrombolytic
therapy for acute iliofemoral deep venous thrombosis
FBL01
Differential effect of dabigatran, rivaroxiban and apixaban on thrombomodulin mediated activation of protein C and thrombin activated fibrinolysis inhibitor
(TAFI) may impact their safety and efficacy profiles
Strijkers RH1, Kuiper G-JA2, van Oerle R3, Wetzels R3, ten Cate
A4, ten Cate H4,5, Wittens CH1,6 and Henskens Y3
1
Vascular Surgery, Cardiovascular Research Institute, Maastricht,
The Netherlands; 2Anesthesiology and Pain Treatment,
Maastricht University Medical Centre, Maastricht, Germany;
3
Central Diagnostic Laboratory, Maastricht University Medical
Centre; 4Laboratory for Clinical Thrombosis and Haemostasis,
Cardiovascular Research Institute; 5Internal Medicine, Maastricht
University Medical Centre, Maastricht, The Netherlands;
6
Vascular Surgery, University Hospital RWTH, Aachen, Germany
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Objectives: The new oral anticoagulant agents Dabigatran, Rivaroxiban and Apixaban target thrombin and factor Xa to mediate their
anticoagulant effects respectively. However significant differences have
been reported in their pharmacodynamics actions. This study was
designed to investigate the effects of active forms of Dabigatran, Rivaroxiban and apixaban on the activation of protein C and TAFI by
thrombin-thrombomodulin complex.
Methods: The active form of Dabigatran was synthesized. While Rivaroxiban and Apixaban were extracted from commercially available
tablets. Both agents were dissolved in appropriate solution matrices at
a stock concentration of 100 lg mL–1. Thrombin-thrombomodulin
mediated activation of protein C and TAFI were measured using specific chromogenic substrate based methods at a concentration of 0–
10 lg mL–1 in different matrices. The activation of Protein C and
TAFI were measured using mass spectrometric and immunoblotting
methods.
Results: Dabigatran produced a strong inhibition of the generation of
both the activated protein C and TAFI (IC50 < 1.0 lg mL) –1 whereas
Rivaroxiban and apixaban did not produce any inhibition of the activation of either of these proteases. Dabigatran also inhibited the amidolytic actions of thrombin-thrombomodulin complex whereas
Rivaroxiban and apixaban did not produce any effect. Neither Dabigatran nor Rivaroxiban produced a direct inhibition of activated protein C or TAFI at concentrations of up to 10 lg mL–1. Mass
spectrometric and immunoblotting methods showed that Dabigatran
blocked the activation of both TAFI and Activation C by thrombinthrombomodulin complex whereas rivaroxaban and apixaban did not.
Conclusion: The persistant inhibition of thrombin and its regulatory
effects by Dabigatran may differentiate its pharmacologic profile from
Rivaroxiban and apixaban. Since thrombin plays several regulatory
functions, its persistent inhibition may compromise hemostatic regulation by this agent.
Disclosure of Interest: None declared.
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Objectives: Accurate determination of fibrinogen levels is important
for decision-making in patients undergoing thrombolytic therapy.
Fibrinogen levels < 1.0 g L–1 require discontinuation of thrombolytic
therapy or administration of plasma to reduce the risk of bleeding.
After experiencing rapidly decreased or immeasurable fibrinogen levels
in some patients undergoing thrombolysis, we compared different
methods.
Methods: A prospective analysis of 55 citrated plasma samples of 7
patients undergoing ultrasound-accelerated catheter directed thrombolysis with urokinase (100K U hr–1) for iliofemoral DVT was performed. Fibrinogen levels (Clauss method) were determined using
three coagulation analyzers: CA-7000 (standard method), CS2100i
(both Dade Thrombin, Siemens), and STA-R Evolution (STA Liquid
Fib, Stago). A functional fibrinogen assay (FIBTEM, Amplitude after
10 min, A10) in whole blood was performed by thromboelastography
(ROTEM).
Results: Fibrinogen could not be determined by CA7000 and CS2100i
in, respectively, 22 and 21 of 55 samples due to a coagulation curve
error report, indicating fibrinogen levels < 1.0 g L–1. The STA-R
method did not result in errors and 2/55 samples resulted in a result
< 1.0 g L–1. The consistent difference between CA7000/STA-R is
shown in Fig 1. Using the FIBTEM assay, 2/55 samples were below
the reference range (A10, 6–22 mm) and no error was reported.
Hoppensteadt D1, Harenberg J2, Lewis B3 and Fareed J1
Pathology, Loyola University Medical Center, Maywood, USA;
2
Ruprecht-Karls University Heidelberg, Mannhein, Germany;
3
Medicine, Loyola University Medical Center, Maywood, USA
1
Conclusion: In plasma samples from patients undergoing thrombolysis,
there are substantial discrepancies in fibrinogen results between Clauss
methods. When compared to a functional fibrinogen assay (FIBTEM)
12/06/2014
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
ABSTRACTS
FBL02
The Netherlands purification and characterization of
novel enzymes with antiplatelet and anticoagulant
activities from snake venom
extent, DD, whereas the second one contained only DD and did not
contain hFDPs. Twenty-two MAbs were tested in pairs with DD and
FDPs in a one-step fluoroimmunoassay format. Most assays had
higher specificity to FDPs and lower to DD. Some assays were more
specific to DD than to FDPs. Only one assay utilizing Mab DD189 as
capture and Mab DD255 as detection equally recognized DD and
FDPs in the range of analyte concentrations from 20 to 1000 ng mL–1.
Conclusion: We believe that for precise analyte measurements, DD
assays should equally recognize both DD and FDPs. Such approach
should significantly simplify the standardization of DD assays.
Disclosure of Interest: None declared.
Jugder B-E1, Dashnyam P1, Buyanbadrakh B2, Byambasuren S1,
Buyanbat A2 and Purev T1
1
Mongols Life Sciences Institutes; 2School of Biomedicine, Health
Sciences University of Mongolia, Ulaanbaatar, Mongolia
FBL04
Introduction of a point of care D-dimer EQA program
to include assessment of testing and postanalytical
interpretation of results
Kitchen DP, Munroe-Peart S, Kitchen S, Jennings I, Woods T and
Walker I
UK Neqas for Blood Coagulation, Sheffield, UK
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Objectives: Point of care (POC) D-dimer testing is increasingly being
used in evaluation of patients with suspected thromboembolism
(VTE). A D-dimer test result below the stated cut-off value together
with a low clinical evaluation score would suggest VTE was unlikely
and allow the clinician to avoid referring the patient for complex and
expensive investigations. The devices used are portable desktop analyzers well suited to non-laboratory areas but still requiring quality
control to ensure reliable test results. Although external quality assessment (EQA) is already available to laboratories for D-dimer testing
(n = 702 centers registered in UKNEQAS BC laboratory program), a
specific program for POC devices was evaluated.
Methods: 9 pilot EQA surveys of POC D-dimer testing have been carried out with the quantitative Cobas h232 method (Roche, n = 94 centers) and with the qualitative Clearview Simplify kits (Alere, n = 40
centers). Three pilot studies have been performed at 28 centers using
the quantitative Triage method (Alere). Participating centers were
asked to test the samples and, using the short patient scenario provided, were asked whether DVT could be excluded.
Results: Interpretations showed full agreement in 5/12 samples (Cobas), 1/6 samples (Clearview), and 1/6 samples (Triage). In one sample
with a patient scenario showing a Wells score of 4, 16/48 Cobas users,
7/14 Triage users, and 1/20 Clearview users stated they would exclude
DVT. Results for this sample were Cobas median = 0.14 lg mL–1
FEU (CV 18.4%), Triage users median = 141 ng mL–1 FEU (CV
24.7%) and Clearview 18/19 centers reported a positive result.
Conclusion: These data identify a clear need not only for quality assessment but also for education to improve the postanalytical interpretation of test results in combination with the clinical scenario. UK
NEQAS BC have introduced a POC D-dimer testing program in 2014
with assessment of both the result obtained and the interpretation of
that result.
Disclosure of Interest: None declared.
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Objectives: Snake venom contains a variety of bioactive proteins and
enzymes possessing a wide range of effects on hemostasis, and antiplatelet and anticoagulant agents play important roles. The present
study presents part of the technological development of several target
proteins purified in parallel from Agkistrodon venom species (A. halys
halys, A. blomhoffi ussuriensis, and A. halys palas), in particular, the
development of manufacturing steps for two novel active enzymes, a
fibrinolytic enzyme and a protein C activator, having both antiplatelet
and anticoagulant effects.
Methods: The enzymes were purified using three different HPLC steps:
affinity chromatography, ion exchange, and gel filtration chromatography. The enzyme characterization was carried out by SDS-PAGE, 2D electrophoresis, enzyme electrophoresis, platelet aggregation assay,
APTT, and MALDI-TOF-MS. Proteolytic activities of the enzymes
were investigated by chromogenic substrates S-2366 and S-2251.
Results: The fibrinolytic enzyme and the protein C activator were purified to homogeneity with approximate purity of 98% and 85%, respectively. The fibrinolytic enzyme was determined to be a dimer serine
protease with molecular weight of 35.3 kDa and pI of 7.0, whereas the
protein C activator had 37.5 kDa molecular weight and pI 5.0. Both
the fibrinolytic enzyme and the protein C activator inhibited ADPinduced rabbit platelet aggregation 85% and 75%, respectively. Both
enzymes also exhibited profound in vitro anticoagulation activities.
The fibrinolytic enzyme and the protein C activator were found to
exhibit high specificity for the chromogenic substrates S-2366 and S2251, respectively.
Conclusion: The purification process of the novel enzymes with anticoagulant and antiplatelet activities comparable with similar commercially available products has been developed. These favorable
antithrombotic profiles together with their anticoagulant properties
make the purified enzymes potential agents for the prevention and
treatment of thrombosis.
Disclosure of Interest: None declared.
49
FBL03
New approach for D-dimer antibody selection
Kogan A, Mukharyamova K, Bereznikova A and Katrukha A
HyTest, Turku, Finland
Objectives: D-dimer (DD) measurements are widely used in clinical
practice. Unfortunately, there is a great discrepancy in DD values
determined by different assays. The absence of agreement between different assays is explained by the fact that patients’ plasmas contain a
broad spectrum of fibrin degradation products (FDPs) and anti-DD
Mabs of different assays show different specificity to them. To solve
this problem, we have selected Mabs with equal specificity to both to
DD and to FDPs with higher molecular weight.
Methods: The solutions of DD and FDP were prepared from fibrin
clot by plasmin digestion. The liquid fraction was withdrawn at the
early step and divided into two samples. In one sample, the reaction
was stopped by PMSF to produce FDP, and in the other sample, the
reaction was continued to produced DD. Thus, FDP and DD preparations contained the same amount of cross-linked fibrin-derived material. All Mabs were from the new generation of HyTest anti-DD
antibodies.
Results: Analysis of FDPs and DD preparations showed that the first
one contained mostly high molecular weight FDPs and, to a lesser
FBL05
MST-188 increases rates of fibrin assembly and subsequent urokinase/TPA-mediated lysis in vitro
Syed D1, Emanuele M2, Hoppensteadt D1 and Fareed J1
1
Pathology, Loyola University Medical Center, Maywood; 2Mast
Therapeutics, San Diego, USA
Objectives: MST-188 (Mast Therapeutics, San Diego, CA) is a purified
form of a non-ionic, triblock copolymer poloxamer 188 capable of
binding to hydrophobic surfaces of damaged cells. In previous studies,
we have demonstrated the ability of this agent to increase fibrin assem-
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
12/06/2014
50
ABSTRACTS
bly in vitro. In this study, we investigated the hypothesis that MST-188
also increases the rate of urokinase/tPA-mediated lysis.
Methods: An optical method was used to monitor the kinetics of fibrin
assembly/lysis in plasma samples supplemented with P188, dextran
40K (D40), a polydeoxyribonucleotide (defibrotide), and unfractionated heparin (UFH) over 30 min on the addition of calcium and thrombin. Samples were supplemented with MST-188 (10–0 mg mL–1), D40
(10–0 mg mL–1), defibrotide (10–0 mg mL–1), UFH (10–0 lg mL–1), or
saline. Urokinase was supplemented to all samples (2500–0 U mL–1).
Results: MST-188 increased both maximum absorbance and rate of
thrombin-mediated fibrin formation compared to saline controls.
Moreover, the lytic profiles of MST-188–supplemented samples demonstrated an increased onset and rate of lysis compared to saline controls. No effect was noted in rates of fibrin assembly or urokinasemediated lysis in UFH, D40, or defibrotide-supplemented samples.
Conclusion: These results suggest that MST-188 facilitates fibrin polymerization and also enhances urokinase and tPA-mediated lysis.
Whether the enhanced lysis is a result of increased fibrin-dependent
urokinase-mediated lysis or whether P188 alters the polymeric composition of the fibrin clot, making it more susceptible to lysis, is to be further investigated. These findings suggest that P188 may facilitate
pharmacologic thrombolysis in vaso-occlusive disorders.
Disclosure of Interest: None declared.
Conclusion: The thrombus targeting system ENP could also target TF
highly expressing C6 glioma in vivo.
Disclosure of Interest: None declared.
Hemostasis and Malignancy
Zhang B and Hu Y
Institute of Hematology, Union Hospital, Wuhan, China
Co
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Objectives: EGFP-EGF1 was isolated from factor VII and has been
shown to bind to tissue factor (TF) highly expressed in thrombosis in
vitro and in vivo. As TF was also abundent in tumor tissues in both
tumor cells and endovascular cells, it occurred to us the drug delivery
system mediated by EGFP-EGF1 for thrombus targeting could also
been applied in tumor drug delivery.
Methods: Unmodified PEG-PLA nanoparticles (NP) were prepared
through an emulsion/solvent evaporation technique. ENP was prepared via a maleimide-thiol coupling reaction. Physical and chemical
parameters of nanoparticles were analyzed by using Malvern Nano ZS
(Malvern Instruments, UK) and the transmission electron microscope.
Glioma-bearing mice were established by intracranial injection of
5 9 105 C6 cells into male nude mice. The dual targeting effect in vivo
was investigated by in vivo imaging and glioma slides.
Results: The diameters for both NP and ENP were around 110 nm
with regular size and smooth surface. ENP accumulated more in the
glioma site than unmodified NP demonstrated by in vivo imaging. In
vivo distribution experiment showed there were only a few green particles distributed in glioma tissues in NP group (Figure 1A–E) and the
nanoparticles mainly sited near neovascular cells (Figure 1D and E),
while a significantly higher and wider distribution of coumarin-6–
labeled ENP in glioma tissues was observed (Figure 1F–J). ENP not
only distributed in neovascular cells but also penetrated vascular to
reach extravascular glioma cells (Figure 1I and J).
Figure 1 In vivo distribution of coumarin-6–labeled nanoparticles in
brain glioma of Balb/c mice Blue: cell nuclei. Green = coumarin-6–
labeled nanoparticles. Red: CD 31 labeled neovascular cells. The white
and yellow arrows in image E and J represented nanoparticles distribution in extravascular glioma cells and in neovascular cells, respectively.
Bar: 100 lm.
Yu Y-B1,2,3, Gau J-P1, Hsu H-C3, Tzeng C-H1 and Lee T-S3
1
Division of Hematology and Oncology, Taipei Veterans General
Hospital; 2Faculty of Medicine; 3Department of Physiology,
National Yang-Ming University, Taipei, Taiwan
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HM01
Factor VII–derived EGFP-EGF1 protein mediate drug
delivery system to TF highly expressing tumors
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HM02
Thrombin-activatable fibrinolysis inhibitor gene polymorphism G-438A is associated with venous thromboembolism in cancer patients: a case-controlled study
Objectives: Cancer patients are susceptible to venous thromboembolism (VTE), and impaired fibrinolysis might further increase the risk of
this complication. Thrombin-activatable fibrinolysis inhibitor (TAFI),
which regulates the conversion of plasminogen to plasmin, is a critical
enzyme in modulating fibrinolytic activities. However, the association
between functional DNA polymorphisms of the TAFI gene (CPB2)
and cancer thrombosis remains unclear.
Methods: Cancer patients with VTE and age- and diagnosis-matched
cancer patients without VTE were enrolled in a case-control study at a
1:2 ratio. The plasma levels of TAFI were measured and four functional polymorphisms of CPB2 (G-438A, G505A, C1040T, and
G1344A) were determined using TaqMan or a 50 nuclease allelic discrimination assay.
Results: From September 2007 to August 2013, 138 cancer patients
with VTE and 276 matched cancer patients without VTE were
enrolled. The median age was 62.0 years (range, 19–92 years) and
48.6% of them were male. The most common cancer subtypes were
gastrointestinal malignancies (27.5%), hematological malignancies
(23.2%), and lung cancer (12.3%). Plasma TAFI levels were identical
in both groups of patients (84.0% vs. 83.4%, P = .881). The distributions of G505A, C1040T, and G1344A genotypes were similar in both
groups, but the GG genotype of the G-438A polymorphism was more
frequently noted in VTE patients (73.7% vs. 61.8%, P = .018). In a
logistic regression analysis, the hazard ratio of VTE in patients with
the GG genotype over the non-GG genotype was 1.74 (95% confidence interval 1.10–2.74, P = .017).
Conclusion: The G-438A polymorphism of CPB2 is associated with
VTE in cancer patients. The resulting interaction between defective
fibrinolysis and cancer thrombosis warrants further study.
Disclosure of Interest: None declared.
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© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
ABSTRACTS
HM03
Study of snake venom–derived antiplatelet agents
chemotherapy, and after 3 weeks. We examined the frequency of
thrombocytopenia (platelet count < 100 9 109/L), frequency of bleeding associated with low platelet counts, and need for platelet transfusions. Patients with preexisting thrombocytopenia were excluded from
the study group.
Results: Initially, 37 patients with breast cancer receiving the DAC regimen were involved in this study, from which 1 patient was excluded
because of preexisting thrombocytopenia; in total, data from 36
patients were analyzed. During the entire study, only 5 (13.89%) of 36
patients developed thrombocytopenia. Of a total of 177 cycles of chemotherapy, there were only 7 cases (3.95%) of grade 1 or grade 2
thrombocytopenia. No grade 3 and 4 thrombocytopenia was noted, as
well as no incidence of bleeding and no need for platelet transfusions.
Conclusion: The results of our study suggest that in breast cancer
patients receiving the DAC regimen, CAT is a relatively rare complication and patients mostly presented with mild thrombocytopenia with
no bleeding and no need for platelet transfusions. Further research is
needed to examine the frequency and risk of CAT in other subgroups
of patients.
Disclosure of Interest: None declared.
Buyanbat A1,2, Jugder B-E1, Dashnyam P1, Buyanbadrakh B2,
Byambasuren S1 and Tseveg T2
1
Mongols Life Sciences Institutes; 2School of Biomedicine, Health
Sciences University of Mongolia, Ulaanbaatar, Mongolia
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HM05
Platelet-microparticles (PMP) in essential thrombocythemia (ET): characterization of procoagulant and
proangiogenic activities
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Russo L1, Gamba S1, Marchetti M1, Vignoli A1, Tartari CJ1, Finazzi
G2, Rambaldi A2 and Falanga A1
1
Division of Immunohematology and Transfusion Medicine;
2
Division of Hematology, Hospital Papa Giovanni XXIII,
Bergamo, Italy
Objectives: Different studies suggested that circulating microparticles
(MP) play a role in the ET pathogenesis and in vitro may promote neoangiogenesis. This study aims to characterize the procoagulant and
proangiogenic properties of MP released in vitro by platelets isolated
from ET patients.
Methods: Platelets from 23 ET and 16 controls (CTR) were isolated
and resuspended in HEPES Tyrode’s buffer at 1 9 109 platelets mL–1.
PMP were isolated by two serial centrifugations, resuspended in different buffers for specific assays, and quantified by flow cytometry (Accuri C6, BD) as CD41+ events. PMP procoagulant properties were
characterized by CAT assay to measure thrombin generation (TG)
capacity and by P-PPL/1 assay to evaluate phospholipid-dependent
procoagulant activity (PCA) (both tests from Stago). The capillarylike tube formation assay in Matrigel was performed to characterize
the PMP proangiogenic potential.
Results: The in vivo study showed that PMP levels in plasma of ET
patients were significantly (P < 0.05) higher compared to CTR. After
correction for platelet counts, ET patients displayed lower rate of circulating PMP/platelets compared to CTR (P < 0.05). Platelets from
ET released in vitro significantly (P < 0.05) lower quantity of PMP
compared to CTR. In addition, ET-PMP showed a lower TG activity
and PCA compared to CTR (P < 0.05). After normalization of the
PMP count, the procoagulant potential of PMP from ET patients
remained lower than that of CTR. Finally, in the capillary-like tube
formation assay, the ET PMP were less proangiogenic compared to
those of CTR (P < 0.05).
Conclusion: In agreement with the observation that, in vivo the rate of
PMP/platelets is significantly lower in ET than in CTR, our data show
that ET platelets release in vitro less PMP. Moreover, ET PMP show a
lower procoagulant and proangiogenic potential. Therefore, the demonstrated higher PCA of MP in vivo might be mainly due to the presence of elevated number of MP in plasma but not to an intrinsic
increased procoagulant potential.
Disclosure of Interest: None declared.
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Objectives: Snake venom is a rich source of a number of proteins of
biotechnological interest due to their wide range of effects on haemostasis. Agkistrodon venom contains a variety of proteins that possess
antiplatelet activities. This study presents recent development in our
laboratory to produce and purify antiplatelet proteins, such as phospholipases A2 (PLA2) enzyme and disintegrin-like protein (DLP),
derived from A. blomhoffi ussuriensis venom.
Methods: Different matrices of HPLC (affinity interaction, anion
exchange, and size exclusion chromatography) were used for purifying
the proteins. The same chromatographic steps (Blue sepharose and Qsepharose chromatography) were used to obtain both proteins followed by separate size exclusion steps for final purity (Superdex-200
for PLA2 and Superdex-75 for DLP). The purity, Mr, pI, and protein
sequence identification were determined by SDS-PAGE, 2-D gel-electrophoresis, and MALDI-TOF-MS. The inhibitory activity on ADPinduced platelet aggregation was examined in the rabbit platelet-rich
plasma (PRP). PLA2 enzymatic activity was investigated by using a
fluorogenic substrate PED6.
Results: The purified DLP was a single-chain glycoprotein with an Mr
of 13 kDa and pI of 4.7, and this was identified as a novel platelet
aggregation inhibitor by MS analysis. The PLA2 was obtained with an
Mr of 14 kDa and pI of 4.17. A dose-dependent activity analysis
showed that both proteins exhibit antiplatelet activity, and the ID50
values of DLP and PLA2 were 0.25 and 0.65 lmol L–1, respectively.
Additionally, the inhibitory activity of PLA2 was demonstrated to act
via enzymatic reaction, and its specific activity with PED6 was
613.0 21.6 U mg–1.
Conclusion: In the present study, bioprocesses to produce and purify
novel antiplatelet agents from A. blomhoffi ussuriensis venom have
been developed, using modern liquid chromatographic matrices. The
purified proteins can be potential candidates for cardiovascular disease
management. Ongoing work to optimize large-scale production process is being undertaken.
Disclosure of Interest: None declared.
HM04
Chemotherapy-associated thrombocytopenia in breast
cancer patients: a retrospective hospital-based cohort
study
Tamamyan GN, Danielyan SH, Zohrabyan DG, Safaryan LL,
Harutyunyan LA, Avagyan AT, Sargsyan LR, Hakobyan LS and
Voskanyan AA
Clinic of Chemotherapy, Muratsan Hospital Complex of Yerevan
State Medical University, Yerevan, Armenia
Objectives and Aim: Although chemotherapy-associated thrombocytopenia (CAT) has been known for decades, but there limited data on
the frequency and relative risk of CAT in patients with different types
of cancers. The aim of the study was to identify the frequency of
thrombocytopenia and thrombocytopenia-associated bleeding in
breast cancer patients receiving chemotherapy according to the DAC
regimen, as well as to assess the need for platelet transfusions of the
same group of patients.
Methods: From 2009 to 2013, adult patients (median age 53.3 years)
with breast cancer receiving chemotherapy according to the DAC regimen (docetaxel, doxorubicin [or epriubicin], cyclophosphamide) at the
Clinic of Chemotherapy of ‘Muratsan’ Hospital Complex of Yerevan
State Medical University were observed during the treatment course.
The platelet counts were assessed before chemotherapy, 5–6 days after
51
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
12/06/2014
52
ABSTRACTS
HM06
Procoagulant properties of prostate cancer cell lines
and the effect of androgen deprivation
Results: Of 666 patients receiving IVCFs during this time period, 247
(37.1%) had active cancer. Of these, 151 (22.7%) had carcinoma, 92
(13.8%) had sarcoma, and 115 (17.3%) had metastatic disease. Follow-up was available for a median of 401.0 (IQR: 107.5–786.5) days.
IVCF- related complications occurred in 17.7% and 19.8% of patients
with and without cancer, respectively (P = 0.50). Patient with cancer
were less likely to have the IVCF retrieved (28.0% vs. 42.0%,
P < 0.001). Excluding death, cancer and non-cancer patients had similar adverse outcomes (Figure). In multivariable analysis, the absence
of anticoagulation after IVCF placement was associated with IVCF
related complications (OR = 0.43, P = 0.003). Localized cancer and
metastatic disease were not associated with IVCF complications
(OR = 0.82, P = 0.47 and OR = 1.09, P = 0.77, respectively). Both
localized (OR = 0.63, P = 0.041) and metastatic cancer (OR = 0.34,
P < 0.001) were associated with reduced IVCF retrieval.
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Conclusion: In a modern cohort of patients undergoing IVCF placement, active malignancy is not associated with a higher rate of IVCFrelated complications but is associated with a reduced rate of filter
retrieval.
Disclosure of Interest: None declared.
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Objectives: Tissue factor (TF) is the main trigger for coagulation. Prostate cancer (PCa) patients show elevated plasma TF-carrying microparticles and appear to be hypercoagulable. The hemostatic properties
of most widely used PCa cell lines are not known. We aim to characterize the expression of TF in the three most common PCa cell lines
including DU-145, LNCaP, and PC3, as well as an LNCaP subtype,
C4-2B2, and to evaluate the effects of bicalutamide (an androgen
receptor antagonist) on TF expression in LNCaP and DU-145 cells.
Methods: TF expression was evaluated by RT-PCR and Western blot.
The activity of TF was measured by a functional ELISA. Cell surface
expressions of TF and TF-MPs in culture media were analyzed by flow
cytometry. In addition, ultracentrifuged culture media samples from
each cell line were used to spike normal blood to test its coagulability
using thrombelastography. Androgen deprivation was mimicked in vitro using bicalutamide.
Results: TF was expressed in DU-145, LNCaP, PC3, and C4-2B2 cells
at the mRNA and protein levels. TF cell surface expression varies significantly among cell types. TF in cell lysates and that carried on MPs
were capable of initiating the coagulation cascade. Bicalutamide-treated LNCaP and DU-145 cells did not differ significantly in TF cell surface or protein expression in comparison to their controls. A
significant difference was found between the percent change of cell surface expression with bicalutamide treatment when comparing LNCaP
and DU-145 cells. The TF was proved to be functional in both cell
types (treated and untreated)
Conclusion: The study provides novel information on the phenotypic
characterization of the most widely used cells in prostate cancer
research with evidence for the effect of androgen ablation on TF
expression in androgen-dependent LNCaP and androgen-independent
DU-145 PCa cells. The information would be useful when using these
cells for xenograft studies.
Disclosure of Interest: None declared.
R
Sterniczuk B1, Toukh M1, Kaur H1, Barr S1, Siemens R2, Graham
C1 and Othman M1,3
1
Biomedical and Molecular Sciences; 2Urology, Queens
University; 3Health Science, St Lawrence College, Kingston,
Canada
HM07
Inferior vena cava filter usage, complications, and
retrieval rate in cancer patients
Abtahian F1, Weinberg I2, Hawkins BM3, Cefalo P4, Nasser NJ5,
MacKay C2 and Jaff MR2
1
Cardiology; 2The Institute for Heart, Vascular and Stroke Care,
Massachusetts General Hospital, Boston; 3Medicine, University
of Oklahoma College of Medicine, Oklahoma City; 4Department
of Medicine and Physical Medicine and Rehabilitation,
Massachusetts General Hospital, Boston, USA; 5Department of
Radiation Oncology, Princess Margaret Cancer Centre, Toronto,
Canada
Objectives: Venous thromboembolism contributes significantly to morbidity and mortality in cancer patients. The effectiveness and safety of
inferior vena cava filters (IVCFs) in these patients have not been established.
Methods: A retrospective review of IVCFs implanted at a tertiary
referral hospital between January 1, 2009, and December 31, 2011,
was conducted. Indications for IVCF placement, anticoagulation practices, complications, and patient outcomes were analyzed for patients
with and without active cancer and for cancer subtypes, including
localized and metastatic cases.
HM08
Time in therapeutic range in subjects with cancer associated thrombosis
Hull R1, Liang J1 and Jarner M2
University of Calgary, Calgary, Canada; 2LEO Pharma, Ballerup,
Denmark
1
Objectives: For vitamin K antagonist (VKA)-treated subjects, the
mean time spent within therapeutic range (TTR) reflects the intensity
of anticoagulation. The Main-LITE Cancer study compared long-term
treatment LMWH (tinzaparin) with usual care VKA therapy (warfarin) for 3 months in subjects with cancer associated thrombosis
(CAT). Our aim was to estimate the TTR for this study.
Methods: For the 100 warfarin-treated subjects, study drug commenced on day 1 at 5-10 mg then adjusted daily to maintain the International Normalized Ratio (INR) between 2.0 and 3.0, overlapping
with heparin to day 6 when heparin was discontinued if the INR was
therapeutic. Thereafter, INR monitoring was performed every 1–
2 weeks until end of therapy.
The TTR is calculated as described by Rosendaal et al. (Thromb Haemost; 69, 236–9, 1993) starting at day 7 until the day of the last dose of
warfarin = TTR = (the sum of number of days with an INR between
2.0 and 3.0 / the sum of number of days on treatment) *100%
Results: The TTR was calculated for 95 subjects from 1500 INR values
measured on/after day 7 and included in calculating the TTRs. The
number of INR values per subject ranged from 2–67, with 5% having
12/06/2014
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
ABSTRACTS
< 5 INR values, and 70% having > 10 INR values. The mean number
of INR values per subject per week was 1.8, ranging from 0.3 to 6.2
INR values/week.
with the use of newer Surgicel hemostat products compared to Surgicel
Original.
Disclosure of Interest: M. Corral is a is a shareholder of Johnson &
Johnson, Employee of: Ethicon, Inc., J. Riebman Is a shareholder of
johnson & johnson, Employee of: Ethicon, Inc., S. Lim Is a shareholder of Johnson & Johnson, Employee of: Ethicon, Inc., J. Godfrey
Scaife Is a consultant for Johnson & Johnson, D. Martyn Is a consultant for Johnson & Johnson, Y. Soneji Is a consultant for Johnson &
Johnson, G. Miyasato: None declared, Y. Rao: None declared, K. Prifti: None declared, R. Kocharian Is a shareholder of Johnson & Johnson, Employee of: Ethicon, Inc.
Conclusion: VKA treatment of subjects with CAT is a challenge: VKA
interactions are likely, and comorbidities may interfere with maintaining the INR within therapeutic range. A TTR of 44.8% in this study
of CAT corresponds well with the TTR calculated in studies in similar
patient populations: CLOT 46% and CANTHANOX 41%. VKA
therapy has been used in a series of recent studies in non-cancer
patients: In four different studies in subjects with atrial fibrillation,
mean TTR was 62.4% (55.0–68.4%), and in five studies in venous
thromboembolism, mean TTR was 60.9% (57.7–63.5%). As expected,
the TTR is lower in subjects with cancer than in patients without
malignancies.
Disclosure of Interest: R. Hull has grant/research support from: LEO
Pharma, Sanofi, Is a consultant for Bayer, Janssen, Portola, J. Liang:
none declared, M. Jarner is an employee of: LEO Pharma.
Kreuziger LB1,2, Richter E3, Gomez-Timana L4, Giever T2 and
Atallah E2
1
BloodCenter of Wisconsin; 2Medicine, Hematology and
Oncology, Medical College of Wisconsin; 3Froedtert Hospital;
4
Medical College of Wisconsin, Milwaukee, USA
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Objectives: The optimal duration of anticoagulation (AC) for catheterrelated thrombosis (CRT) has not been studied. Consensus guidelines
recommend 3 months of AC, but treatment in patients with hematologic malignancies (HM) is often abbreviated to balance thrombotic
and bleeding risks. HM patients with CRT were reviewed to evaluate
CRT treatment and incidence of recurrent thrombosis and hemorrhage.
Methods: Retrospective cohort study of hospitalized patients with HM
and CRT between 2009 and 7/2013. Recurrent thrombosis was defined
as documented new deep vein thrombosis (DVT), pulmonary embolism (PE) or CRT thrombus extension.
Results: There were 141 upper extremity CRT events in 132 patients.
Mean age was 54 years (range 19–82 years) with median follow-up of
432 days. HM diagnosis included acute leukemia (39%), plasma cell
dyscrasias (27%), and non-Hodgkin lymphoma (21%). CRT occurred
primarily due to PICC lines (128/141, 91%). Table 1 denotes CRT
characteristics based on treatment category.
Table 1
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Corral M1, Riebman J2, Lim S1, Scaife JG3, Martyn D3, Soneji Y3,
Miyasato G3, Rao Y4, Prifti K4 and Kocharian R4
1
Global Health Economics and Market Access, Ethicon, Inc.;
2
Medical Affairs, Ethicon, Somerville, MA; 3Consultant; 4Trinity
Partners, Boston, MA, USA
HM10
Duration of anticoagulation treatment for catheterrelated thrombosis in hematologic malignancy
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HM09
Hospital resource use and cost reduction with the use
of advanced topical absorbable hemostats in surgical
procedures
53
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Objectives: The use of hemostatic agents has increased over time for
all surgical procedures. Previous studies have analyzed the use of hemostatic agents and transfusion rates. The objective of this study was
to compare product utilization, healthcare resource use (HCRU),
costs, and outcomes in patients 18 years of age or older associated
with the use of the newer Surgicel topical absorbable hemostat products (Surgicel Fibrillar and Surgicel SNoW) compared to Surgicel Original for specific surgical procedures.
Methods: A retrospective database analysis was conducted utilizing
Premier Perspectives database during the time periods Q1 2011-Q4
2012. Patients who underwent Brain/Cerebral (BC), Cardiovascular
(CV) and Carotid Endarterectomy (CEA) procedures were analyzed
using Propensity Score Matching to compare the use of Surgicel Original vs. Surgicel Fibrillar/SNoW across a range of metrics.
Results: Matched cohorts (N = BC: 758; CV: 3388, CEA: 2,041); Surgicel Fibrillar/SNoW cohort had statistically significant lower all-cause
costs per discharge (*P-values based on a non-parametric Wilcoxon
signed rank sum test; **P-values based on a non-parametric Wilcoxon-Mann-Whitney test. Both compare the distribution of values,
rather than the medians: *P < 0.05, **P < 0.0001) (BC: 18%**, CV:
17%**, CEA: 12%**), units per discharge (BC: 2%, CV: 25%**,
CEA: 8%**), and mean LOS (BC: 14%*, CV: 15%**, CEA: 14%**).
ICU costs were also lower (BC: 20%*, CV: 19%**, CEA: 8%**), and
mean ICU stay was also lower in BC (19%*) and CV (11%). Units of
blood transfused were reduced (BC: 61%*, CV: 23%, CEA: 20%)
compared to the Surgicel Original cohort.
Conclusion: By controlling for patient characteristics and hospital factors, we observed a reduction of HCRU and hospital costs associated
CRT, n (%)
Central vein CRT,
n (%)
Median platelet count
(9103 ll–1) at CRT
Duration AC, median
(range) days (n = 91)
Recurrent thrombosis,
n (%)
AC
AC + catheter
removal
Catheter
removal
No
treatment
13 (9%)
10 (12%)
91 (65%)
59 (68%)
32 (23%)
16 (18%)
5 (3%)
2 (2%)
73
74
48
60
57 (3–539)
42 (2–537)
3 (23%)
24 (26%)
6 (19%)
1 (20%)
Catheters were removed due to thrombosis (76%) and infection
(14%). Also, 34 recurrent thrombotic events occurred (13 CRT, 7
DVT, 9 PE, 2 clot progression), 3 while on therapeutic AC for CRT.
Duration of anticoagulation was not significantly different between
patients with and without recurrence (median 56 vs. 41 days, Wilcoxon P = 0.3). Age predicted recurrent thrombosis but use of AC therapy and CRT location were not associated with recurrence. Bleeding
events occurred in 18 (18%) of CRT treated with anticoagulation with
four major hemorrhages.
Conclusion: Duration of AC therapy was not associated with risk of
recurrent thrombosis. Shorter course AC therapy appears safe for
CRT and should be studied prospectively.
Disclosure of Interest: None declared.
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
12/06/2014
54
ABSTRACTS
Lupus and Anticoagulant/
Phospholipid
were retested after 12 or more weeks. Patients under anticoagulation
were excluded. Screen and confirm steps of DRVVT (VRR-Diagnostica Stago) were carried out. Following SSC-ISTH recommendations,
we established that R³ = 1.12, which did not correct with normal
plasma (1:1) and decreased by ³10% when using excess of phospholipids was diagnostic of LA. Patients were divided, according to first
DRVVT Rs value, in A: 1.12–1.19 (median 1.18; n = 32), B: 1.20–1.39
(median 1.29; n = 69) and C: > 1.40 (median 1.44; n = 25). Chi square
test was applied to detect differences in the proportion of patients in
which LA was confirmed in the second study.
Results: The proportion of positive DRVVT in the 2nd study was A:
28/32 (87.5%) (29/32 were positive considering also APTT results), B:
66/69 (95.6%), and C: 23/25 (92.0%) patients. No significant difference (P = 0.329) was found between the groups; in addition, non-significant differences were observed between either A and B (P = 0.408)
or A and C (P = 0.762). Moreover, 2/9 patients negative in the second
study become positive in a third study (A:1/4; B:1/3).
Conclusion: The number of patients in whom LA was confirmed in the
second study was independent of the first DRVVT R value; low
DRVVT R (< 1.20) showed similar chances than those with more prolonged time. These data suggest that ‘weak’ effects have to be considered as well as those with stronger effect on prolongation of DRVVT,
in order to analyze clinical behavior.
Disclosure of Interest: None declared.
LA01
Solid phase assays in antiphospholipid syndrome
(APS): variation between results amongst UK Neqas
(blood coagulation) participants
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‘Weak’ effects showed similar chances of being confirmed as positive lupus anticoagulant (LA) as those
with strong effects on DRVVT
Grosso S, Ingratti M, Remotti L, Vera Morandini MP, Luceros AS,
Meschengieser S, Lazzari MA and Blanco A
de Hemostasia y Trombosis, IIHEMA, Academia Nacional de
Medicina, Buenos Aires, Argentina
Objectives: Diluted Russell viper venom time (DRVVT) is applied
for diagnosis of LA. In order to investigate if patients with low
(‘weak’ effect) patient/normal DRVVT ratio (R) have a low chance
of having positive results in a second study, we retrospectively analyzed a cohort of 125 patients who ere DRVVT positive in a first
study.
Methods: Over a 4-year period, we analyzed 93 females and 33 males
(mean = 43 years old; 7–81) with clinical criteria for antiphospholipid
syndrome (APS) and LA (positive DRVVT) in the 1st study, which
CD
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LA03
Activated protein C resistance detected by the calibrated automated thrombogram as the most prevalent
prothrombotic factor in patients with early manifestations of venous thromboembolism
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Objectives: National and international guidelines recommend use of
solid-phase assays as part of screening for APS; studies have shown
that triple positivity [lupus anticoagulant (LA)+, anticardiolipin antibody (ACLA)+, b-2 glycoprotein 1 antibody (b2GP1)+] is associated
with a higher risk of venous thromboembolism than single or double
positivity. Accurate assays are therefore important for diagnosis of
APS.
Methods: We report here two UK NEQAS (BC) exercises in which
participants were asked to perform solid-phase assays for ACLA and
b2GP1 on four samples along with LA screening, to evaluate betweencenter agreement for these assays.
Results: There were 310 centers that returned LA results, with 100
returning solid-phase assay results. For sample LA12:01, 95.3% of
centers reported LA negative. Solid-phase assay interpretations
were ACLA (IgG) 92/92 normal, (IgM) 71/73 normal; b2GP1
(IgG) 50/50 normal, (IgM) 32/33 normal. For sample LA12:02,
97% of centers reported LA positive. Solid-phase assay results
were: ACLA (IgG) 89/92 abnormal, (IgM) 4/74 abnormal; b2GP1
(IgG) 45/51 abnormal, (IgM) 0/33 abnormal. Samples LA13:01 and
LA13:02 were identical samples with weak lupus anticoagulant positivity. 88.9% and 90.7% of centers reported a positive LA screen
respectively. For solid-phase assays, 81% reported abnormal ACLA
(IgG) results, 6% reported abnormal ACLA (IgM) results, 73%
reported abnormal b2GP1 (IgG) results and 5% reported abnormal
b2GP1 (IgM) results. Ranges of reported results for solid-phase
assays were large, with a 10-fold difference in results between methods (CVs >50%).
Conclusion: Evaluation of thrombotic risk may depend on accuracy of
results with solid-phase assays. This exercise revealed marked variation in results reported both between- and within-method groups for
each assay, and a lack of concordance in interpretation of solid-phase
assay results. Further study is required to evaluate the accuracy and
precision of these methods, and to determine the degree of further
standardization required.
Disclosure of Interest: None declared.
R
Jennings I, Kitchen S, Kitchen DP, Woods TAL and Walker ID
UK Neqas (Blood Coagulation), Sheffield, UK
Shmeleva V1, Polyakova A2, Matvienko O1, Kobilyanskaya V1,
Morozova T1, Namestnikov Y1, Golovina O1, Kapustin S2 and
Papayan L1
1
Laboratory of Blood Coagulation; 2Biochemistry Laboratory,
Russian Research Institute of Haematology and Transfusion,
St-Petersburg, Russian Federation
Objectives: Activated protein C resistance (APCR) is associated with
high risk of venous thromboembolism (VTE). After the factor V Leiden (FVL) mutation was found as the main cause of APCR, little
attention is paid to the role of acquired APCR to VTE. The aim of our
study was to determine prevalence of APCR and its reasons in patients
with VTE manifestation under the age of 45 years.
Methods: Calibrated automated thrombography (CAT) was done in
platelet-poor plasma (PPP) with PPP plasma TM reagent according
to Hemker et al. The study involved 30 patients (M/F 13/17, mean age
35.0 8.0 years) and 30 controls. Patients 8, 4, and 1 had heterozygous factor V Leiden mutation (FVL), lupus anticoagulant (LAC),
and prothrombin G20210A mutation, respectively. STATISTICA 6.1
was used.
Results: Endogenous thrombin potential (ETP) and peak thrombin
(PT) were markedly reduced in the presence of TM. Normal ranges of
reduce defined as 5–95th percentile of ↓ETP and ↓PT calculated in
controls were 21–62% and 14–51%, respectively. Values below 21%
for ↓ETP and/or 14% for ↓PT (i.e., APRC) were found in 17 (56%) of
samples. All patients with FVL and LAC demonstrated APCR.
Importantly abnormal ↓PT was found in all these patients and ↓ETP
only in 50% cases with FVL and 75% with LAC. From 6 patients with
increased FVIII activity APCR was found in 3 cases (50%). So, APCR
was the most prevalent (56%) prothrombotic factor in our patients
with early manifestations of VTE. In 26% of cases APCR was due to
FVL mutation; LAC and increased FVIII could explain 23%, and the
reasons for APCR in the remaining 7% of cases need further elucidation.
Conclusion: The results of our study make us conclude that CAT is
useful tool to evaluate haemostatic abnormalities in thrombophilia
12/06/2014
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
ABSTRACTS
patients and in cost-effectiveness aspect could be preferable to some
genetic and functional tests. From parameters derived from the thrombin generation curves measured both with and without TM, abnormal
↓PT seems the most sensitive to detect APCR in patients with FVL
and LAC.
Disclosure of Interest: None declared.
55
Pediatric/Neonatal Hemostasis and
Thrombosis
PHT01
Venography (VE) in children with clinical assessment
of post-thrombotic syndrome (PTS) following upper
venous system thrombotic event (UVSTE)
LA04
Abstract withdrawn.
Sciuccati G1, Cervio C1, Sierre S2, Hepner M1, Montanari A1,
Frontroth JP1, Pieroni G1, Annetta SE1, Torres AF1 and Bonduel M1
1
Hematologia / Oncologia; 2Radiologıa Intervencionista,
Hospital de Pediatria Dr Juan P Garrahan, Buenos Aires,
Argentina
LA05
Abstract withdrawn.
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Objectives: Assays detecting anticardiolipin (aCL) and anti-b2glycoprotein I antibodies (ab2GPI) classifying patients with antiphospholipid syndrome (APS) show a high interassay variability. Coatings of
b2GPI on the solid phase differ in exposure of the domain I G40-R43
epitope and may therefore influence the performance of the assays.
Methods: Two human-derived monoclonal antibodies were used to test
the specificity and sensitivity of aCL and ab2GPI IgG assays. The antibody P2-6 recognizes b2GPI independently of its conformation, while
P1-117 only recognizes b2GP in its open conformation binding to the
G40-R43 epitope. Serial dilutions of antibodies (0-10 lg mL–1) were
tested in duplicate in two different automated systems that use magnetic beads as the solid phase. HemosILâ AcuStar (Instrumentation
Laboratories) applies chemiluminescent technology and Bio-Rad BioPlexâ 2200 System (Bio-Rad Laboratories) utilizes multiplex flow
immunoassay technology. The 99th percentile cut-off values of 20 U
mL–1 were applied for all assays.
Results: For both aCL and ab2GPI, BioPlex showed a comparable
reactivity to both antibodies while AcuStar showed an increased affinity to P2-6 compared to P1-117. The AcuStar and BioPlex ab2GPI
assays were positive at 0.16 and 0.31 lg mL–1 P2-6, respectively.
Despite the reduced exposure of G40-R43 in the ab2GPI AcuStar
assay the threshold for positivity of P1-117 was identical for both systems (0.31 lg mL–1). Both aCL assays showed a comparable threshold
for positivity of P2-6 (0.63 lg mL–1). AcuStar and BioPlex showed
positivity for P1-117 at 5.00 and 0.32 lg mL–1, respectively.
Conclusion: Both aCL and ab2GPI IgG BioPlex assays demonstrated
a comparable affinity for both antibodies, while both AcuStar assays
had higher affinity for P2-6. The threshold for positivity of P1-117
was equal in both ab2GPI systems but differed in the aCL assays.
Besides the exposure of the epitope, the threshold for positivity of
the antibody concentration may contribute to the sensitivity of the
assay.
Disclosure of Interest: None declared.
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Devreese K1, Kelchtermans H2 and de Laat B2
1
Coagulation Laboratory, Department of Clinical Chemistry,
Microbiology and Immunology, Ghent University Hospital, Gent,
Belgium; 2Department of Biochemistry, Synapse BV, Maastricht
University, Maastricht, The Netherlands
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LA06
Differences in sensitivity of two automated panels for
anticardiolipin and anti-beta2glycoprotein I antibodies
in the laboratory diagnosis of antiphospholipid syndrome due to the exposure of the domain I epitope of
beta2glycoprotein I on the solid phase
Objectives: PTS following UVSTE is a chronic, potentially disabling
complication. Recent recommendations of the Perinatal and Paediatric
SSC of the ISTH concerning the standardization of clinical assessment
of this syndrome in pediatric practice were published, but few data
about radiographic evaluation have been reported. To evaluate the
radiographic findings in those infants and children with UVSTE who
underwent both clinical assessment and VE measurement of PTS.
Methods: From January 2011 to December 2012, 59 consecutive neonates and children < 18 years old with objectively confirmed UVSTE
were diagnosed in our center. Eight patients died of their underlying
diseases. The remaining 51 patients were clinical assessed using
Manco-Johnson instrument (MJI) and modified Villalta scale (MVS);
in 15 of them VE was also performed at least 3 months after the UVSTE. A retrospective evaluation of the radiographic findings was done.
At the time of the VE, no patient had central venous catheter (CVC)
in place.
Results: In 15 children [9 (60%) females, median age at thrombosis
0.5 year (14 days–14.8 years)], median time from UVSTE to PTS
assessment was 12 months (3.2–17.1). All but one patient had CVCrelated thrombosis. Clinical assessment according MJI showed PTS
was presented in 12 patients, 2 of them had PTS that was clinically
important. MVS disclosed moderate PTS, 1 patient; mild PTS, 6
patients and absent, 8 patients. VE showed involvement of the subclavian veins in 14 patients (right vein, 6 patients; left vein,4 patients and
bilateral, 4 patients), > 1 veins involved, 4 patients, complete occlusion, 10 patients; subocclusion, 2 patients; stenosis, 2 patients and collateral circulation = moderate, 7 patients and severe, 5 patients.
Conclusion: We conclude that clinical assessment using MJI and MVS
detected disabling presentation in the minority of the patients evaluated during this short-term follow-up. However, extensive vein
involvement by VE was found in most of them. Imaging studies and
the timing of its assessment may be also taken into account for the
complete standardization of UVSTE PTS in children.
Disclosure of Interest: None declared.
PHT02
Development of a new risk model for hospital-associated venous thromboembolism (HA-VTE) in the pediatric intensive care unit (PICU): findings from a large
single-institutional case-control study
Atchison 1, Arlikar S2, Amankwah E2, Ayala I3, Barrett L2,3,
Branchford BR4, Streiff M5, Takemoto C3,6, Smith M7 and
Goldenberg NA2,3,5,6
1
Undergraduate Medical Education, University of South Florida
Morsani College of Medicine, Tampa; 2Clinical and Translational
Research Organization, All Children’s Research Institute, St
Petersburg; 3Johns Hopkins Medicine Pediatric Thrombosis
Program, All Children’s Hospital and Johns Hopkins Children’s
Center, St Petersburg and Baltimore; 4Section of Hem/Onc/BMT,
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
12/06/2014
56
ABSTRACTS
Dept. of Pediatrics, University of Colorado School of Medicine
Anshutz Medical Campus and Children’s Hospital Colorado,
Aurora; 5Division of Hematology, Department of Medicine;
6
Division of Hematology, Department of Pediatrics, Johns
Hopkins University School of Medicine, Baltimore; 7Division of
Critical Care, All Children’s Hospital and Johns Hopkins
Children’s Center, St Petersburg and Baltimore, USA
Tuttle A1, Grabell J1, Clark DS2, Moorehead P3, Blanchette VS2,
Wu J4, Steele M5, Klaassen R6, Silva M1, Rand ML2 and James
PD1
1
Queen’s University, Kingston; 2The Hospital for Sick Children,
Toronto; 3Janeway Child Health and Rehabilitation Centre, St.
John‘s; 4The Child and Family Research Institute, Vancouver;
5
Alberta Children’s Hospital, Calgary; 6Children’s Hospital of
Eastern Ontario, Ottawa, Canada
Objectives: Expert-administered bleeding assessment tools (BATs)
have been developed and prospectively validated for the diagnosis of
VWD. Our overall objective is to generate, pre-test, optimize, and validate a self-/parent-administered PBQ.
Methods: Following an initial phase of pre-testing and optimization,
the Self-PBQ was administered in five bleeding disorders clinics (Vancouver, Calgary, Toronto, Kingston, Ottawa) to two populations: (i)
healthy children identified as having no significant past bleeding history and taking no medications; and (ii) children previously diagnosed
Control
(n = 21)
P-value
10 (3–16)
19 (54)
5 (0–13)
5 (0.5–15)
11 (52)
0 (0–2)
0.005
1.000
<0.001
5 (0–13)
0 (0–3)
<0.001
17/24 (71)
0.36 (0.09–0.64)
2/8 (25)
–
0.017
–
0.29 (0.08–0.48)
–
–
0.59 (0.10–1.15)
–
–
Conclusion: Based on our results, no items were removed. We can now
move to prospective validation of the optimized questionnaire as a
screening tool for children referred to Hematology for investigation of
a bleeding disorder.
Disclosure of Interest: None declared.
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PHT03
Item reduction analysis of the self-PBQ (self-administered pediatric bleeding questionnaire): healthy children and children previously diagnosed with type 1
von Willebrand disease (VWD)
Affected
(n = 35)
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Age, median (range)
Females (%)
ISTH-BAT, median
(0 to 4 scoring)
PBQ, median
(1 to 4 scoring)
Blood group O (%)
VWF:Ag (IU mL–1),
mean (range)
VWF:RCo (IU mL–1),
mean (range)
FVIII:C (IU mL–1),
mean (range)
iza
Objectives: Hospital-associated venous thromboembolism (HA-VTE)
risk is increased when comparing critically ill to non–critically ill pediatric subpopulations. We sought to develop a novel and distinct HAVTE risk model for children admitted to the Pediatric Intensive Care
Unit (PICU).
Methods: In a retrospective analysis from April 2013–January 2006,
HA-VTE cases were identified via the electronic health record at All
Children’s Hospital Johns Hopkins Medicine using ICD-9 discharge
diagnosis codes, with subsequent validation in radiologic records.
Cases were restricted to PICU admissions. For each case, three nonHA-VTE PICU controls were randomly selected. Data were
abstracted on putative risk factors, and associations between risk factors and HA-VTE were estimated using odds ratios (ORs) and Wald
95% confidence intervals (95%CIs). A P-value threshold ≤ 0.2 was
used for inclusion into a multivariate (adjusted) model.
Results: There were 55 HA-VTE cases and 165 controls. Incidence was
3 per 1000 PICU admissions. Statistically significant risk factors in
univariate analyzes included mechanical ventilation (OR: 10.13, 95%
CI 5.04–20.36), the presence of a short-term or long-term central
venous catheter (CVC) (OR: 46.22, 95% CI 18.86–113.28 and OR:
3.19, 95% CI 1.32–7.72), infection (OR: 12.94, 95% CI 6.04–27.75),
malignancy (OR: 6.99, 95% CI 2.62–18.61), length of stay (LOS)
≥ 7 days (OR: 82.81, 95% CI 19.31–355.17), and cardiac catheterization (OR:4.48, 95% CI 1.48–13.57). After adjustment, only short-term
CVC (OR: 130.33, 95% CI: 20.02–848.55) and LOS ≥ 7 days (OR:
68.13, 95% CI: 8.66–536) remained independently significant. Risk of
VTE was > 2% (threshold for anticoagulant thromboprophylaxis [TP]
in hospitalized adults) with short-term CVC, and achieved a risk of
> 1% but < 2% (risk zone for mechanical TP in hospitalized adults)
when LOS was ≥ 7 days.
Conclusion: If prospectively validated, these findings form the basis for
a novel VTE risk model and help guide TP in the PICU.
Disclosure of Interest: None declared.
with Type 1 VWD. Two bleeding scores were generated for each child
using (i) the PBQ Scoring Key and (ii) the ISTH–BAT Scoring Key.
Category-total, category-category, and discrimination analyses were
performed with the intent of item reduction by Spearman, Pearson
and chi-square correlations, respectively.
Results: For this portion of the study, a total of 56 subjects were
recruited. Bleeding scores and laboratory results are shown in Table 1
The category-total analysis showed no reported symptom had a strong
correlation (> 0.90) to the cumulative bleeding score. No significant
correlations (> 0.90) were found in the category-category analysis to
indicate category redundancy. No items were identified to be discriminatory (P < 0.05) in the identification of affected and unaffected children.
PHT04
Idiopathic thrombosis in teenagers: the role of thrombophilia
Ryan A1,2, Monagle P3 and Newall F4
1
Haematology Research, Royal Children’s Hospital; 2Melbourne
Medical School, University of Melbourne; 3Haematology;
4
Clinical Haematology, Royal Children’s Hospital, Melbourne,
Australia
Objectives: This study investigated the aetiology of spontaneous
venous thrombosis and or pulmonary embolism in teenagers who presented to the Royal Children’s Hospital (RCH), Melbourne, over the
period of 2003–2014.
Methods: This single centre retrospective cohort design study aimed to
identify which markers of thrombophilia were identified in the specified population. The project determined which thrombophilia testing
was performed on the subjects and the results of these tests. Patient
information was taken from the warfarin database at the RCH. This
information was cross-referenced with patient medical records to
ensure that the patients met the study eligibility criteria. The RCH laboratory results database was used to collect data relevant to the study.
The data were analyzed as follows: for continuous data, the appropriate estimate of central tendency was employed, while proportions were
used for non-continuous variables. Rates of thrombophilia testing
were determined and described descriptively, as outlined previously.
Other factors contributing to thrombosis risk in the subjects were also
considered in data analysis. These included vascular anomalies, dehydration, significant exercise, and use of the oral contraceptive pill.
Results: Fifty-two patients (36 with deep vein thrombosis (DVT), 7
with pulmonary embolism (PE), 8 with DVT/PE, and 1 with portal
vein thrombosis) were identified from the warfarin database as fulfill-
12/06/2014
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
ABSTRACTS
PHT07
Acquired FVII deficiency and glomerulopathy in Egyptian children: a report of five cases: an association
and/or an immune triggered phenomenon
ing study eligibility requirements. The additional data previously outlined will be obtained from these participants.
Conclusion: This ongoing study will provide further evidence as to the
contribution of thrombophilia vs. other acquired risk factors for spontaneous DVT/PE in a population of young adults. This will help determine the appropriate investigation strategy for such patients.
Disclosure of Interest: None declared.
Abdelwahab M1 and Bazaraa H2
1
Pediatric Hematology; 2Pediatric Nephrology, Cairo University
Pediatric Hospital, Cairo, Egypt
PHT05
Abstract withdrawn.
PHT06
FV deficiency patients: no correlation of phenotype
with factor activity and report of three new mutations
in Egyptian children
Objectives: To report five Egyptian children with acquired factor VII
(FVII) deficiency and glomerulopathy.
Methods: Five children (P1–P5) who presented over a 3-year period
with a severe bleeding tendency had their bleeding and coagulation
profile done. Then they underwent renal biopsy for their persistent
hematuria and were managed accordingly.
Results: Four males and 1 female with age ranging from 1.5 to 7 years
initially presented with a generalized mucous membrane bleeding triggered by a stressful event. All bleeding symptoms were controlled on
fresh frozen plasma/8 h except massive hematuria with subsequent
development of proteinuria, increased albumin creatinine ratio, and
hypertension. Coagulation profile showed prolonged PT, normal INR,
and decreased plasma FVII activity (30–50%, N = 70–120%) with
negative family history of bleeding and screening of family members
.Renal biopsy showed Alport syndrome (AS) in four children and mesangioproliferative glomerulonephritis in one case. Four children were
positive for anti-neutrophil cytoplasmic antibody (ANCA). Hematuria
subsided temporarily on activated FVII (FVIIa) and then recurred
with complete resolution as well as remission of the renal manifestations on immunosuppressive therapy. P4 has associated epilepsy,
asymmetrical muscle hypertrophy, arthritis responding to colchicines,
and history of very late onset bleeding tendency and epilepsy in the
grandfather. P5 has undescended testis and the father has renal problems.
Conclusion: Normal family laboratory testing and control of bleeding
on FVIIa is with the diagnosis of acquired FVII deficiency. Massive
gross hematuria is atypical of AS, implying that the bleeding tendency
augmented the problem and resolution of bleeding tendency and
remission of proteinuria and hypertension on immunosuppressive
therapy, all atypical of AS further confirms the underlying immune
nature of the whole process.This is the first report of this association
and immune triggered and mediated FVII deficiency.
Disclosure of Interest: None declared.
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Abdelwahab M1, Stefano D2, Asselta R3, Paraboschi EM3 and
Peyvandi F4
1
Pediatric Hematology, Cairo University Pediatric Hospital,
Cairo, Egypt; 2Medical Biotechnology and Translational
Medicine, University of Milan, Milan; 3Medical Biotechnology
and Translational Medicine; 4Department of Medicine and
Medical Specialities, IRCCS Maggiore Hospital, Mangiagalli and
Regina Elena Foundation, University of Milan, and Luigi Villa
Foundation, University of Milan, Milan, Italy
Objectives: To correlate factor V (FV) activity with the severity of
bleeding spectrum in Egyptian FV deficiency patients and study the
underlying mutation of FV gene in some.
Methods: All patients enrolled had their FV activity level measured in
plasma and were classified as severely affected, if FV level was < 10–
15%, or mildly to moderately affected if FV level was 20–30%. Four
children were screened for mutations in the F5 gene by DNA sequencing.
Results: The study included 16 patients; 12 males and 4 females; 9 children (mean age: 8.5 years) and 7 adults (mean age: 31.6 years). Fourteen patients were severe and 2 had mild deficiency. Oral bleeding and
ecchymoses were reported in 62.5% of cases, nosebleeds in 43.8%, post
circumcision bleeding in 25%, joint bleeds in 18.8%, intracranial hemorrhage (ICH) and bleeding per rectum each in 12.5%, hematuria and
muscle bleeds each in 6.3%, menorrhagia in the 2 women during the
childbearing period and other bleeds in 37.5%. All patients presented
with symptoms in the first 2 years of life, except three adults. Patients
with FV 8–15% and the patient with FV 30% bled more frequently
than those with lower levels; 25% of our patients had congenital
anomalies and four had delayed wound healing. The four genetically
tested children (P1–P4) showed three new mutations. Two unrelated
patients P1-P2 were homozygous for the Gly628Glu mutation
(c.1972G>A, Gly600Glu without the signal peptide) and both presenting in the first year of life with recurrent ICH. P3 was homozygous for
the Glu2217Lys mutation (c.6739G>A, Glu2189Lys) and presented
with post-injury bleeding in infancy. P4, with epistaxis as main bleeding symptom, was found to be homozygous for the Trp947ter nonsense
mutation (c.2931G>A, Trp919ter). P1–P3 had a very mild bleeding
spectrum after their initial episodes though not on prophylaxis.
Conclusion: There is no correlation between the FV activity and the
degree and severity of bleedings in our Egyptian cohort. This is the
first report of three new mutations for FV deficiency in Africa.
Disclosure of Interest: None declared.
57
PHT08
Effect of 22Q11.2 deletion on bleeding risk following
cardiac surgery in children with congenital heart disease
Brenner MK1, Clarke S2, Bercovitz RS1, Tomita-Mitchell A3,
Mitchell M4 and Newman DK1
1
Blood Research Institute, Blood Center of Wisconsin; 2Critical
Care and Cardiology, Children’s Hospital of Wisconsin;
3
Cardiothoracic Surgery, Medical College of Wisconsin;
4
Cardiothoracic Surgery, Children’s Hospital of Wisconsin,
Milwaukee, USA
Objectives: Approximately 80% of patients with 22q11.2 deletion syndrome (DS) have congenital heart defects (CHDs) that require surgical
intervention. The deleted segment of chromosome 22q11.2 encompasses the gene encoding glycoprotein (GP) Ibb. GPIbb is required for
expression of the GPIb-V-IX complex on the platelet surface, where it
functions as the receptor for von Willebrand factor (VWF). Binding of
GPIb-V-IX to VWF is important for platelets to adhere to damaged
blood vessels, initiate hemostasis, and stimulate vessel repair. It is not
known whether hemizygosity for GPIbb increases the risk for bleeding
of patients with 22q11.2 DS in the setting of cardiac surgery.
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
12/06/2014
58
ABSTRACTS
Methods: We performed a retrospective chart review of 136 pediatric
patients who underwent cardiac surgery for repair of CHD from 2004
to 2011 at Children’s Hospital of Wisconsin. Patients with 22q11.2 DS
and controls were matched for demographic features including age
and type of cardiac abnormality and compared for mean platelet volume (MPV) and platelet count. Primary outcomes included surgical
bleeding and red blood cell (RBC) transfusions.
Results: Median MPV was significantly higher and median platelet
count was significantly lower in patients with 22q11.2 DS relative to
controls (P < 0.0001). Patients with 22q11.2 DS received a larger volume of RBCs peri- and post-operatively relative to controls
(P < 0.05). A significantly higher incidence of unidentified hematologic defects and surgical complications that led to bleeding was observed
in patients with 22q11.2 DS relative to controls (P < 0.005).
Conclusion: These data suggest that pediatric patients with 22q11.2 DS
exhibit mild macrothrombocytopenia and experience more bleeding
than do patients with other causes of CHD when undergoing cardiac
surgery. Future studies will characterize platelet function in patients
with 22q11.2 DS and determine whether prophylactic platelet transfusion corrects excessive bleeding in this patient population.
Disclosure of Interest: None declared.
Disclosure of Interest: S. O’Brien is a consultant for a member, Pediatric Apixaban Studies Committee (Bristol Myers Squibb), R. Kulkarni:
none declared, A. Wallace: none declared, F. Hamblin = none
declared, S. Burr: none declared, N. Goldenberg had grant/research
support from: This project was funded by Eisai via a IIS grant mechanism. Dr. Goldenberg receives research support from CPC Clinical
Research through his trial oversight roles in pharmaceutical industry–
sponsored research.
PHT10
Height and age have more impact on anti–vitamin K
antagonist dose requirement than VKORC1 genotype
in children/pediatric patients
Vera Morandini MP1, Remotti L1, Chuit R2, Grosso S1, Ingratti
M1, ALberto F1, Bermejo E1, Meschengiesser S1, Luceros AS1,
Lazzari M1 and Blanco A1
1
Instituto de Investigaciones Hematologicas Mariano R Castex Academia Nacional de Medicina; 2Instituto de Investigaciones
Epidemiologicas - Academia Nacional de Medicina, CABA,
Argentina
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O’Brien SH1, Kulkarni R2, Wallace A3, Hamblin F4, Burr S3 and
Goldenberg NA4
1
The Research Institute at Nationwide Children’s Hospital,
Columbus; 2Michigan State University, East Lansing; 3Children’s
Hospital of Colorado, Aurora; 4All Children’s Hospital, St
Petersburg, USA
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PHT09
Dalteparin treatment for acute pediatric venous thromboembolism: a multicenter dose-finding, safety, and
efficacy study
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Objectives: Low molecular weight heparins (LMWH) are the mainstay
of anticoagulant therapy for pediatric venous thromboembolism
(VTE). The safety and effectiveness of dalteparin, an LMWH, in children has not been established, and pediatric data on dalteparin for
VTE therapy are limited to one single-center experience. Our objective
was to establish dose-finding (primary end point) and efficacy/safety
outcomes (secondary end points) in children treated with dalteparin in
a substudy of the Kids-DOTT trial.
Methods: A prospective multicenter trial using dalteparin subcutaneously twice-daily for acute VTE treatment in children ≤ 18 years old
was conducted under an investigator-held Investigational New Drug
application registered with the U.S. Food and Drug Administration.
Initial weight-based dosing per protocol was: infants (< 12 months):
150 IU kg–1; children (1–12 years): 125 IU kg–1; teens (13–18 years):
100 IU kg–1. Starting doses were adjusted by 10–20%, if needed, to
achieve goal anti-Xa activity 0.5–1.0 U mL–1. Bleeding events were
categorized using International Society on Thrombosis and Haemostasis criteria. Descriptive non-parametric statistics were employed for
all analyzes.
Results: Eighteen patients (67% males) were enrolled from January
2010 to October 2013 across four U.S. centers. VTE sites included
extremities (10), central sinus vein (4), jugular (3), and hepatic (1). No
supratherapeutic levels were observed. Median (range) therapeutic
doses by age group were: infants (n = 3): 180 IU kg–1 (146–181 IU kg–
1
); children (n = 7): 125 IU kg–1 (101–175 IU kg–1); teens (n = 8):
100 IU kg–1 (91–163 IU kg–1). Median duration of dalteparin use was
48 days (range: 2–169 days), and median follow-up was 10.5 months
(range: 2–35 months). There were no related serious adverse events,
no clinically relevant bleeding events, and no symptomatic recurrent
VTEs.
Conclusion: Dalteparin successfully achieved targeted anti-Xa levels in
18 children with acute VTE using a standardized age-based dosing regimen, with a favorable safety and efficacy profile.
Objectives: Polymorphisms (SNPs) in vitamin K epoxide reductase
gene (VKORC-1), 1173C>T and –1639G>A, are associated with different sensitivity to oral anticoagulants (OA). The aim of the study
was to test the impact of the genotype and other characteristics as age
and height on mean OA dose requirements to achieve appropriate
INRs (2–3) in a cohort of pediatric patients.
Methods: 30 patients on OA, 18 males and 12 females, aged ≤ 18 years
old were selected. The mean age was 11 years old (2–18) and the height
was 1.37 m (0.90–1.70). Genotyping was performed using RFLPs:
PCR, followed by MspI (-1639G>A) or StyI (1173C>T) digestion.
Influence of age, height, and SNPs in the mean OA dose was determined by linear regression model.
Results: SNPs frequencies were 33.3% CC, 60% CT, and 6.7% TT for
1173C>T and 36.7% GG, 50% GA, and 13.3% AA for –1639G>A.
The mean dose (mg/week) was higher in carriers of 1173CC (12.95)
compared to that required in CT (11.03) or TT (9.75); however, the
differences were not significant (P > 0.05). In the case of –1639G>A,
the dose was higher in GG (13.14) compared to CT (11.30) or CC
(8.38), but the differences were not significant (P > 0.05). In this
cohort, age, height, and genotype account for 30.9% of dose variation.
SNPs frequencies in OA children were similar to that of controls (children or adults). Contrary to expectations, no significant differences
were observed between the mean dose required and the genotype
(1173C>T. P = 0.302; –1639G>A. P = 0.122). According to the regression model, age (P = 0.006) and height (P = 0.009) made a significant
contribution to the variability in dose requirement. Both were found
to be good predictors of the square root of dose, but in a model where
genotypes were also included, age was found to be the superior predictor.
Conclusion: These data provide evidence that in contrast to adults,
assessment of SNPs has no significant impact on OA dosing in children. Moreover, age and height are the variables with more impact on
OA dosing.
Disclosure of Interest: None declared.
PHT11
Utility of thrombophilia screening in children awaiting
kidney transplantation
Bhat R1, Bock M2 and Bobrowski A2
1
Hematology; 2Nephrology, Lurie Children’s Hospital, Chicago,
USA
Objectives: Graft thrombosis is a common cause of early allograft loss
in pediatric kidney transplantation (KTx). The utility of screening for
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© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
ABSTRACTS
acquired and inherited thrombophilia is controversial. We posited that
universal pre-transplant screening for thrombophilia was of low predictive value for postoperative outcomes.
Methods: We reviewed 100 transplants in children aged 1–18 years
between the years 2005–2013, who underwent thrombophilia evaluations prior to KTx for short- and long-term postoperative outcomes
of bleeding, thrombosis and graft survival.
Results: Preoperative screening demonstrated thrombophilia in 52%
of patients (52/100). A pre-KTx thrombosis event occurred in 11/100
patients (11%). Patients were followed on average 3.6 years (SD: 2.1)
after KTx. In 14/100 (14%) children, a bleeding (7/100, 7%) or thrombotic (10/100, 10%) event occurred after KTx. Of the 7 bleeding events
(major or minor), 3 occurred during perioperative anticoagulation.
Postoperative thrombotic event was associated with history of pre-Ktx
thrombotic event (P = 0.043), diagnosis of focal segmental glomerulosclerosis (P = 0.002), FVL mutation (P = 0.034), and low AT level
(P = 0.037). History of a pre-KTx thrombosis was associated with
worse graft survival at 3 years (P < 0.001). Similarly, postoperative
thrombotic event was associated with poorer graft survival at 3 mo, 1
and 3 years (P = 0.001 and P = 0.003). FVL mutation was associated
with poorer graft survival at 3 mo (P < 0.0001) and 1 year
(P = 0.023); low AT levels were associated with poorer graft survival
at both 1 and 3 years (P < 0.0001 for both). Two of 100 grafts were
lost to thrombotic events.
Conclusion: Analysis of long-term follow-up data demonstrates that
the value of universal thrombophilia evaluation before KTx is low.
Focused universal investigation in addition to more detailed testing in
those with a previous TE event, however, may be advisable, as these
patients may benefit from tailored perioperative anticoagulation.
Disclosure of Interest: None declared.
per 1,000 NICU admissions. In univariate analyzes, mechanical ventilation (OR = 7.27, 95% CI = 2.02–26.17, P = 0.002), central venous
catheter (CVC; OR = 25.27, 95% CI = 5.54–115.4, P < 0.001), infection (OR = 7.24, 95%CI = 2.66–19.72, P < 0.001), major surgery
(OR = 5.60, 95% CI = 1.82–17.22, P = 0.003) and length of stay
≥15 days (OR = 6.67, 95% CI = 1.85–23.99, P = 0.004) were associated with HA-VTE. Only CVC (OR = 11.88, 95% CI = 2.18–64.60,
P = 0.004) remained an independent risk factor in the multivariate
analysis. Based on this result, the estimated risk (post-test probability)
of HA-VTE in NICU patients with a CVC was 0.8%.
Conclusion: This study identifies CVC as an independent risk factor
for HA-VTE in critically ill neonates. However, the level of risk associated with CVC is below the conventional threshold for primary anticoagulation thromboprophylaxis. Larger studies are needed to
substantiate these findings and identify novel putative risk factors to
further distinguish NICU patients at highest HA-VTE risk.
Disclosure of Interest: None declared.
PHT13
Site, recurrences and outcomes of deep vein thrombosis in neonates and children
CD
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Pergantou H, Kapsimali Z, Avgeri M, Komitopoulou A, Adamtziki
E and Platokouki H
Haemophilia Centre/Haemostasis and Thrombosis Unit, Aghia
Sophia Children’s Hospital, Athens, Greece
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Objectives: The aim was to investigate morbidity and outcomes in neonates and children diagnosed with deep vein thrombosis (DVT) in our
center.
Methods: We retrospectively analyzed the data of 57 children, whereof
fourteen neonates (mean age 8 years for children and 3 days for neonates) with non–central venous line–related DVTs referred from 1/
1999 to 1/2014.
Results: The primary location of DVT was: lower extremities (n = 29,
whereof 3 neonates), upper extremities (n = 3), portal vein (PVT) (n =
7), spleen (n = 9), mesenteric (n = 8, whereof one neonate), renal veins
(n = 10, all neonates), and lung (n = 3). Lower extremity DVTs were
extended to inferior vena cava in 9 patients, while concomitant pulmonary embolism was present in three patients, all non-neonate children.
The mean follow-up period was 7 years (3–14 years). All patients survived from thrombosis; one patient succumbed because of an underlying congenital heart disease. Recurrence rates were 21% (9/43) in
children and 7% (1/14) in neonates. All children who suffered a recurrence had either a strong congenital (n = 2) or acquired (n = 1) thrombophilic factor or an underlying disease predisposing to DVT (n = 7).
The outcomes were: chronic portal hypertension and esophageal varices (n = two children/PVT), renal atrophy and mild renal insufficiency
(n = 5 neonates/RVT), hypertension (n = 1 neonate/RVT), severe
chronic renal failure (n = 1 neonate/RVT) while 9 children with DVT
in lower extremities (31%) developed post-thrombotic syndrome
(PTS) after a mean period of 3 months following DVT. Additionally,
one neonate with DVT suffered amputation of both his limbs due to
necrosis. Long-term outcome of PTS after a mean observational period of 6 years and application of anticoagulation plus graduated elastic compression stockings was: complete recovery (n = 1),
improvement (n = 1) and unchanged clinical status (n = 7).
Conclusion: Recurrent DVTs and PTS were more common in older
children (mean age: 8.9 years) in both groups while neonates suffering
RVT are vulnerable to renal morbidity.
Disclosure of Interest: None declared.
or
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PHT12
Risk factors for hospital-associated venous thromboembolism in the neonatal intensive care unit
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Amankwah EK1,2, Atchison CM3, Arlikar S1, Ayala I4, Barrett L1,
Branchford BR5, Streiff M6, Takemoto C4,7 and Goldenberg
NA1,4,6,7
1
Clinical and Translational Research Organization, All Children’s
Hospital Johns Hopkins Medicine, St. Petersburg; 2Department of
Pediatrics, Johns Hopkins University School of Medicine,
Baltimore; 3Department of Pediatrics, University of South
Florida, Tampa; 4Johns Hopkins Medicine Pediatric Thrombosis
Program, All Children’s Hospital and Johns Hopkins Children’s
Center, St. Petersburg; 5Section of Hematology/Oncology/Bone
Marrow Transplantation, Department of Pediatrics, University of
Colorado School of Medicine Anschutz Medical Campus and
Children’s Hospital, Aurora; 6Division of Hematology,
Department of Medicine; 7Division of Hematology, Department
of Pediatrics, Johns Hopkins University School of Medicine,
Baltimore, USA
Objectives: Knowledge on hospital-associated venous thromboembolism (HA-VTE) risk factors in critically ill neonates is limited. In this
study, we determined HA-VTE risk factors in the neonatal intensive
care unit (NICU) of a large hospital.
Methods: We conducted a case-control study in the NICU of All Children’s Hospital Johns Hopkins Medicine (St. Petersburg, FL), from
January 1, 2006–April 10, 2013. HA-VTE cases were identified using
electronic health record. Four NICU controls were randomly selected
for each HA-VTE case. Associations between putative risk factors and
HA-VTE were estimated using odds ratios (ORs) and ninety-five percent confidence intervals (95%CIs) from univariate and multivariate
regression analyzes.
Results: Twenty-three HA-VTE cases and 92 controls were included.
The annual HA-VTE incidence was approximately 1.4 HA-VTE cases
59
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
12/06/2014
60
ABSTRACTS
PHT14
Whole blood vs. reconstituted whole blood for cardiopoulmonary bypass surgery in infants
PHT15
Use of warfarin oral suspension for antithrombotic
therapy and prevention of thrombosis in infants and
children
Niebler RA1,2, Scott JP3, Cole R1,2, Stuth E3, Shah T1,2, Benson
D1,2 and Tweddell J2,4
1
Pediatrics, Medical College of Wisconsin; 2Herma Heart Center,
Children’s Hospital of Wisconsin; 3Anesthesiology;
4
Cardiothoracic Surgery, Medical College of Wisconsin,
Milwaukee, USA
Bonduel MM1, Hepner M1, Buontempo F2, Sciuccati G1, Pieroni
G1, Annetta SE1, Penuto RF2, Cervio C1, Torres AF1 and Frontroth
JP1
1
Thrombosis and Haemostasis; 2Farmacia, Hospital Garrahan,
Buenos Aires, Argentina
0.778
0.483
0.475
90.7 52.1
91.1 47.4
0.971
58/121 (0.48)
21/35 (0.60)
43.3 (0–421.8)
42.0 (0–222.9)
0.649
60.9 (12.0-457.8)
0.808
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8 (1–56)
3.2 0.6
173.8 62.4
or
9 (0–56)
3.1 0.8
165.3 61.7
or
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0.209
52.3 (11.8-229.1)
1
Median (range) reported for non-normally distributed variables.
Mean standard deviation reported for normally distributed variables.
2
Conclusion: The use of whole blood to prime the cardiopulmonary
bypass circuit was not superior to a prime consisting of pRBC and
FFP in this cohort of young infants in measures of postoperative
transfusion requirements and blood loss.
Disclosure of Interest: None declared.
Objectives: Vitamin K antagonists (VKA) are only commercially available in tablets. Infants and young children are unable to swallow tablets. Furthermore, splitting tablets leads to inaccurate doses. Although
oral liquid formulation compounded from warfarin tablets (WT) was
previously described, no data about its use in children have been
reported. Our aim was to describe the laboratory monitoring data,
recurrent thrombosis and bleeding in children with thrombosis or
mechanical prosthetic heart valve (PV) who received warfarin oral suspension (WOS) prepared from WT.
Methods: From February 2012 to December 2013, infants and children
with venous or arterial thrombosis and PV receiving WOS were retrospectively evaluated. WOS was prepared from tablets by the Pharmacy
Department of our hospital based on a previous report (Sharley NA,
et al. J Pharm Pract Res2007;37:95–7). Therapy intensity and duration
depended on the underlying disorders. Children received an initial
loading dose of 0.2 mg/kg. Dose adjustment was based on international normalized ratio (INR).
Results: Sixteen patients (patients), 2.6 patients-yrs, 11 males (69%),
median age 0.47 years (range 0.25–4.8), venous thrombosis 10
patients, arterial thrombosis 5 patients and PVR 1 patient were evaluated. No patient discontinued the controls. Ten patients (62.5%) with
this regimen achieved their therapeutic target INR range (TTR) in
< 8 days. The results are expressed as median (range), length of treatment 2.2 months (1.3–22.9), dose to maintain TTR: 0.21 mg kg–1
(0.15–0.5). The INR values at the ranges of 2.0–3.0 or 1.5–1.9 were
observed for 30.8% and 59.1% of the follow-up time, respectively. No
patient had serious bleeding or recurrent thrombosis.
Conclusion: We conclude that is the first report about the use of WOS
in infants and children. The thrombotic and bleeding rates observed in
these patients are similar to the data published in previous pediatric
studies using WT. A prospective study, including a large number of
pediatric patients, is ongoing in our center to confirm these results.
Disclosure of Interest: None declared.
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pRBC and
FFP Prime
n = 35
Co
Age (days)1
Weight (kg)2
Bypass Time
(minutes)2
Cross Clamp Time
(minutes)2
Use of Deep
Hypothermic
Circulatory Arrest2
24 h pRBC volume
transfused (mL/kg)1
24 h chest
tube output
(mL/kg)1
Fresh Whole
Blood Prime
n = 121
pi
Objectives: To determine the impact of using whole blood to prime the
cardiopulmonary bypass circuit on postoperative transfusion requirement and blood loss in infants.
Methods: A retrospective chart review of all infants < 60 days of age
undergoing surgery requiring cardiopulmonary bypass at Children’s
Hospital of Wisconsin from 11/2009 to 4/2012 was completed. Patients
with a prior surgery requiring cardiopulmonary bypass or on mechanical circulatory support pre-operatively were excluded. The cardiopulmonary bypass circuit was primed with either whole blood or a
combination of packed red blood cells (pRBC) and fresh frozen
plasma (FFP) as determined by availability from the blood bank. Volumes of chest tube output for 24 h post-operatively and transfused
volumes of pRBC within 24 h postoperatively were compared. Other
variables known to be associated with increased perioperative blood
loss were collected to insure the groups were comparable.
Results:
Plasma Coagulation Inhibitors
PCI01
Corn trypsin inhibitor non-loop regions are required for
the specific inhibition of factor XIIa
Korneeva V and on behalf of Mikhail M. Trubetskov, Alena V.
Korshunova, Vladimir N. Kolyadko, Mikhail A. Panteleev, and
Fazoil I. Ataullakhanov
The Laboratory of the Molecular Mechanisms of Hemostasis,
Center for Theoretical Problems of Physicochemical
Pharmacology RAS, Moscow, Russian Federation
Objectives: An activated form of factor XII (FXIIa) is selectively
inhibited by corn trypsin inhibitor (CTI) among other plasma proteinases. CTI is believed to be a canonical serine protease inhibitor and
interact with FXIIa through its protease-binding loop. Our objective is
to determine whether the CHFI protease-binding loop alone is sufficient for the selective inhibition of FXIIa or whether other regions of
canonical inhibitors are also involved.
Methods: Six recombinant mutants that were truncated from the Nand/or C-terminus were generated and expressed in E. coli. A synthetic cyclic 25 amino acid peptide containing residues 20–45 (1BEA,
12/06/2014
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
ABSTRACTS
PDB) was designed to exclude interactions outside the active site of
FXIIa. Unpaired Cys residues were replaced by Asn to avoid
unwanted disulfide bond formation between unpaired Cys in all
mutants. The inhibitory potency of the mutants was tested in vitro
using the chromogenic substrates H-D-Pro-Phe-Arg-pNA-2HCl and
Z-D-Arg-Gly-Arg-pNA-2HCl for FXIIa and bovine pancreatic trypsin, respectively.
Results: The CTI-234 mutant, which lacks the first and fifth disulfide
bonds and 11 and 19 amino acid residues at the N- and C-termini,
respectively, exhibited no significant changes in the ability to inhibit
FXIIa (Ki = 3.2 0.4 nmol L–1). The CTI-123 mutant, which lacks
34 amino acid residues at the C-terminus and the fourth and fifth
disulfide bridges inhibited FXIIa with a Ki of 116 16 nmol L–1.
Unexpectedly, the isolated protease-binding loop exhibited no potency
for the inhibition of FXIIa but retained partial inhibitory activity
against trypsin (Ki = 11.7 1.2 lmol L–1). Full-length CTI inhibited
trypsin with a Ki of 1.3 0.2 lmol L–1.
Conclusion: Our results suggest that the protease-binding loop is not
the only region required for the interaction between FXIIa and CTI;
other regions of the inhibitor also contribute to specific inhibition.
Nevertheless, the isolated protease-binding loop acts as an independent structural element that retains its inhibitory activity against trypsin.
Disclosure of Interest: None declared.
Disclosure of Interest: P. Ellery: none declaredfeclared, C. Augustsson
is an employee of: Novo Nordisk, S. Maroney: none declaredfeclared,
J. Wood: none declaredfeclared, L. Peterson is an employee of: Novo
Nordisk, I. Hilden is an employee of: Novo Nordisk, A. Mast has
grant/research support from: Novo Nordisk.
PCI03
Unfractionated and low molecular weight heparins, but
not fondaparinux, block inhibition of prothrombinase
by TFPI-alpha
Wood J1, Baumann Kreuziger LM2,3, Camire RM4,5 and Mast
AE1,6
1
Blood Research institute, Blood Center of Wisconsin;
2
Department of Medicine, Hematology, and Oncology, Medical
College of Wisconsin; 3Blood Center of Wisconsin, Milwaukee;
4
Division of Hematology, Children’s Hospital of Philadelphia;
5
Department of Pediatrics, University of Pennsylvania,
Philadelphia; 6Department of Cell Biology, Neurobiology, and
Anatomy, Medical College of Wisconsin, Milwaukee, USA
CD
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Objectives: Tissue factor pathway inhibitor (TFPI)a regulates the initiation phase of thrombin generation in part by inhibiting early forms of
prothrombinase, the complex of factor Xa (FXa) and factor Va (FVa).
Prothrombinase inhibition requires a charge-dependent interaction
between the TFPIa basic C-terminus and an acidic region of the FVa
B-domain, present in FXa-activated FVa and FVa released from activated platelets, but not in thrombin-activated FVa. Large negatively
charged molecules, including unfractionated heparin (UFH), block
this interaction, preventing prothrombinase inhibition and promoting
thrombin generation; however, the effect of low molecular weight heparin (LMWH) or the antithrombin binding pentasaccharide, fondaparinux, on this interaction is unknown.
Methods: TFPIa inhibition of thrombin generation by prothrombinase, assembled with a form of FVa containing the acidic region of the
B domain, was measured in the absence or presence of UFH, enoxaparin, dalteparin, or fondaparinux at therapeutic concentrations. The
effect of these compounds on the direct inhibition of FXa by TFPIa
was also measured using an FXa chromogenic substrate.
Results: TFPIa inhibited prothrombinase activity (IC50 = 6.8 nmol
L–1), and 0.5 U mL–1 or 1 U mL–1 UFH blocked this inhibition
(IC50 = 12.5 nmol L–1 or 14.9 nmol L–1, respectively). Enoxaparin
(0.8 U mL–1; IC50 = 30.3 nmol L–1) and dalteparin (1 U mL–1;
IC50 = 29.7 nmol L–1) were more effective at reversing TFPIa inhibition, though the reasons for this enhanced effect are not clear, as UFH
and the LMWHs similarly enhanced the direct inhibition of FXa by
TFPIa. In contrast, fondaparinux (0.66 mg L–1) had no effect in either
assay.
Conclusion: Therapeutic doses of UFH or LMWH have a procoagulant activity that is mediated by blocking prothrombinase inhibition
by TFPIa, while fondaparinux does not. This may be important clinically under conditions in which antithrombin is either deficient or
compromised, and the anticoagulant effects of heparins are diminished.
Disclosure of Interest: J. Wood: none declared, L. Baumann Kreuziger:
none declared, R. Camire has grant/research support from: Pfizer and
Alnylam Pharmaceuticals, A. Mast has grant/research support from:
Novo Nordisk
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PCI02
Measurement of plasma and platelet tissue factor
pathway inhibitor (TFPI) in blood donors
Ellery P1, Augustsson C2, Maroney S1, Wood J1, Peterson L2,
Hilden I2 and Mast A1
1
Blood Center of Wisconsin, Wauwatosa, USA; 2Novo Nordisk,
Malov, Denmark
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or
Objectives: Tissue factor pathway inhibitor (TFPI) is an anticoagulant
protein produced by endothelium and megakaryocytes. It circulates in
plasma as TFPIa (‘free’ TFPI; ‘full-length’ TFPI) and variably truncated forms bound to lipoproteins. TFPIa is within platelets and
released following activation to dampen platelet accumulation. Plasma
TFPI concentration poorly correlates with thrombotic risk. We
hypothesize that platelet TFPI will provide a more accurate measure
of thrombotic risk. As an initial step to test this hypothesis, high-affinity monoclonal antibodies were used to develop ELISAs for plasma
and platelet TFPI.
Methods: Plasma total TFPI and TFPIa, and platelet TFPIa, were
determined in 427 blood donors (209 men, aged 18–87; 218 women,
aged 19–89). Data were stratified by gender, age, race/ethnicity, and
oral contraceptive (OC) use, and reference ranges established.
Results: The reference ranges for platelet and plasma TFPI are broad,
varying approximately 5-fold (Table). Females aged 19–50 and not on
oral contraceptives (OC) had significantly lower plasma total TFPI
and TFPIa than females over 51, likely due to an effect of endogenous
estrogen on plasma TFPI levels. OC users had decreased plasma
TFPIa compared to non-OC users. Interestingly, platelet TFPIa in
females did not vary by age or OC use. Platelet and plasma TFPI in
males did not vary by age.
61
Conclusion: This study is the first to determine platelet TFPI concentration in a large cohort of normal donors. Since platelet TFPI is not
affected by age or estrogen, its measurement may be more useful than
plasma TFPI in assessing thrombotic risk.
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
12/06/2014
62
ABSTRACTS
PCI06
A new report of FVII-inhibitor in a patient suffering
from severe congenital FVII deficiency
PCI04
Risk factors of thrombosis in an African population
Toure AO
Medecine, Cheikh Anta Diop University, Dakar, Senegal
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PCI05
Protein Z intron FG79A polymorphisms are not
associated with hyperlipidemia
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Hua H-Y1, He S-l2 and He M-x1
1
Henan Academy of Medical and Pharmaceutical Sciences,
Zhengzhou University, Zhengzhou; 2Xiangya Medical College,
Changsha, China
Objectives: We investigated the distribution of protein Z intron
FG79A polymorphisms in in the Chinese Han population in Henan Province and the association of protein Z FG79A SNPs with
hyperlipidemia.
Methods: The 325 healthy controls and the 357 hyperlipidemic subjects
were enrolled from Nov.2011 to March 2012. All subjects were Chinese Han population in Henan Province. The diagnostic criteria of
hyperlipemia is that total cholesterol (TC) > 6.0 mmol L–1 or low-density lipoprotein cholesterol (LDL-C) > 3.36 mmol L–1 or triglycerides
(TG) > 1.78 mmol L–1 or high-density lipoprotein cholesterol (HDLC) < 0.82 mM. The protein Z intron FG79A polymorphisms were
analyzed by the method of polymerase chain reaction-based DNA
analysis.
Results: In the case subjects, the frequencies of the GG, GA and AA
genotypes were 19.3% (69/357), 51.5% (184/357) and 29.1% (104/
357), respectively. There were no significant differences compared with
controls (GG: 16.6% [54/325]; GA: 60.3% [196/325] and AA: 32.1%
[75/325]), and no differences were seen allele frequencies of PZ gene
between two groups (P > 0.05).
Conclusion: Protein Z intron FG79A polymorphisms are present in
Chinese population in Henan Province. Protein Z intron FG79A
polymorphismswere not associated with hyperlipidemia.
Disclosure of Interest: None Declared.
Objectives: Here we report a case that suffered from severe FVII deficiency who developed inhibitors directed against FVII after recombinant activated FVII (rFVIIa) replacement therapy.
Methods: An 8-year-old girl sustained a fracture around the left wrist
joint 2 years back. After removal of POP she started to develop hemarthrosis and uncontrolled bleeding after tooth extraction. A diagnosis
of severe isolated FVII deficiency was made. rFVIIa treatment was
started at a dose of 15 lg kg–1 6 hourly. After 2 weeks of rFVIIa treatment, she developed hematomas and multiple hemarthrosis. Then
hematuria, gums bleeding and bruises despite 30 lg kg–1 of rFVIIa.
She was transfused with red cells and started with FFP but showed no
response to treatment. Screening for anti-FVII inhibitors was performed using an adapted Bethesda assay (BU). Then, isotypic analysis
of the antibodies directed against FVII was performed using an immunoassay based on the x-MAP technology.
Results: Screening for FVII Inhibitor revealed titer of 11 BU leading
us to stop rFVIIa infusions and started treatment with Steroid’s,
immunosuppressant’s, Immunoglobulin, and FEIBA to treat acute
bleeding episodes. Her last titer is 1.5BU and she has improved clinically but still has episodes of bleeding on & off. Isotypic analysis of
the antibodies directed against rFVIIa showed a polyclonal response
with a large predominance of IgG1 and in a less extent IgG3. The
kinetics revealed a decrease of 43 and 41% of IgG1 and IgG3 respectively.
Conclusion: Here, we reported the first case of Isolated Factor VII deficiency with inhibitor in Pakistan. By contrast to other published FVIIinhibitor cases, who could be successfully treated by rFVIIa, our
patient did not respond to rFVIIa therapy. Therefore, management of
these patients remains a challenge.
Disclosure of Interest: None Declared.
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Objectives: In Black Africans, biological, epidemiological and clinical
risk factors for thrombosis and venous thromboembolic disease (VTE)
are not well known. We undertook a study of the prevalence of VTE
risk factors in the occurrence of thrombosis in the Senegalese’s population.
Methods: We conducted a 3-year comparative descriptive case/control
study involving 105 cases and 200 controls in different hospitals in Dakar (Senegal).
Results: Our results demonstrate that the use of pills (oestro-progestative drugs), casted immobilization, surgery and blood group were significantly associated with VTE occurrence. Additionally, 16 cases and
two controls had Protein S (PS) values < 48.4% (M-2SD), with a
highly significant difference (P < 1 9 104). The number of cases
exhibiting a low Protein C (PC) level was significantly higher than the
respective number of controls. We established a correlation, by regression logistic methods, of significantly associated variables to deep
venous thrombosis (DVT) occurrence. Age, obesity, sickle cell disease
and decreased PC levels were not significantly associated with thrombosis. In contrast, gender, decreased PS levels, varices, surgery, non-O
blood group and the presence of antiphospholipids antibodies were
significantly and independently associated with DVT.
Conclusion: To our knowledge this is the first study highlighting the
various epidemiological, clinical and biological risk factors for both
thrombosis and DVT in a black African country. This information is
extremely important for the clinical management of patients suffering
from DVT since it allows reduction of the high recurrence rate
observed in our study.
Disclosure of Interest: None Declared.
Borhany M1, Delbes C2, Giansily-Blaizot M2, Zubair M3, Fatima
N4 and Shamsi TS1
1
Haematology, Haemostasis & Thrombosis, National Institute of
Blood Disease & Bone Marrow Transplant (Nibd), Karachi,
^ pital Saint
epartement d’h
ematologie biologique, Ho
Pakistan; 2D
Eloi, Montpellier, France; 3Military Hospital Rawalpindi,
Rawalpindi; 4Research and Development, National Institute of
Blood Disease & Bone Marrow Transplant (NIBD), Karachi,
Pakistan
PCI07
Major bleeding events with dabigatran versus warfarin
in patients with acute venous thromboembolism: a
pooled analysis of RE-COVER and RE-COVER II
Schulman S1, Eriksson H2, Goldhaber SZ3, Kakkar A4, Kearon C1,
Mismetti P5, Schellong S6, Kreuzer J7, Feuring M7 and Friedman J8
1
Department of Medicine, McMaster University, Hamilton,
Canada; 2Department of Medicine, Sahlgrenska University
€
Hospital-Ostra,
Gothenburg, Sweden; 3Cardiovascular Division,
Brigham and Women’s Hospital, Harvard Medical School,
Boston, USA; 4Thrombosis Research Institute and University
College London, London, UK; 5Department of Vascular
Pathology, Bellevue Hospital, St Etienne, France; 6Medical
Division 2, Municipal Hospital Friedrichstadt, Dresden;
7
Boehringer Ingelheim GmbH & Co KG, Ingelheim am Rhein,
Germany; 8Friedman Consulting, Portland, USA
Objectives: In the acute venous thromboembolism (VTE) RE-COVER
and RE-COVER II trials, dabigatran etexilate (DE) compared with warfarin (W) resulted in similar rates of recurrent VTE/VTE-related death
12/06/2014
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
ABSTRACTS
PCI08
Timing of corn trypsin inhibitor to platelet poor plasma
alters thrombin generation
with fewer bleeding events. We further assessed major bleeding events
(MBEs) according to ISTH and TIMI criteria and bleeding locations.
Methods: An analysis on the pooled results from the RE-COVER
studies to assess the incidence, characteristics, and sites of MBEs during the 6-month double-dummy treatment period (treatment with DE
or W alone).
Results: MBEs overall, for each ISTH criterion, and for key anatomical sites were numerically less frequent with DE vs. W. Rates of TIMI
and life-threatening bleeds were low, and no difference between treatments was detected (Table). Although the rate of any gastrointestinal
(GI) bleeds was numerically higher with DE vs. W, the frequency of
major GI bleeds was lower with DE vs. W.
24 (1.0)
22 (0.9)
2.1
40 (1.6)
37 (1.5)
3.6
1 (0.0)
4 (0.2)
2 (0.1)
11 (0.4)
20 (0.8)
30 (1.2)
2 (0.1)
10 (0.4)
6 (0.2)
9 (0.4)
4 (0.2)
354 (14.4)
70 (2.9)
4 (0.2)
12 (0.5)
12 (0.5)
11 (0.4)
6 (0.2)
503 (20.4)
55 (2.2)
Objectives: Both tissue factor (TF; extrinsic pathway) – and contact
(intrinsic pathway)-mediated thrombin generation appear to be important in animal models of thrombosis. Contact pathway inhibition, typically with corn trypsin inhibitor (CTI), is essential for quantifying
plasma thrombin generation solely mediated by TF. However, the
effect of timing and sequence of CTI addition to stored plasma on contact pathway inhibition is uncertain.
Methods: Nine volumes of whole blood were collected by antecubital
venipuncture into glass tubes containing one volume of (1) 3.2% trisodium citrate and CTI [final CTI concentration = 25 lg mL–1; ‘CTIBefore’] or (2) 3.2% citrate only [‘CTI-After’]; platelet poor plasma
was harvested from both samples and stored at 80 °C. For the CTIAfter samples, CTI (50 lg mL–1 plasma) was added before or after
plasma thaw in a 37 °C water bath. Plasma thrombin generation to
5 pmol L–1 TF/4 lmol L–1 phosphatidylserine (PPP) or 1 pmol L–1
TF (PRP) was assayed using the Calibrated Automated Thrombogram
(CAT) and reported as lagtime (minutes) and peak height (nmol L–1
thrombin), expressed as mean (SD).
Results: For both PPP and PRP, the peak height was significantly
increased for CTI-After compared to CTI-Before plasmas. The lagtime
to PRP was significantly shorter in CTI-After vs. CTI-Before plasmas
but no different to PPP. There were no significant differences in CAT
parameters when CTI was added before vs. after plasma thaw
(P > 0.05).
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N = 2462
n (%)
Park MS1, Xue A2, Rosedahl JK3, Harmsen WS3, Kuntz MM1 and
Heit JA2,3
1
Surgery, 2Hematology; 3Health Sciences Research, Mayo Clinic,
Rochester, Rochester, USA
iza
ISTH MBEs
Patients with 1 MBE
MBEs/100 patient years
MBE criteriaa
Fatal
Symptomatic, in a critical
area or organ
Fall in Hb ≥ 20 g L–1, or ≥ 2 units
transfusion of blood or red cells
MBE siteb
Intracranial
GI
Urogenital
TIMI MBEsc
Life-threatening bleeds
Any bleeds
Any GI bleeds
DE
N = 2456
n (%)
63
An MBE can belong to > 1 criterion; bPatients can have > 1 site of
bleeding; cEvents causing a fall in Hb level of > 50 g L–1, or in hematocrit of > 15%, or intracranial bleeding.
Conclusion: This analysis confirms the favourable safety profile of DE
for the treatment of acute VTE. The detailed assessment of MBEs by
ISTH criteria showed a lower frequency with DE vs. W and very low
rates of TIMI and life-threatening bleeds. Although a higher rate of
any GI bleeds was observed with DE, major GI bleeds were less frequent with DE vs. W.
Disclosure of Interest: S. Schulman Consultant for: Boehringer Ingelheim honoraria for work in study-related committees, H. Eriksson
Consultant for: Boehringer Ingelheim, Bayer and Pfizer on advisory
boards, Speakers Bureau: speaker fees from Bayer, Boehringer Ingelheim, Pfizer and LeoPharma, S. Goldhaber has grant/research support
from: Daiichi and BMS, Consultant for: Boehringer Ingelheim, Daiichi, BMS, Janssen, Merck, Pfizer, Portola, and Sanofi-Aventis, A.
Kakkar has grant/research support from: Boehringer Ingelheim, Pfizer, BMS, Daiichi, Bayer, Sanofi, and Eisai, Consultant for: Boehringer
Ingelheim, Pfizer, BMS, Daiichi, Bayer, Sanofi, and Eisai, C. Kearon
Consultant for: Boehringer Ingelheim and Bayer on advisory boards,
P. Mismetti Consultant for: Bayer, BMS, Pfizer, Daiichi Sankyo, Boehringer Ingelheim, Astra Zeneca on advisory boards and honoraria
for work, S. Schellong Consultant for: Boehringer Ingelheim, Bayer
HealthCare, Daiichi Sankyo and BMS/Pfizer on advisory boards,
Speakers Bureau: Boehringer Ingelheim, Bayer HealthCare, Daiichi
Sankyo and BMS/Pfizer, J. Kreuzer is an employee of: Boehringer Ingelheim, M. Feuring is an employee of: Boehringer Ingelheim, J.
Friedman Consultant for: Boehringer Ingelheim
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PPP
Mean (SD)
PRP
Mean (SD)
CTI-Before
CTI-After
CTI-Before
CTI-After
Lagtime
(min)
Lagtime
(min)
P value
Peak Height
(nmol L–1)
Peak Height
(nmol L–1)
P value
3.0 (0.5)
2.9 (0.5)
0.300
229.9 (36.7)
247.9 (42.4)
< 0.010
11.4 (3.0)
8.4 (2.3)
< 0.001
22.6 (11.5)
44.6 (21.5)
< 0.001
Conclusion: We have observed an elevation in thrombin Peak Height
and shortened Lagtime when whole blood samples are not collected
into CTI-pretreated tubes; this needs to be taken into account when
interpreting CAT results.
Disclosure of Interest: None Declared.
PCI09
Interim results from the prospective observational
study on Novosevenâ room temperature stable (VII25)
in patients with hemophilia A or B
Kavakli K1, Arkhammar P2, Benson G3, Chambost H4, De Martis
F5 and Rosholm A6
1
Pediatric Hematology, Ege University Children’s Hospital, Izmir,
Turkey; 2Medical and Science, Novo Nordisk Haemophilia R&D,
Soborg, Denmark; 3Northern Ireland Haemophilia
Comprehensive Care Centre, Belfast City Hospital, Belfast, UK;
4
Centres des Hemophiles, Hopital de la Timone, Marseille,
France; 5University of Firenze, Azienda Ospedaliera, Firenze,
Italy; 6Bioistatistics Biopharma, Novo Nordisk, Soborg, Denmark
Objectives: A formulation (VII25) of recombinant activated factor VII
(rFVIIa), allowing storage ≤ 25 °C, was developed. Bioequivalence to
rFVIIa was demonstrated in healthy human subjects leading to EU
approval in April 2008. Neutralising antibodies to rFVIIa has not been
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
12/06/2014
ABSTRACTS
Disclosure of Interest: Z. Wang is an employee of: Celsus Laboratories,
a subsidiary company of Celsus Glycoscience, Inc., L. Cole is an
employee of: Celsus Laboratories, a subsidiary company of Celsus
Glycoscience, Inc., G. Li: none declared, R. Taylor: none declared, R.
Schubert is an employee of: Celsus Laboratories, a subsidiary company of Celsus Glycoscience, Inc., R. Linhardt: none declared.
PCI11
Technoclotâ protein s – a new simplified assay for the
determination of proteins activity
Binder N, Leitner M and Riha M
Technoclone GmbH, Vienna, Austria
aa
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PCI10
Characterization of a high potency heparin prepared
from porcine intestinal brine
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Wang Z1, Cole L1, Li G2, Taylor R3, Schubert R1 and Linhardt R2
Celsus Glycoscience, Inc., Cincinnati; 2Rensselaer Polytechnic
Institute, Troy, NY; 3Miami University, Oxford, OH, USA
1
Objectives: Protein S stimulates the proteolytic inactivation of coagulation factors V and VIII as a cofactor of activated protein C, increasing the risk of thrombosis. For determination of Protein S levels
various tests based on different methods are available. Aim of our
study was to evaluate the new, simplified clotting assay Technoclotâ
Protein S for quantification of Protein S activity.
Methods: Samples were diluted in Protein S deficient plasma (1:10),
followed by addition of TECHNOCLOTâ Protein S Reagent. After
incubation of 120 s, clotting was initiated by addition of CaCl2. Calibration plasma with assigned value of Protein S activity was used for
generation of a calibration curve. Clotting times measured were converted into Protein S activity (%). Normal and abnormal controls
were used for assay control. All tests were performed on the fully automated coagulation analyzer Ceveronâ alpha.
Results: Using standard plasma dilution, measuring range was found
between 10 and 150%. Repeatability within run was ≤ 5% and within
lab was ≤ 6% for controls. Method comparison was performed using
various plasma samples of different Protein S activity levels: A correlation r > 0.9 was found with three major competitors. The assay is insensitive to heparin up to 1 U mL–1. Stability of reagent on board was ≥ 8 h.
Conclusion: Our data demonstrate that TECHNOCLOTâ Protein S is
suitable for the determination of Protein S activity. Reduction of kit
components – no need of extra diluent – make it a simplified and robust
assay, optimized for running on automated coagulation analyzers.
Disclosure of Interest: N. Binder is an employee of: Technoclone
GmbH, M. Leitner is an employee of: Technoclone GmbH, M. Riha is
an employee of: Technoclone GmbH
or
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confirmed in patients with hemophilia A/B with inhibitors. This postapproval study investigates any potential change in immunogenicity
related to the VII25 formulation in this patient population. The primary endpoint is structured reporting of cases of reduced therapeutic
response and potential FVII neutralising antibodies.
Methods: This is a prospective, observational, single-arm, multicenter,
multinational study to detect antibodies to FVII during treatment with
VII25. Recombinant FVIIa binding is first tested in a double radioimmunoassay and, if confirmed, tested in an FVII:C assay for neutralizing
antibodies against endogenous FVII and FVIIa. All bleeding episode
information, including home therapy with VII25, is captured in a
patient diary and adverse events (AEs) recorded. Patients are expected
to remain in the study for up to 2 years and are offered testing for FVII
binding and neutralizing antibodies at study entry, study end, and
when clinically warranted. Study recruitment has been finalized.
Results: The study is ongoing. Interim data from 2 November 2010–3
September 2013 will be presented. Patient status: 51 enrolled, 24 completed, 3 discontinued, and 24 ongoing. Data comprise 1275 exposure
days and 511 treatment episodes. Thirty AEs have been reported in 17
patients (18 serious AEs in 11 patients, none related to study drug).
No thromboembolic events have been reported. Two AEs of decreased
response in relation to bleed treatment have been received. Both were
considered mild in intensity with outcome ‘recovered’. No patients
have tested positive for binding antibodies.
Conclusion: To date, half of the patients have completed this safety
study. No safety issues have been identified and no antibodies to
rFVIIa have been detected.
Disclosure of Interest: K. Kavakli has grant/research support from:
Novo Nordisk, Consultant for: Novo Nordisk, Speakers Bureau:
Novo Nordisk, P. Arkhammar is an employee of: Novo Nordisk, G.
Benson: none declared, H. Chambost has grant/research support from:
Novo Nordisk, Consultant for: Bayer, Baxter, Behring, Novo Nordisk, Pfizer, F. De Martis: none declared, A. Rosholm is an employee
of: Novo Nordisk
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Objectives: Elucidate the structure characteristics of a heparin prepared from porcine intestinal brine that contribute to its high anti-factor Xa and IIa activities, and test its conformity to the USP Heparin
Sodium Monograph.
Methods: The molecular weight of the brine heparin was analyzed by
size exclusion chromatography (SEC). Nuclear magnetic resonance
(NMR) was used to analyze its structure and impurities. SAX-HPLC
analysis was applied on the heparinases digested samples to map its
oligosaccharide fingerprint. Capillary electrophoresis and HPLC was
used to detect the potential impurities. HPLC-MS was used to identify
heparin samples’ antithrombin III-binding oligosaccharide sequences.
Results: The brine heparin displayed > 1.5-fold higher anti-factor Xa
and IIa activities than porcine mucosal heparins. NMR analysis indicated increased amount of antithrombin III-binding sites in the brine
heparin. SEC analysis showed that the brine heparin had higher MW
than porcine mucosal heparins. HPLC-MS showed the brine heparin
had higher level of trisulfated disaccharide and higher level of antithrombin III-binding sequences compared to porcine mucosal heparins. SAX-HPLC indicated more antithrombin III-binding sites in the
brine heparin as well. No significant amount of impurities was detected
in the brine heparin sample.
Conclusion: The increased amount of antithrombin III-binding sites
and higher molecular weight of the brine heparin contribute to its
higher anti-factor Xa and IIa activities. The brine heparin conforms to
the current USP Heparin Sodium Monograph.
PCI12
Pilot study of the safety of rivaroxaban for VTE
prophylaxis in non-orthopedic surgical patients
Thurm CA1, Schubl S2, Vakil A1, Klein T2, Raynor J3 and
Cervellione KL3
1
Department of Medicine; 2Department of Surgery; 3Department
of Clinical Research, Jamaica Hospital Medical Center, Jamaica,
USA
Objectives: Rivaroxaban is an oral direct inhibitor of factor Xa that is
currently approved for VTE prophylaxis after hip and knee replacement surgeries. Data is lacking on its use in other post-operative settings. In this pilot study, we explored the safety of rivaroxaban for
VTE prophylaxis in non-orthopedic surgical patients.
Methods: Post-operative adult patients with a Caprini score ≥ 3 who
were able to begin rivaroxaban within 72 h of surgery were eligible.
Exclusion criteria included: contraindication to rivaroxaban; acute or
high risk of bleed; further planned surgical intervention during current
admission; recent trauma; neurological, ocular, or spinal cord surgery.
Patients received 10 mg rivaroxaban daily beginning between 6 and
72 h post-operation. They were allowed other forms of medical prophylaxis before beginning rivaroxaban.
Results: Thirteen patients were enrolled; average age was 61 years
(range 41–82) and BMI of 27.1 (19.5–33.8). Six (46%) had large bowel
12/06/2014
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
ABSTRACTS
65
PCI14
Investigating determinants of inhbitor development to
factor VIII molecules in previously treated hemophilia
A patients
surgery, 3 (23%) thoracic, and 4 (31%) hernia, appendectomy or cholecystectomy. Caprini scores ranged from 3 to 10 (median = 5).
Patients received a total of 1–11 doses (median = 4). No patients experienced major bleeding. One experienced minor, clinically significant
bleeding at a chest tube site; that patient had received concomitant
aspirin. One patient experienced non-traumatic bruising of the arm.
No patients experienced VTE.
Conclusion: This small pilot study provides evidence supporting the
use of rivaroxaban for VTE prophylaxis in carefully selected, nonorthopedic post-operative patients. Rivaroxaban may be an attractive
alternative for patients preferring once daily oral medication to subcutaneous injections up to three times daily. Only large randomized, controlled trials can fully assess the efficacy and safety of rivaroxaban for
VTE prophylaxis in this population. Generalizability of this approach
will be limited by the inability of many post-operative patients to take
oral medications.
Disclosure of Interest: C. Thurm Speakers Bureau: Janssen Pharmaceuticals, S. Schubl: none declared, A. Vakil: none declared, T. Klein: none
declared, J. Raynor: none declared, K. Cervellione: none declared.
Barbara AMG1, Aledort LM2, Mathew P3,4, Mauser-Bunschoten
E5, Rivolta GF6, Romanov V7, Tagliaferri A6, Roussel-Robert V8,
Windyga J9 and Iorio A1
1
Clinical Epidemiology & Biostatistics, McMaster University,
Hamilton, Canada; 2Mount Sinai Hospital, New York; 3Bayer
HealthCare Pharmaceuticals, San Francisco; 4University of New
Mexico, Albuquerque, USA; 5Hematology, University Medical
Center Utrecht, Utrecht, Netherlands; 6University of Parma,
Parma, Italy; 7Baxter Healthcare Corporation, Westlake Village,
^pital Cochin, Paris, France; 9Institute of Haematology &
USA; 8Ho
Transfusion Medicine, Warsaw, Poland
R
PCI13
Establishment of method for determining heparin
content of human antithrombin concentrate
Objectives: The development of inhibitors continues to be the most
serious challenge in hemophilia A treatment. Several cohorts of previously treated patients (PTPs) have been studied to understand factor
VIII immunogenicity. The overall incidence of inhibitors, or neutralizing alloantibodies, in PTPs has been found to be three per 1000 person-years (Xi et al, J Thromb Haemost 2013). Very little is known
about the characteristics of de novo inhibitors in PTPs by analyzing
cases of de novo inhibitors in PTPs reported in the scientific literature;
international hemophilia registries and other sources.
Methods: A systematic review of prospective or retrospective studies
was undertaken to identify hemophilia A PTPs who developed new
inhibitors during treatment. Study authors were contacted to contribute to the study by providing additional patient data. We developed a
case report form (CRFs) to extract the relevant patient level data;
including details of inhibitor detection and testing, inhibitor course
and treatment, factor VIII products used, and details of events that
can trigger inhibitor development (surgery, vaccination, immune disorders, malignancy, product switch). The CRF will also be used to
gather data from hemophilia treatment centers that have reported
cases to a hemophilia registry, surveillance system.
Results: As a result of our systematic review, we identified 19 publications
that reported 39 new inhibitors in PTPs with hemophilia A. To date, individual patient data has been collected for 29 (74%) inhibitor cases: 14
(36%) from CRFs completed by study investigators and 15 (39%)
extracted from patient-level information available in the published literature. Data collection from other sources will commence shortly. We will
present descriptive analyses and narrative reports of the case series.
Conclusion: The interim results confirm the feasibility of the study. We
will present summary reports of commonly reported determinants and
suggest guidelines for optimal reporting of inhibitors.
Disclosure of Interest: None Declared.
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Objectives: Human antithrombin (AT) concentrates are used as therapy for congenital or acquired AT deficiencies. AT is a physiological
anticoagulant that can inactivate several coagulation factor proteins,
and heparin cofactor increases AT activity more than 100-fold. Since
human AT concentrates are obtained by heparin affinity chromatography, it may contain heparin. Therefore the risk of unexpected bleeding
or heparin-induced thrombocytopenia by the residual heparin should
be considered. In this study, as a national control laboratory, we plan
to establish an assay method for heparin content of human plasmaderived AT concentrates.
Methods: European Pharmacopoeia, United States Pharmacopoeia or
other in-house regulatory requirements have described heparin content
assay, and set limit for allowable levels of heparin impurities present in
AT. We design and conduct a preliminary examination of the accuracy
and precision with respect to assay methods noted above to adopt a
superior and most suitable test method for laboratory conditions.
Then validation study for the method is performed according to International Conference on Harmonization (ICH) Q2 guidelines. Finally a
collaborative study involving Korean AT manufacturers will be carried out to share the heparin content assay method, and to confirm the
level of heparin in final AT products.
Results: We developed an assay method for heparin content of AT by
modifying an in-house method, and it was validated according to the
ICH Q2 guidelines. Collaborative study demonstrated that Korean
AT concentrates products meet the criterion of EP or USP (Maximum
0.1 IU of heparin activity per International Unit of AT activity). Further experiments with more batches of AT will be done in order to set
up a standard specification of AT and improve its manufacturing process.
Conclusion: Heparin content assay of AT can be applied to the quality
control of human plasma-derived AT concentrate by manufacturers as
well as the national control laboratory.
Disclosure of Interest: None Declared.
or
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Kang YS, Kang HE, Kim J-H, Park SM, Kim H-O, Oh H-K,
Kim Y-L, Nam K and Ahn C-Y
Blood Products Division, NiFDS, Ministry of Food and Drug
Safety, Cheongwon-gun, Korea
Platelet Immunology
PI01
The effects of different B-cell activating factor receptors on lymphocyte function and secretion of cytokines
in immune thrombocytopenia
Min Y and Shi Y
Department of Hematology, Qilu Hospital, Shandong University,
Jinan, China
Objectives: B-cell activating factor (BAFF), a member of the TNF family, is a vital homeostatic cytokine for B cells. BAFF exerts its effect by
binding to three receptors – B-cell activation factor receptor (BAFFR), transmembrane activator and calcium modulator and cyclophilin
ligand interactor (TACI) and B cell maturation antigen (BCMA). We
aimed to elucidate the functional roles for those three receptors and
related pathogenic mechanisms in immune thrombocytopenia (ITP)
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
12/06/2014
66
ABSTRACTS
PI02
Standardization of the NOD SCID mouse model for the
assessment of antibody-mediated thrombocytopenia
Methods: We have collected peripheral blood mononuclear cells
(PBMCs) from patients with ITP, and established a dendritic cell (DC)
–PBMC co-culture system pulsed with acid-treated platelets. With the
utility of blocking antibodies for BAFF-R, TACI, and BCMA, the cocultured cells are divided in to five groups. The effects of rhBAFF and
BAFF receptors on lymphocyte function, anti-apoptotic genes and
secretion of cytokines were measured by flow cytometry, RT-PCR and
ELISA, respectively.
Results: The apoptotic rates of CD19+ cells and CD8+ cells in the
rhBAFF group and the BAFF-R group are significantly lower than that
in the baseline group. Accordingly, the proliferation rates of CD19+
cells and CD8+ cells in those two groups are higher than that in the baseline group. Moreover, the proliferation rates of CD4+ cells and CD8+
cells in the TACI group are also higher than in the baseline group. Additionally, elevated bcl-2/bax ratio and mcl-1 expression along with elevated secretion of IFN-c are seen in the rhBAFF group and BAFF-R
group whilst higher secretion of IL-4 and IL-6 found in the TACI group.
€llner H, Wesche J, Greinacher A and Bakchoul T
Fuhrmann J, Zo
Institute for Immunology and Transfusion Medicine, ErnstMoritz-Arndt- University Greifswald, Greifswald, Germany
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Objectives: Evaluation of human platelet (hPLT) survival in the NOD
SCID mouse model provides important insights into the pathology of
antibody- mediated thrombocytopenia. Recently, distinct methods
have been introduced by different groups. Standardization of the
methods of the NOD SCID mouse model is crucial to reliably compare
the results.
Methods: In this study, hPLTs were injected either retro-orbitally or
via lateral tail vein into NOD SCID mice. The survival of hPLTs was
determined at 0.5, 1, 2, 5 and 24 h after injection by flow cytometry
(FC) using two different preparation methods: isolation of platelets by
gradient centrifugation or whole blood preparation with PerFix-nc
Kit. hPLTs count at 0.5 h post injection was defined as 100%. Moreover, we evaluated the use of a new mouse strain, the SCID beige, in
the investigation of survival of hPLTs in vivo.
Results: Similar recovery and survival of hPLTs in murine circulation
was achieved with tail vein injection compared to retro-orbital injection (mean hPLTs clearance 3% per h vs. 6% per h respectively). Density gradient centrifugation and whole blood preparation yielded
comparable mean hPLTs percentage. However, standard deviation
(SD) was considerably lower with PerFix-nc Kit (SD 1 h post injection
105% vs. 27% respectively; SD at 5 h post injection 65% vs. 24%
respectively).
Clearance of hPLTs from circulation was faster in SCID beige compared to NOD SCID (median hPLTs after 1 h: 62%, range 39–84%
vs. 100%, range 97–132% respectively; median hPLTs after 24 h 15%,
range 8–21% vs. 42%, range 25–82% respectively).
Conclusion: The results of our study demonstrate that tail vein injection is a simple, noninvasive method with consistent results. The use of
PerFix-nc Kit enables fast and accurate determination of hPLTs survival with minimal effect on platelets.
Disclosure of Interest: None Declared.
PI03
P-selectin based functional platelet flow cytometry
assay for heparin-induced thrombocytopenia (HIT):
platelet donor variability and laboratory selection criteria for identifying adequate donors
Warad D, Miller R, He R, Chen D, Nichols WL and Pruthi RK
Special Coagulation Laboratory, Mayo Clinic, Rochester MN,
Rochester, USA
Conclusion: BAFF-R is the major conductor of pathogenic effects of
BAFF in ITP, which promotes survival and proliferation of B and
CD8+ T cells, by up-regulating anti-apoptotic genes expression and
IFN-c secretion. Cotrary to the previous conclusion that TACI is a
negative immune regulator, we find it an important promoter for T cell
proliferation and Th2 cytokine secretion in ITP.
Disclosure of Interest: None Declared.
Objectives: Functional HIT assays are based on the detection of platelet activation by heparin-PF4-antibody immune complexes. Our objective was to examine platelet donor variability in a P-selectin based flow
cytometry (FC) HIT functional assay and to identify donors with adequate platelet reactivity.
Methods: Citrated platelet-rich plasmas from 67 normal donors were
tested individually by incubating with a lab pooled HIT-positive serum
control, a lab pooled HIT-negative serum control, or donor selfserum, respectively, in the presence of 0, 0.3, and 100 IU mL–1 heparin
for 30 min at room temperature. Fluorescein-labeled anti-CD61
(platelet GP IIIa) and phycoerythrin-conjugated anti-CD62P (P-selectin) monoclonal antibodies were used to identify platelets and activated platelets respectively after incubation. Samples were analyzed by
the BD FACSCalibur flow cytometer and Cellquest program (Becton
Dickinson). Percent activated (CD61+ CD62P+) platelets were used
to calculate the Activation Ratio (AR, 0.3 IU mL–1: 0 IU mL–1 heparin) and Inhibition Ratio (IR, 0.3 IU mL–1: 100 IU mL–1 heparin).
Good responder criteria were: activated platelets > 14% at 0.3 IU
12/06/2014
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
ABSTRACTS
67
mL–1 heparin and AR ≥ 2 with the pooled positive control. Student’s
t-test was used for statistical analysis.
Results: Twenty two good responders were identified. Responders demonstrated significantly higher AR (2.8 0.8 vs. 1.3 0.4, mean SD, P < 0.001) and IR (3.8 1.5 vs. 1.9 0.7, mean SD,
P < 0.001) with the pooled positive control, while their ranges of negative pooled serum were 0.8–1.3 and 0.9–1.4 (mean 2 SD) respectively.
Conclusion: As with other functional HIT assays, including the gold
standard serotonin release assay, donor variability is observed in the
P-selectin-based FC HIT functional assay. It is therefore important to
establish criteria to identify adequately responsive platelet donors to
ensure consistent results. In this study we have developed a laboratory
donor selection mechanism based on testing parameters for platelet
reactivity to accomplish this goal.
Disclosure of Interest: None Declared.
PI04
Regulation of platelet and neutrophil interaction in the
formation of DNA extracellular traps
da
p
or
CD
Conclusion: In conclusion our data show that hyperthermia and acidosis exert an opposite regulation on NET generation, although the
effect is counterbalanced under both conditions. The inhibition of
platelets could be a possible target for avoiding the exacerbated NET
formation.
Disclosure of Interest: None Declared.
PI05
Variable impact of normal IgG subclasses on heparindependent platelet aggregation induced by a HIT
monoclonal antibody according to FccRIIa H131R polymorphism
Co
pi
aa
ut
or
iza
Objectives: Neutrophils secrete DNA extracellular traps (NETs) that
kill bacteria and also exert proinflammatory and prothrombotic activities. Through the interaction with neutrophils, platelets appear to be
key elements in NET formation. Considering that hyperthermia and
acidosis are characteristic features of inflammation, NET generation
in the presence of platelets was evaluated under these conditions. We
also analyzed the effect of endothelial-derived platelet inhibitors prostacyclin (PGI2) and nitric oxide (NO), as well as the anti-inflammatory
acetyl salicylic acid (ASA).
Methods: Washed platelets (WP) and polymorphonuclear leukocytes
(PMN) were isolated from peripheral blood of normal donors. WP
were stimulated with either Gram negative (LPS 2 lg mL–1) or Gram
positive (Pam(3)CSK(4) (Pam 1 ng mL–1)) bacteria wall components
and then cultured with PMN at 37, 40 and 42 °C and at pH 7.4, 7 or 6.5.
Results: Confocal microscopy studies and quantification of free DNA
showed that platelets potentiated NET formation triggered by LPS or
Pam (PMN:0.2 0.02; PMN + WP:0.2 0.02; PMN + LPS:
0.4 0.05; PMN + WP + LPS:0.8 0.1*; PMN + Pam:0.3 0.05;
PMN + WP + Pam:0.6 0.1*; lg mL–1 of DNA X SEM, n = 3,
*P < 0.05 vs. PMN + LPS or PMN + Pam). NET generation was
augmented by hyperthermia and decreased by acidosis (Figure), and
under both conditions, the effects were compensated, reaching basal
values. NETs were inhibited when platelets were preincubated with
PGI2 (3 nmol L–1), SNP (0.1 mM) or ASA (1 mM), (PGI2:76 and
65%, SNP: 95 and 82% and ASA:87 and 35%of inhibition for LPS
and Pam, respectively).
R
Carestia A, Rivadeneyra L, D’Atri LP, Negrotto S and Schattner M
Laboratory of Experimental Thrombosis, Institute of Experimental
Medicine, CONICET-National Academy of Medicine, Buenos
Aires, Argentina
Rollin J1,2, Pouplard C1,2, Saada A1, Gouilleux-Gruard V2,3,
Thibault G2,3 and Gruel Y1,2
1
Haemostasis, University Hospital of Tours; 2UMR 7292, CNRS;
3
Immunology, University hospital of Tours, Tours, France
Objectives: The response of platelets to anti-platelet factor 4/heparin
complexes (PF4/H) IgG developed in heparin-induced thrombocytopenia (HIT) is variable from one subject to another. This variability,
partly explained by gene polymorphisms affecting FccRIIa signaling
(Rollin et al, Blood 2012), could also depend on variations in plasma
proteins. Recently, we developed a chimeric monoclonal antibody to
PF4/H, 5B9, which has a human IgG1 Fc and fully mimics the effects
of HIT antibodies, and we investigated whether normal IgG may influence its ability to promote platelet aggregation according to FccRIIa
H131R polymorphism.
Methods: The platelet response of 59 healthy donors to 5B9/H was
thus studied using platelet aggregation tests performed with platelet
rich plasma (PRP) or washed platelets (WP).
Results: No aggregation was obtained when the PRP of 20/59 donors
was incubated with 5B9/H, vs. 9/59 when WP were tested. In addition,
the lag time was significantly longer when FccRIIa 131H platelets were
activated in PRP by 5B9/H compared to RR donors (P = 0.03). This
variable response related to FccRIIa polymorphism was confirmed
when HIT plasma was tested in PRP but not with WP or when collagen was used as inducer. Importantly, the platelet response to 5B9/H
in PRP was fully restored in non-responders when the IgG fraction
was removed from the plasma. Alternatively, the addition of human
polyvalent IgG to IgG-depleted PRP inhibited the aggregation
induced by 5B9/H, but lower concentrations of IgG were required to
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
12/06/2014
68
ABSTRACTS
achieve this effect in FccRIIa 131HH subjects, compared to RR
donors. Finally, we demonstrated that this variable effect of IgG on
platelet aggregation induced by HIT antibodies mainly depends on
IgG2, which presumably bind more efficiently the H131 isoform,
whereas IgG1 exert a similar inhibitory effect in RR and HH homozygous donors.
Conclusion: These results demonstrate that endogenous IgG differently
regulate platelet activation induced by HIT antibodies according to
FccRIIa H131R polymorphism.
Disclosure of Interest: None Declared.
of abs is pathogenic is not fully understood but it is generally accepted
that abs positive in the serotonin release assay (SRA) are most likely
to cause disease. We addressed this issue by studying PF4-dependent
platelet binding of abs positive in PF4 ELISA.
Methods: Binding of HIT abs and platelet activation (p-selectin expression) in the presence and absence of PF4, without heparin addition,
was characterized using 57 identity-blinded SRA-positive and negative
sera from ab positive HIT cases.
Results: IgG in SRA-positive sera preferentially bound to platelets and
induced p-selectin expression when PF4 was present but SRA-negative
abs failed to bind or activate platelets (P < 0.0001). PF4-dependent
binding of SRA-positive abs to platelets was significantly reduced
(P < 0.05) following treatment of platelets with chondroitinase ABC.
Conclusion: The findings are consistent with the possibility that the
main role of heparin in HIT pathogenesis is to induce abs that recognize epitopes created in the PF4 tetramer when it complexes with a glycosaminoglycan (GAG). All such abs recognize PF4 in a complex with
heparin, but only a subset recognizes more subtle epitopes created
when PF4 reacts with less highly charged chondroitin-4 sulfate, the
major GAG expressed on platelets. We propose that the latter abs are
capable of activating platelets and possibly other target cells whenever
PF4 is present and could in part explain the high risk of thrombosis
that persists for several weeks-months after heparin withdrawal in
patients recovered from HIT. They could also account for ‘delayed
HIT’ in which thrombocytopenia and thrombosis occur after heparin
has been discontinued.
Disclosure of Interest: None Declared.
PI06
Rapid detection of IgG, IgA, IgM antibodies associated
with heparin-induced thrombocytopenia (HIT)
Co
pi
aa
ut
PI07
Platelet-activating HIT antibodies bind preferentially to
PF4 treated platelets in the absence of heparin: implications for HIT pathogenesis
Padmanabhan A1, Jones C2, Bougie D2, Curtis B2, McFarland J2,
Wang D2 and Aster R2
1
Transfusion Medicine; 2BloodCenter of Wisconsin, Milwaukee,
USA
Objectives: Antibodies (abs) specific for platelet factor 4 (PF4) in a
complex with heparin, i.e., those positive in PF4 ELISA, are the hallmark of heparin-induced thrombocytopenia/thrombosis (HIT) but
many antibody-positive patients do not have HIT. Why only a subset
CD
or
PI08
Rationale for the use of newer oral anticoagulants in
the long term management of heparin induced thrombocytopenia
da
p
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iza
Objectives: The platelet-factor 4 (PF4) ELISA is a first line diagnostic
test for HIT because it can be done rapidly on-site. However, inhibition of a positive test by high dose heparin is recommended to confirm
specificity, increasing cost and performance time. Although many
believe only IgG abs cause HIT, some reports claim IgM and IgA abs
can cause severe disease. Until this controversy is settled, test platforms that can distinguish between these Ig isotypes are desirable, but
this can also increase cost. Here, we describe a rapid flow cytometry
bead assay (FBA) for simultaneous detection of IgG/A/M HIT abs
with high sensitivity and specificity.
Methods: The FBA uses beads coated with heparin-PF4 complexes
created by adding PF4 to heparin fragments end-linked covalently to
the bead. Beads are incubated with test serum and washed once. Beadbound IgG, IgA and IgM abs are detected simultaneously using Ig
class-specific probes labeled with different fluorophores.
Results: Sera from 300 HIT suspects (150 serotonin release [SRA] positive, 150 negative) were tested by PF4 ELISA and FBA. FBA results
showed good sensitivity (87% IgG, 63% IgA, 36% IgM) for IgG, and
specificity (96% IgG, 91% IgA, 93% IgM) for all isotypes compared
to ELISA, and high sensitivity/specificity (93%/91%) compared to
SRA. Testing of 100 normal serum samples by PF4 ELISA and FBA
showed significantly more false positive results for IgM antibodies by
ELISA (19) compared to FBA (4). All samples positive by FBA inhibited with high dose heparin.
Conclusion: FBA has sensitivity comparable to PF4 ELISA but
improved specificity, since inhibition with high dose heparin appears
to be unnecessary. Moreover, IgG, A and M aantibodies are reliably
detected in a single assay rapidly and at low cost. Superiority of the
FBA over PF4 ELISA may reflect use of end-linked heparin, passively
associated with PF4, a configuration that may mimic the target recognized by HIT abs involved in PF4-dependent platelet activation.
Disclosure of Interest: None Declared.
R
Sullivan MJ, Grady S, McFarland JG and Curtis B
Platelet & Neutrophil Immunology Lab, BloodCenter of
Wisconsin, Milwaukee, USA
Fareed J1, Hoppensteadt D1, Kalodiki E2, Walenga JM3 and
Lewis B4
1
Pathology, Loyola University Medical Center, Maywood, USA;
2
Josef Pflug Vascular Surgery, Ealing Hospital & Imperial College,
London, UK; 3Throacis and Cardiovascular Surgery; 4Medicine,
Loyola University Medical Center, Maywood, USA
Objectives: Heparin induced thromboycytopenia (HIT) is a catastrophic complication of heparin therapy. While parenteral antithrombin agents such as argatroban, bivalirudin and hirudin can be used for
the acute intravenous management of this syndrome, there is an unmet
need for long term outpatient antithrombotic management of HIT
patients. The purpose of this study is to validate the use of newer oral
anticoagulant agents (NOACs) as substitute anticoagulants for HIT
patients.
Methods: Individual (n = 25) and pooled (n = 5) HIT sera were used
to investigate their effects on platelet aggregation, PF4 release and
thrombin generation. The effect of rivaroxaban, apixaban and dabigatran were compared with unfractionated heparin (UFH) and low
molecular weight heparins (LMWHs) in platelet rich plasmas from
normal donors with thrombin generation markers including prothrombin fragment (F1.2) and thrombin antithrombin complex (TAT)
were also measured. The relative immunogenic potential of each of
these agents was also compared with heparins (UFH and LMWHs) in
a rabbit model.
Results: In contrast to UFH and LMWHs none of the NOACs produced any aggregation of platelets (7 3) vs. (64 9%) While heparin resulted in a release of PF4 from platelets (179 + 11) the NOACs
produced marginal release (< 30 ng mL–1). In the thrombin generation
assays variable results were obtained with NOACs. The relative immunogenicity of NOACs was marginal in comparison to heparin in rabbits.
Conclusion: These results suggest that the NOACs do not interact with
or release PF4 from platelets. Moreover, none of these agents produce
12/06/2014
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
ABSTRACTS
formed. In accordance with the FCA described by Tomer A, 10 lL of
controls fresh platelet-rich plasma were incubated with 10 lL of
patient plasma in presence of heparin (0.3 or 100 IU mL–1) or saline.
Eeach sample was incubated with a mix of platelet marker PE-conjugated anti CD41 and activation marker FITC-CD62P mAb. Platelet
suspensions were analyzed on a two colours flow cytometer BD Accuri
C6.
Results: The sensitivity and the specificity of SRA were respectively:
94% (CI 95%: 88–100) and 84% (CI95%: 73–94) and the sensitivity
and the specificity of FCA were 98% (CI95%: 95–100) and 82%
(CI95%: 71–92). When experts blinded to the SRA results did not
have doubt about HIT diagnosis, they did not change their opinion
even if SRA was not in accordance with their judgement in 6 cases
among 59 cases. When the experts blinded to the SRA results had
doubt about HIT diagnosis, they followed for their final decision the
results of SRA in 100% of cases (44/44).
Conclusion: FCA displayed similar results than SRA in HIT diagnosis.
Taken into account the potential advantages of this method, FCA
seems represent a promiscuous method in routine practice. SRA and
possibly FCA seem to have a big influence in HIT diagnosis only when
initial diagnosis is doubtful
Disclosure of Interest: None Declared.
activation or aggregation of platelets. These observations warrant
bridged clinical trials with parenteral anticoagulant agents to demonstrate the efficacy and safety of NOACs for management of HIT following parenteral anticoagulant treatment.
Disclosure of Interest: None Declared.
PI09
Evidence that clopidogrel metabolites can trigger
drug-induced immune thrombocytopenia (DITP)
Bougie D, Nayak D and Aster RH
Blood Research Institute, BloodCenter of Wisconsin, Milwaukee,
USA
CD
R
Platelet Physiology
or
PP01
Morphological distinction unravels mechanisms of
platelet biogenesis from bone marrow megakaryocytes
da
p
Nishimura S1, Eto K2 and Nagai R3
1
Jichi Medical University, the University of Tokyo, Tochigi,
Tokyo; 2Kyoto University; 3Jichi Medical University, Kyoto, Japan
Co
pi
aa
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or
iza
Objectives: The platelet inhibitor clopidogrel has been implicated as a
cause of acute thrombocytopenia (TP) in isolated instances. Clinical
findings in these cases suggested an immune etiology, but clopidogreldependent, platelet-reactive antibodies have not been described. Possible reasons for failure to detect such antibodies (DDAbs) include the
possibility that a clopidogrel metabolite could be the sensitizing agent.
Methods: We recently described a NOD/SCID mouse model in which
metabolite-specific DDAbs can be detected by showing that in vivo
clearance of transfused human platelets by candidate metabolite-specific antibodies is accelerated when mice, injected with the implicated
drug, produce metabolites in vivo (Blood 116:3033, 2010). We used this
tool to screen serum samples from 50 patients in which clopidogrel,
taken with or without other drugs was implicated as a possible cause
of thrombocytopenia.
Results: One of these individuals had a recurrence of profound TP
when treated with the structurally similar inhibitor, prasugrel. Seven
of the 50 patient samples shortened the survival of transfused platelets
(P < 0.01) only when mice were injected with clopidogrel. To further
define mechanisms, human platelets were transfused to mice given
clopidogrel and were then removed and tested with patient serum.
Two of the 7 sera reacted with these targets (P < 0.05) and 5 did not.
Conclusion: These findings are consistent with the possibility that clopidogrel caused TP in these patients by two different mechanisms: 1) in
5 patients, a DDAb was induced that recognized platelets when a clopidogrel metabolite, possibly the glucuronide conjugate, was present in
soluble form; 2) in 2 patients, TP was caused by an antibody specific
for the active metabolite of clopidogrel covalently linked to the P2Y12
ADP receptor, a process known to account for the platelet-inhibiting
function of this drug. These observations add to evidence that drug
metabolites may be relatively common triggers for DITP.
Disclosure of Interest: None Declared.
PI10
Serotonin release assay and flow cytometric assay in
the diagnosis of hit based on experts’ opinion
Tardy B1, Tardy B2, Montmartin A2, Piot M3 and on behalf of HIT
Study Group
1
CHU Nord; 2Inserm CIE3; 3EA3065, CHU Nord, Saint Etienne,
France
Objectives: Background: Having a rapid, widely available, independent
of use of radioactive reagents, and reliable functional assay is one of
the major wishes for clinicians face to heparin induced thrombocytopenia (HIT) suspicion. Our objectives were 1) to evaluate the performance of a functional flow cytometric assay (FCA) 2) to evaluate the
influence of SRA results on the experts’ opinion.
Methods: Plasmas of 103 patients included in a multicenter study ‘HIT
Score’ (NCT00748839) were randomly selected: 54 with positive HIT
diagnosis and 49 with a negative HIT. In the HIT score study, experts’
opinion was recorded first blinded to the SRA and then with the
knowledge of SRA results. SRA and FLCA assays were centrally per-
69
Objectives: Blood platelets are generated in the bone marrow (BM)
from their precursors, megakaryocytes (MK). Although we know that
MKs produce platelets throughout life, precisely how platelets are produced in vivo remains uncertain, largely because of the rarity of MKs
in the BM and the lack an adequate visualization technique. In the
present study, we were able to visualize MK dynamics leading to platelet release in living animals at high resolution.
Methods: To clearly understand the nature of thrombopoiesis in BM
MKs, we optimized an in vivo imaging technique based on two-photon
microscopy that enabled us to visualize living BM in CAG- enhanced
green fluorescent protein (eGFP) mice.
Results: By visualizing living bone marrow in vivo, we observed that a
second thrombopoietic process, rupture-like MK fragmentation, can
be ongoing simultaneously with proplatelet formation in the same
mouse BM. Short proplatelets predominated in the steady state, but
highly elongated proplatelets were apparent when thrombopoietin
(TPO) levels were high (e.g., after BM transplantation). Conversely,
following blood loss, 5-FU administration, antibody-based platelet
depletion or acute inflammation, there was accelerated release of larger
platelets from mature MKs mediated via the interleukin-1 (IL-1)
alpha-type1 IL-1 receptor axis and ERK-dependent MK apoptosis.
Moreover, microtubule assembly contributed to proplatelets in TPOstimulated MKs but did not in IL-1a-stimulated MKs due to uncoordinated expression of alpha- and beta-tubulin.
Conclusion: These findings support the idea that IL-1alpha acts acutely
as a platelet releasing factor, coordinating with TPO to dynamically
modulate the cellular programming of MKs that regulates platelet
counts.
Disclosure of Interest: None Declared.
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
12/06/2014
70
ABSTRACTS
PP02
Phosphorylation of CLEC-2 by Src-family kinases is
essential for anti-CLEC-2 antibody-induced receptor
internalization
remained elusive. However, it was speculated that vasoactive mediators released from platelet granules might play an important role.
Our aim was to investigate the relevancy of platelet degranulation in
maintaining vascular integrity.
Methods: Mice lacking both Nbeal2 and Munc13-4 were generated
and platelet function was assessed in vitro and in vivo.
Results: Mice deficient in both Munc13-4 and Nbeal2 are viable and fertile and show no signs of spontaneous bleeding. Platelets of these mice
were not able to secrete ATP even after strong stimulation and P-Selection recruitment to the platelet surface after stimulation was further
reduced compared to Nbeal2-deficient platelets. Double-deficient platelets
showed impaired aggregation and adhesion under flow ex vivo, which
translated into severely defective hemostasis and thrombus formation in
vivo. Interestingly, when subjected to the reverse passive Arthus reaction
the mice showed no signs of hemorrhage. Additionally, we did not
observe any sign of blood-lymphatic mixing in adult mice.
Conclusion: Here we demonstrate for the first time that defective
release of both major platelet granule types has no effect on vascular
integrity during inflammation thereby excluding platelet granule
derived factors as major regulators in this setting.
Disclosure of Interest: None Declared.
Lorenz V1, Stegner D1, Stritt S1, Walzog B2, Kiefer F3 and
Nieswandt B1
1
Department of Experimental Biomedicine, Rudolf Virchow
€rzburg, Wu
€rzburg; 2Walter Brendel Centre of
Center Wu
Experimental Medicine, Ludwig Maximilians University
M€
unchen, M€
unchen; 3Department of Vascular Cell Biology, Max
€nster, Germany
Planck Institute for Molecular Biomedicine, Mu
CD
R
PP04
An essential role of the inhibitory Fc gamma receptor
IIb in antibody-induced glycoprotein VI ectodomain
shedding in vivo
Co
pi
aa
ut
PP03
Combined deficiency of platelet alpha and dense granule release abrogates platelet aggregation and adhesion but has no effect on vascular integrity upon
inflammation
Deppermann C, Wolf K, Stegner D and Nieswandt B
€ rzburg,
Chair of Vascular Medicine, Rudolf Virchow Center, Wu
Germany
Objectives: Platelet activation results in the release of dense and agranule content, which amplifies the activation response and promotes
thrombo-inflammation. We have previously shown that deficiency of
either a- or dense granules resulted in impaired platelet adhesion and
aggregation in vitro, which translated into a profound protection from
arterial thrombosis and ischemic stroke as well as in a dramatic hemostatic defect. Remarkably, no intracranial hemorrhage was observed
after stroke. Under conditions of inflammation, platelets prevent hemorrhage via (hem)ITAM signaling but the downstream effectors
or
€ tting S1, Lorenz V1, Gessner JE2 and
Stegner D1, Popp M1, Du
1
Nieswandt B
1
Department of Experimental Biomedicine, University of
W€
urzburg, W€
urzburg; 2Molecular Immunology Research Unit,
Hannover Medical School, Hannover, Germany
da
p
or
iza
Objectives: The C-type lectin-like receptor 2 (CLEC-2) plays an important role in hemostasis and thrombosis. Clustering of CLEC-2 induces
phosphorylation of the hemITAM motif of the receptor and the
recruitment of Syk, thereby initiating a signaling cascade involving
LAT cumulating in phospholipase Cc2 activation. Treatment of mice
with the CLEC-2-specific antibody, INU1, leads to a depletion of
CLEC-2 in circulating platelets. Such CLEC-2-depleted mice are protected from occlusive thrombus formation but do not show a major
bleeding defect.
The mechanisms underlying anti-CLEC-2 antibody-induced thrombocytopenia and receptor depletion were assessed in vivo using pharmacological inhibitors and mouse models deficient in central components
of the CLEC-2 signaling pathway.
Methods: To deplete CLEC-2, mice were treated with the monoclonal
antibody INU1 (100 lg i.v.). Platelets were analyzed up to 7 days after
injection using flow cytometry, biochemical and functional assays.
Results: INU1-treatment induced a severe transient thrombocytopenia
in wild-type and Lat/ mice. In sharp contrast, in INU1-treated mice
lacking CLEC-2 or Syk (PF4-Cre/loxP) no thrombocytopenia was
observed. Further, INU1 provoked the irreversible loss of CLEC-2 in
wild-type, Lat/ and Syk-deficient platelets. In Syk-deficient mice,
INU1-induced CLEC-2 down-regulation occurred through internalization and intracellular degradation, demonstrating for the first time
an active mechanism of CLEC-2 down-regulation in platelets. Strikingly, in vitro blockade of Src family kinases by PP2 inhibits the
INU1-induced internalization in both wild-type and Syk-deficient
platelets revealing a crucial role of hemITAM phosphorylation for
CLEC-2 internalization.
Conclusion: Our results reveal that anti-CLEC-2 antibody induced
CLEC-2 loss occurs through Src family kinase-dependent internalization and can be mechanistically uncoupled from the undesired thrombocytopenia in vivo. This may lead to the development of therapeutic
agents modulating CLEC-2 function.
Disclosure of Interest: None Declared.
Objectives: The activatory collagen receptor glycoprotein (GP) VI contributes to normal hemostasis, but also to occlusive thrombus formation. The anti-GPVI antibody JAQ1 induces down-regulation of the
receptor in circulating platelets through ectodomain shedding and/or
internalization, accompanied by a transient thrombocytopenia. The
metalloproteinases ADAM10 and ADAM17 mediate GPVI shedding
in vitro, however, JAQ1-induced effects in vivo were unaltered in mice
lacking both proteases in platelets.
Fc gamma receptors (FccR) bind IgG antibodies and might thereby
contribute to the in vivo effects of JAQ1. Mice bear three different activating FccRs (I, III and IV), which are expressed on different immune
cells. FccRIIb, the only inhibitory FccR in mice, is present on various
immune cells and some endothelial cells, but not on platelets.
We speculated that another cell-type might be involved in mediating
JAQ1-triggered effects in vivo via FccRs.
Methods: We studied the in vivo effects of JAQ1 in mice pretreated
with the FccRIIb/RIII blocking antibody 2.4G2 and in genetically
modified mice.
Results: In contrast to control mice, 2.4G2 pre-treated mice developed
no thrombocytopenia upon JAQ1-injection and exhibited abolished
GPVI shedding. Studies in FccRIII- and FccRIIb-deficient mice unexpectedly revealed that FccRIIb, but not FccRIII, mediated JAQ1induced thrombocytopenia and GPVI shedding. Histological analyzes
revealed that the transient thrombocytopenia was caused by FccRIIbdependent platelet sequestration to the liver. Kupffer cell depletion did
not prevent the JAQ1-induced effects, indicating a contribution of
endothelial cells in this process. We speculate that FccRIIb positions
JAQ1-bound GPVI for trans-shedding. This process appears to
involve a sheddase other than ADAM10 since lack of endothelial
ADAM10 did not affect GPVI down-regulation in vivo.
Conclusion: Our data demonstrate that anti-GPVI antibody triggered
thrombocytopenia and GPVI shedding in vivo occur through a novel
FccRIIb-dependent mechanism.
Disclosure of Interest: None Declared.
12/06/2014
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
ABSTRACTS
71
PP06
PSI domain of b3 integrin contains endogenous thiol
isomerase function that can be inhibited by a PDI
inhibitor bacitracin and anti-PSI monoclonal antibodies
Nishimura S1, Nagai R2 and Eto K3
1
Department of Cardiovascular Medicine, TSBMI, The University
of Tokyo, Jichi Medical University, Tokyo; 2Jichi Medical
University, Tochigi; 3Kyoto University, Kyoto, Japan
Reddy EC1, Zhu G1, Chen P2, Reheman A3, Lei X3, Xu X4,
Petruzziello T3, Rui M5, Freedman J6 and Ni H7
1
Department of Laboratory Medicine, Keenan Research Centre
for Biomedical Science, St. Michael’s Hospital; 21Department of
Laboratory Medicine, Keenan Research Centre for Biomedical
Science, St Michael’s Hospital and Canadian Blood Services;
3
Department of Laboratory Medicine, Keenan Research Centre
for Biomedical Science, St Michael’s Hospital; 4Department of
Laboratory Medicine, Keenan Research Centre for Biomedical
Science and Department of Laboratory Medicine and
Pathobiology, St Michael’s Hospital and University of Toronto;
5
Department of Laboratory Medicine, Keenan Research Centre
for Biomedical Science, St Micheal’s Hospital; 6Department of
Laboratory Medicine, Keenan Research Centre for Biomedical
Science and Department of Medicine; 7Department of Laboratory
Medicine, Keenan Research Centre for Biomedical, Canadian
Blood Services, Department of Laboratory Medicine and
Pathobiology, Department of Medicine, Department of
Physiology, Department of Medicine, St Michael’s Hospital and
University of Toronto, Toronto, Canada
aa
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A
Objectives: Integrin aIIbb3 is essential for platelet adhesion and aggregation, key events in hemostasis and thrombosis. Recently, endogenous thiol isomerase activity in integrin b3 has been reported, though
its location has not been identified. There is also accumulating evidence to support roles for protein disulfide isomerase (PDI) in thrombosis. While the highly conserved integrin PSI domain contains two
CXXC motifs, the active site of PDI, whether b3 integrin PSI domain
contains thiol isomerase activity is unknown.
Methods: Here, we first generated recombinant murine b3 PSI domain
(rPSI). Refolding of reduced, denatured RNase was employed to
examine the PDI activity of this rPSI. We also examined the impact of
bacitracin on this activity. We also developed four mouse anti-mouse
b3 PSI domain monoclonal antibodies (anti-PSI mAbs) in b3-/- mice.
These mAbs also crossreact with human and other animal b3 PSI
domains.
Results: With the exception of PSI A1, all mAbs inhibited PDI-like
activity of both murine rPSI and purified human platelet b3 integrin.
All mAbs inhibited murine and human platelet aggregation along with
murine thrombus formation. In a cell-free system (ELISA), bacitracin
blocked both fibrinogen and PAC-1 binding to human platelet b3 integrin. These data suggest that PDI-like activity is critical for b3 integrin
ligand binding. Since, PSI A1 inhibited platelet aggregation and
thrombus formation but not PDI-like activity, it is likely the PSI
domain also regulates integrin conformational changes, independent
of PDI-like activity.
Conclusion: In conclusion, we identified integrin PSI domain has PDI
activity and is a novel target for anti-thrombotic therapies. Since the
PSI domain is highly conserved across all the b subunits of the integrin
family, our finding may have broad implications for cell biology and
multiple human diseases.
Disclosure of Interest: None Declared.
iza
Objectives/Aim: The thrombotic cellular mechanisms associated with
cardiovascular events remains unclear, largely because of an inability
to visualize thrombus formation. Thus, we developed in vivo imaging
technique based on single- and multi-photon microscopy to revealed
the multicellular processes during thrombus development.
Methods: We visualized the cell dynamics including single platelet
behavior, and assessed dynamic cellular interplay in two thrombosis
models using two photon microscopy. (Figure a, b).
Results: First, we visualized that rapidly developing thrombi composed
of discoid platelets without EC disruption was triggered by ROS photochemically induced by moderate power laser irradiation. In this
model, thrombus consisted by discoid platelet aggregations without
leukocyte recruitment. The second model is, thrombus with EC disruption. High power laser induced EC erosion and extravasations of circulating leukocytes with thrombus development. Inflammatory
cytokine, adhesion molecules dynamically control these two processes.
(Figure d)
As for the thrombus formation with EC disruption, chemokine expressions in endothelium and leukocyte (especially neutrophils) recruitment played a significant role in these processes. Leukocyte was
immediately recruited into the subendothelial layers with bleeding and
hemostatic reactions. TLR4 signaling also contributed to these steps,
and pretreatmet of LPS markedly enhanced these steps. Thrombus
included calcium activated cores and deformed platelets. Immigrated
leukocyte also showed the increase of intracellular calcium.
R
PP05
Thrombus development processes dependent on
endothelial injuries: visualized by in vivo two-photon
imaging
D
Conclusion: Summary: These results indicated that endothelial function, especially inflammatory status, determined the thrombotic reaction. Leukocyte also contributed with TLR4 signaling. In sum, using
our imaging system can be a powerful tool to analyze thrombus formation and evaluate the therapeutic strategies.
Disclosure of Interest: None Declared.
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
12/06/2014
72
ABSTRACTS
PP07
Arginase II KO reduces platelet aggregation while
sparing coagulation in aged mice
of SCIT in platelet cytoskeleton reorganization, thrombin-induced
platelet response and signaling pathways.
Methods: Washed platelets were incubated with different concentrations of SCIT (0–200 lg mL–1). Modifications in cytoskeletal proteins
were evaluated by electrophoresis and ultrastructural techniques.
Thrombin induced platelet response (0.1 U mL–1) was assessed by
aggregometry, and the resulting activation of signalling pathways
(PKC, phospho-tyrosines) by western-blot.
Results: Ultrastructural analysis revealed loss of platelet discoid shape
towards spheres in the presence of SCIT. The morphometric analysis
confirmed statistically significant increases on platelet circularity with
SCIT concentrations ≥ 50 lg mL–1. Thrombin-induced activation
caused actin polymerization with association of contractile proteins to
the polymerized cytoskeleton, associations that were totally prevented
with SCIT ≥ 100 lg mL–1. Platelet aggregation to thrombin was statistically inhibited with SCIT (50 lg mL–1) (36.6 11.3% vs.
79.6 3.8%; P < 0.01, n = 7), and was totally blocked with SCIT
concentrations ≥ 100 lg mL–1. Thrombin activation of signalling
pathways, PKC and phospho-tyrosines, decreased in a dose-dependent
manner with SCIT.
Conclusion: Our data demonstrate that supra-therapeutic concentrations of SCIT interfere with the cytoskeletal arrangement, in a dosedependent manner, and inhibit thrombin-induced platelet response.
This effect could be involved in the antithrombotic actions associated
with SSRIs. Our results reinforce previous hints that SSRIs or modifications of SSRIs could constitute future therapeutic antithrombotic
strategies.
Disclosure of Interest: None Declared.
Co
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Objectives: Arginase II is expressed by mitochondria of several cell
types including platelets and competes with nitric oxide synthase for
its substrate L-arginine, which in turn is transformed to NO. It has
been shown that inhibition of arginase is associated with increased levels of NO. Furthermore, NO is known to inhibit platelet aggregation.
Therefore, we hypothesised that arginase II deletion reduces platelet
aggregation due to an increased arginine and subsequent NO bioavailability.
Methods: Platelet aggregation was assessed in whole blood from aged
(19 months) female arginase II KO and WT mice; platelet aggregomety in response to thrombin (1 U mL–1) was performed and maximal
aggregation, area under the curve (AUC) and lagtime were determined. Furthermore, we investigated plasma tissue factor (TF) concentration by tissue factor activity assay using ELISA. In addition,
thrombin generation was measured in murine plasma by calibrated
automated thrombogram (CAT). Full blood counts were obtained.
Results: Thrombin-induced platelet aggregation was reduced in KO
compared to WT mice (AUC, KO 51.19 2.9 ohmxmin, WT
64.31 2.1 ohmxmin; maximal aggregation, KO 17.25 0.9 ohm,
WT 20.50 1.0 ohm; both, n = 8, P < 0.05). However, thrombin
generation in murine plasma did not differ in WT and KO rodents
(ETP, KO 456.7 19.0 nmolxmin, WT 414.0 32.23 nmolxmin,
n = 9/10, P < 0.05) and also plasma TF activity showed comparable
results (KO 23.56 3.5 pM, WT 25.61 5.8 pM, n = 10, P < 0.05).
WT
Platelet
count
(KO
1073 71.1 9 103/mm3,
1054 108.2 9 103/mm3, n = 9) as well as red blood cell and leukocyte count were comparable.
Conclusion: Deletion of arginase II blunts thrombin-induced platelet
aggregation but not plasma thrombin generation or tissue factor activity thus, suggesting an interference with platelet activatability, which
may be due to increased NO bioavailability. Results from this study
raise further questions with respect to the physiological relevance of
arginase II gene in arterial thrombosis, which will be further investigated in vivo.
Disclosure of Interest: None Declared.
R
€scher TF1,3, Camici GG1, Xiu-Fen
Reiner MFF1,2, Stivala S1,2, Lu
M4, Yang Z4 and Beer JH2,5
1
€rich, Schlieren;
Center for Molecular Cardiology, Universit€
at Zu
2
Internal Medicine, Cantonal Hospital Baden, Baden;
3
Cardiology, University Heart Center, Zurich; 4Medicine/
Physiology, University of Fribourg, Fribourg; 5Center for
€rich, Zurich, Switzerland
Molecular Cardiology, Universit€
at Zu
PP08
Escitalopram modifies platelet cytoskeletal assembly
and has a direct inhibitory effect on thrombin-induced
activation
Lopez-Vilchez I1, Diaz-Ricart M1, Molina P1, Sanz V1, White JG2,
Gasto C3 and Escolar G1
1
Department Hemotherapy-Hemostasis. Hospital Clinic of
Barcelona. CDB. IDIBAPS. Univ Barcelona, Barcelona, Spain;
2
Department Lab and Clinical Medicine and Paediatrics, Univ
Minnesota, Minneapolis, USA; 3Institute Clinic of Neurosciences.
Hospital Clinic, Barcelona, Spain
Objectives: We reported a prothrombotic phenotype in platelets
caused by serotonin and its correction with the selective serotonin reuptake inhibitor (SSRI) escitalopram (SCIT). Murine models of
depression show that SSRIs also affect the synaptic plasticity likely
through cytoskeleton related mechanisms. We investigated the effects
PP09
Elevated immature platelet fraction (IPF) is useful for
identifying MYH9 related disorders: a report of two
cases
Higgins RA, Moritz A, Holder K and Olson J
Pathology, University of Texas Health Science Center San
Antonio, San Antonio, USA
Objectives: Giant platelets with thrombocytopenia found in MayHegglin anomaly, Fechtner syndrome, Sebastian Syndrome, and
Epstein syndrome all derive from mutations in MYH9 gene which
encodes non-muscle myosin heavy chain A. Symptoms include selflimited bleeding episodes, glomerulonephritis progressing to end-stage
renal failure, presenile cataracts, and sensorineuronal hearing loss.
Recognition of MYH9 related disorder (MYH9-RD) is important in
order to avoid confusion with immune thrombocytopenic purpura,
which may lead to inappropriate medical therapies or splenectomy.
We aim to improve the detection of MYH9-RD by proposing IPF as a
tool to identify potential cases.
Methods: We report the clinical and laboratory features of two
patients with MYH9-RD with markedly elevated IPF. To evaluate the
distribution of IPF values in unselected hospital patients, data
was retrospectively reviewed over a 5 day period in a hospital laboratory setting. IPF was measured using a Sysmex XE-5000 hematology
cell counter.
Results: Both patients have a predominance of giant platelets, thrombocytopenia, and histories consistent with MYH9-RD. Patient #1 is a
heterozygote for a known MYH9 mutation, and patient #2 is a heterozygote for an rare allele c.136 C>T in exon-2 previously unassociated
with disease. Patient #1 demonstrates neutrophil D€
ohle body-like
inclusions by Wright stained peripheral blood film, and both patients
demonstrate abnormal aggregates of membrane complexes within
platelets by electron microscopy. Both patients with MYH9-RD have
extremely high IPF (> 50%). Immature platelet fraction on 441 consecutive unselected patients identifies only one patient with an IPF
> 50% (Figure). This patient is also thrombocytopenic (platelets
< 100K/lL).
12/06/2014
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
ABSTRACTS
73
PP11
Evaluation of platelets surface morphology and their
local elastic properties in patients with implanted heart
ventricle in the early and late postoperative period by
AFM
Drozd L1, Kukharenko L2, Schimmel T3, Chizhik S4 and MarozVadalazhskaya N5
1
Neuroengineering and Bionanotechnology Group, Department
of Informatics, Bioengineering, Robotics and System Engineering,
University of Genova, Genova, Italy; 2Belorussian State Medical
University, Minsk, Belarus; 3Instiut f€
ur Angewandte Physik und
€r Funktionelle Nanostrukturen, Karlsruhe, Germany;
Centrum fu
4
A.V. Luikov Heat and Mass Transfer Institute of National
Academy of Sciences of Belarus; 5Scientific-practical Centre of
Cardiology, Minsk, Belarus
Conclusion: These data suggest that markedly elevated IPF (> 50%)
and thrombocytopenia may be useful in identifying MYH9-RD. Additionally, we report the first case of c.136 C>T in association with typical features of MYH9-RD and demonstrate frequent membrane
complexes in MYH9-RD.
Disclosure of Interest: None Declared.
Harbert J, Pagano K, Heartfield D and Ero M
Machaon Diagnostics, Oakland, USA
Co
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Objectives: The omega-3 fatty acids contained in fish oils have been
extensively studied and proven to have measureable cardiovascular
benefits even though a mechanism of action remains unclear and
highly disputed. To further research the clinical utility of fish oil, this
study examined in vitro addition of three substances and their effect on
thrombin generation measured using platelet-rich plasma on the calibrated automated thrombogram.
Methods: Whole blood from six normal volunteers was drawn by syringe into 3.2% sodium citrate. Citrated whole blood was centrifuged
at 150 g after which the PRP was harvested and remaining packed
cells where centrifuged at approximately 2500 g to obtain cell-free
plasma which was used to adjust the PRP to a concentration of
150,000 platelets per microliter. Fish oil (15% EPA/10% DHA), corn
oil or 1% bovine serum albumin in tyrode’s buffer (BSA) was added
to PRP to achieve a final concentration of 20%. These solutions were
incubated at 37 °C for 2 h. Each sample was then tested using the calibrated automated thrombinogram as previously described by Hemker
(Thromb Haemost, 2003). Endogenous thrombin potential was
recorded after 60 min. Corn oil was selected as a control to mimic the
lipid properties of fish oil within the test reaction, and 1% BSA in tyrode’s buffer was selected as an inert volume control.
Results:
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PP10
Comparison of fish oil and corn oil on the thrombin
generation of platelet-rich plasma as measured by the
calibrated automated thrombogram
Objectives: The goal of study was to estimate the intravascular platelet
activation and determine the platelet local elastic modulus (E) by optical and atomic force microscopy (AFM) in patients with the atrioventricular valveplasty and heart ventricle implantation before surgery
and intrasurgical.
Methods: 10 healthy volunteers (control group), 30 patients with the
atrioventricular valveplasty (I group) and 25 individuals, whose heart
ventricle were implanted (II group) were studied. The optical and
AFM were used to control the intravascular platelet activation: before
surgery and during surgery at the following stages: before the heparin
injection, after the heparin injection, after neutralization of heparin
with protamine, after 30 days and 6 months of surgery.
Results: In the AFM images the intravascular platelet activation was
presented by a dramatically increase of the platelet height, filopodia
formation and platelet aggregates visualization. For patients of I and
II groups the platelet surface morphology changes and platelet aggregation were determined to a greater extend than those for the control
group. It was found that for I and II patient groups at the presurgicale
stage platelet local elastic modulus was significantly greater in comparison with healthy persons group. Moreover platelet local elastic modulus in control group differs considerably from that in patients with
heart ventricle implantation in the early and late postoperative period.
Thus, the local modulus of elasticity in 30 days after surgery was 2.4
times higher than that in the group of healthy individuals.
Conclusion: Our approach allows to assess the intravascular platelet
activation and determine the local elastic modulus of the platelet in
patients with implanted heart ventricle in the early and late postoperative period thus to control platelet pathophysiology.
Disclosure of Interest: None Declared.
20% v/v
1% BSA
Corn Oil
Fish Oil
ETP (nmol L–1min)
2142 225
2027 112
1728 155*
is SD. *P < 0.05 from 1% BSA and Corn Oil groups.
The average ETPs of our experiment are shown in table 1. There was a
significant difference between the BSA group and fish oil group. There
was also a significant difference between the corn oil group and fish oil
group.
Conclusion: The reduction in platelet-associated thrombin generation
in the fish oil group suggests that the omega-3 fatty acids EPA and
DHA have a greater effect than the fatty acids in corn oil. This
research supports platelet involvement in fish oil’s antithrombotic
effect.
Disclosure of Interest: None Declared.
PP12
NOD2 receptor is expressed in platelets and enhances
platelet activation and thrombosis
Zhang S1, Zhang S1, Hu L1, Zhai L1, Ye J1, Chen G2, Mruk J3,
Kunapuli SP4 and Ding Z1
1
Department of Biochemistry and Molecular Biology, Fudan
University Shanghai Medical College, Shanghai, China; 2Thoracic
Oncology Research Laboratory, University of Pennsylvania,
Philadelphia; 3Department of Internal Medicine, University of
Kansas School of Medicine, Witchita; 4Department of
Physiology, and Sol Sherry Thrombosis Research Center, Temple
University School of Medicine, Philadelphia, USA
Objectives: Pattern recognition receptor NOD2 (nucleotide binding
oligomerization domain 2) is well investigated in immunity, its expression and function in platelets has never been explored. Here we investigated the expression and function of NOD2 in platelets.
Methods: Using RT-PCR and Western blot we show that both human
and mouse platelets express NOD2, and its agonist MDP induced
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
12/06/2014
74
ABSTRACTS
PP14
Effects of garlic (Allium sativum) on some hematological parameters using New Zealand White rabbits
NOD2 activation as evidenced by receptor dimerization. The platelet
aggregation and secretion, clot retraction and in vivo thrombosis was
also investigated.
Results: NOD2 activation potentiates platelet aggregation and secretion induced by low concentration of thrombin or collagen, as well as
clot retraction. These potentiation effects of MDP were not seen in
platelets from NOD2-deficient mice. Using intravital microscopy, we
found that MDP administration accelerated in vivo thrombosis in
FeCl3-injuried mesenteric arteriole thrombosis mouse model. Platelet
depletion and transfusion experiments confirmed that NOD2 from
platelets contributes to the in vivo thrombosis in mice. NOD2 activation also accelerates platelet-dependent hemostasis. Finally, we found
that platelets express RIP2 (receptor-interacting protein 2), and provided evidence suggesting that MAPK and cGMP/PGK pathways
downstream of RIP2 mediate the role of NOD2 in platelets.
Conclusion: NOD2 is expressed in platelets and functions in platelet
activation and arterial thrombosis, possibly during the conditions of
inflammation. To our knowledge, this is the first study on NOD-like
receptors in platelets which links thrombotic events to inflammation.
Disclosure of Interest: None Declared.
Rose AA
Medical Laboratory Science, College of Medicine, Ambrose Alli
University, Ambrose Alli University, Ekpoma, Edo State, Nigeria,
Benin, Nigeria
Objectives: To evaluate the effects of garlic consumption on some
hematological parameters.
Methods: 20 rabbits were divided into 5 groups, each group consisting
of 4 rabbits. The baseline parameters of the rabbits were obtained
before the rabbits were fed with garlic. Group A (control group) was
kept on conventional diet. Group B, C, D and E rabbits were given the
conventional diet along with garlic at the dosage of 100 mg, 200 mg,
500 mg and 1000 mg respectively for a period of 4 weeks. Hematological parameters which include Hb estimation, PCV, Plt count, TWBC
and differential leucocyte count was studied using standard methods.
Results: The result obtained showed that there was no significant difference (P < 0.05) in the hematological parameter except for group C
where there was significant increase (P < 0.05) in absolute monocyte
count. There was reduction in platelet count of the group although not
statistically significant (P < 0.05).
Conclusion: This study revealed that garlic concentration or dosage
does not have effect on some hematological parameters.
Disclosure of Interest: None Declared.
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Objectives: Murine paired immunoglobulin-like receptors B (PIRB), as
the ortholog of human leukocyte immunoglobulin-like receptor B2, is
involved in a variety of biological functions. However, the expression
and function of PIRB in platelets remain unknown.
Methods: We assessed the effects of PIRB on platelet activation and
thrombosis in PIRB intracellular domain deletion mice (PIRB-TM).
Inside-out signaling was assessed by measuring fibrinogen binding by
flow cytometry after stimulation of platelets with thrombin and the
collagen related peptide (CRP). Outside-in signaling was examined by
measuring platelet spreading and clot retraction. Susceptibility to
thrombotic occlusion of the carotid artery in response to ferric chloride injury was also measured.
Results: We found PIRB and LILRB2 were highly expressed in mouse
and human platelets respectively. An increasing megakaryocytes ratio
in bone marrow and thrombocythemia were observed in PIRB-TM
mice. Agonist-induced platelet aggregation and spreading on immobilized fibrinogen (Fg) were facilitated in PIRB-TM platelets. The rate
of clot retraction in platelet-rich plasma containing PIRB-TM platelets
was also increased. Characterization of signaling confirmed that PIRB
associated with Shp1 and Shp2 in platelets and the levels of tyrosine
phosphorylation of LAT, SLP-76 and PLCc2 were enhanced in PIRBTM platelets stimulated with CRP. The levels of phosphorylation of
FAK Y397 and integrin b3 Y784, but not b3 Y772, were also
enhanced in PIRB-TM platelet spread on Fg. Furthermore, the PIRB
(LILRB2) ligand angiopoietin-like-protein 2 (ANGPTL2) was highly
expressed and stored in platelet a-granules. Purified ANGPTL2 inhibited agonist-induced human platelet aggregation and spreading on
immobilized Fg.
Conclusion: The data presented here reveal that PIRB and its ligand
ANGPTL2 possess the antithrombotic function by suppressing GPVI
and integrin aIIbb3 mediated outside-in signaling.
Disclosure of Interest: None Declared.
PP15
Identification of the mechanosensory domain in the
platelet mechanosensor GPIb-IX complex
da
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Fan X, Shi P, Dai J and Liu J
Department of Biochemistry and Molecular Cell Biology,
Shanghai Key Laboratory of Tumor Microenvironment and
Inflammation, Shanghai Jiao Tong University School of Medicine,
Shanghai, China
R
PP13
Paired immunoglobin-like receptor b regulates platelet
activation
Zhang W1, Deng W2, Wang Y1, Zhou L2, Yang W3, Liang X2, Cho
S2, Kulman JD4, Zhang XF1 and Li R2
1
Lehigh University, Bethlehem 2Emory University, Atlanta;
3
University of Texas Health Science Center at Houston, Houston;
4
Biogen Idec, Cambridge, USA
Objectives: Elevated shear stress induces binding of von Willebrand
factor (VWF) to glycoprotein (GP)Ib-IX complex on the platelet surface and induces platelet activation. Although GPIb-IX complex has
long been recognized as the primary platelet mechanosensory receptor,
how it transmits the signal of shear-sensitive VWF binding into the cell
remains unclear. We seek to address this fundamental question.
Methods: The association of A1 domain of VWF (VWF-A1) with fulllength GPIb-IX complex has been characterized by single-molecule
force spectroscopy.
Results: With the GPIb-IX complex captured via a biotin group specifically placed at the junction of transmembrane and cytoplasmic
domain of GPIba, pulling on the N-terminal domain of GPIba using a
VWF-A1-coated bead resulted in a stretching or unfolding of a
sequence of about 50–60 residues before detachment of VWF-A1 from
GPIb-IX. The same unfolding events in GPIb-IX were observed when
the specific biotinylation site was moved to the C-terminal end of
GPIX cytoplasmic domain or pulling was performed using a bead
coated with monoclonal antibody WM23 that recognizes the macroglycopeptide region of GPIba. However, pulling on WM23-captured
GPIb-IX using a VWF-A1-coated bead did not produce unfolding
before the detachment. Additional mutagenesis analysis identified the
sequence undergoing unfolding is the juxtamembrane stalk region of
GPIba.
Conclusion: We have demonstrated that the engagement and pulling
of VWF-A1 induces unfolding of the juxtamembrane stalk region of
GPIba in the GPIb-IX complex. Such force-induced conformational
change in GPIb-IX underlies the molecular mechanism of platelet mechanosensing, and illustrates the interplay between blood and flow,
two of the three components of Virchow’s Triad, at an unprecedented
level. The identification of the GPIba stalk region as the mechanosen-
12/06/2014
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
ABSTRACTS
sory domain in GPIb-IX has significant implications for the pathogenesis, diagnosis and treatment of many GPIb-IX-related bleeding diseases.
Disclosure of Interest: None Declared.
PP16
Macrothrombocytopenia of cases with
mutations identified in the BRIDGE cohort
(MCF), and lysis onset time (LOT) were evaluated using rotation
thromboelastometry.
Results: Thrombocytopenia was characterized by reduction of a-Angle
and MCF and fibrinogen partly reversed these alterations. Further
increase of a-Angle and MCF was observed using fibrinogen combined with factor XIII or FEIBA. In the presence of tPA, fibrinogen
enhanced a-Angle while MCF was increased by either fibrinogen,
FXIII, FEIBA or TAFI. LOT values were prolonged by TAFI and to
less extent by combining FXIII with FEIBA. Dilution of thrombocytopenia blood was followed by further reduction of clot formation and
increased susceptibility to tPA. Clot strength was enhanced by fibrinogen, TAFI and FEIBA and fibrinolysis was inhibited by TAFI and
FXIII. Joining fibrinogen with FXIII or FEIBA further increased clot
strength. Increasing fibrinogen concentration from 300 to 600 mg dL–
1
separately or together with other agents did not further improve clot
quality and stability.
Conclusion: The results of this study allow suggesting that simultaneous stimulation of clot formation by fibrinogen and/or FEIBA and
inhibition of fibrinolysis by TAFI or FXIII may be beneficial for treatment of severe thrombocytopenia patients especially complicated with
hemodilution following introduction of liquids to compensate massive
blood loss.
Disclosure of Interest: None Declared.
TUBB1
R
Nurden P on behalf of the BRIDGE Bleeding and Platelet Disorder
(BPD) programme
PTIB, Institut Hospitalo-Universitaire LIRYC, Pessac, France
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PP18
Evaluation of light transmission aggregometry (LTA)
procedures and variations between laboratories in the
Netherlands
da
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Henskens Y1, Verhezen P1, van Wijk E2, Hudig C3, Stroobants A4
and van de Kerkhof D5
1
Central Diagnostic Laboratory, Maastricht University Medical
Centre, Maastricht; 2Clinical Laboratory, St. Elisabeth Ziekenhuis,
Tilburg; 3Lab West, Hagaziekenhuis, The Hague; 4LAKC,
Academic Medical Centre, Amsterdam; 5Clinical Laboratory,
Catharina Hospital, Eindhoven, Netherlands
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Objectives: Tubulin ß1, the major component of microtubules, is
important for proplatelet formation and maintaining platelet shape.
Mutations in the TUBB1 gene have been reported to give rise to macrothrombocytopenia. Here we focus on TUBB1 mutations after classical causes (GP1BA/1BB/9, MYH9) have been excluded.
Methods: The BRIDGE program has enrolled 716 unexplained BPD
cases from 10 centers in Europe and the USA. Whole exome sequencing (WES) has been performed on 480 probands and coding variants
identified that are absent from the 1000 Genomes, UK10K and ESP
reference cohorts.
Results: A look-up was performed for TUBB1 and 11 probands were
identified with coding variants with consequences (amino acid
replacement, premature stop, splice site), which were absent from
the control cohorts. One of the 11 variants was considered a Pertinent Finding, because the same F206S variant has recently been
reported in a similar case of macrothrombocytopenia by Kunishima
et al (EJH, 2013). The BRIDGE case with the F206S mutation had
a platelet count of 114 9 109/L and a MPV of 13.5 fl and macrothrombocytopenia was confirmed by light and electron microscopy.
The latter showed enlarged round platelets, heterogeneous distribution of granules and characteristic elongated abnormal large channels formed from the internal membranes. Four of the 10 TUBB1
variants were Possible Pertinent Findings because the clinical phenotype was compatible, but further studies are required to substantiate
these findings. The remaining 6 cases concern Variants of Unknown
Clinical Significance.
Conclusion: WES reveals the entire variation spectrum in the coding
fraction of the vast majority of genes. For BPDs with dominant inheritance, as is the case for TUBB1 evidence of causality of a variant can
only be obtained by co-seggregation studies in informative pedigrees
complemented by extensive clinical phenotype analysis, which ideally
should include EM analysis.
Disclosure of Interest: None Declared.
75
PP17
In vitro effect of procoagulant and antifibrinolytic
agents on blood clot quality and stability in a model of
severe thrombocytopenia
Shenkman B1, Einav Y2, Livnat T1 and Martinowitz U1
1
National Hemophilia Center, Chaim Sheba Medical Center,
Tel-Hashomer; 2Holon Institute of Technology, Holon, Israel
Objectives: The treatment options in severe thrombocytopenia patients
with bleedings are limited. The aim of this study was to improve blood
clotting and stability in reconstituted thrombocytopenia blood.
Methods: Thrombocytopenia [(16 4) 9 106 mL–1] was created by
differential centrifugation of normal blood followed by reconstitution
of whole blood. Blood was subjected to clotting by CaCl2 and tissue
factor, and to fibrinolysis by tissue plasminogen activator (tPA). To
mimic the situation that may occur in patients subjected to massive
transfusion of plasma substitutes, blood was diluted by 40% of TRIS/
saline buffer. Clotting time (CT), a-Angle, maximum clot firmness
Objectives: Light Transmission Aggregometry (LTA) is considered the
golden standard method for evaluation of platelet function. The SSC
Platelet Physiology published a guideline (JTH 2013) in order to standardize LTA. The society of hematological laboratories in The Netherlands (VHL) has set a goal for 2015, namely to reduce variances in
LTA results by investigating the present differences between LTA
methods and by stimulating all laboratories to uniform their methods.
Methods: We undertook a national survey of platelet function assays
and LTA practices, based on the advices in the SSC guideline. Secondly, we asked 9 LTA users to send us a minimum of 10 results of different healthy volunteers using their current method for all
recommended agonists.
Results: In total 47/70 hospital laboratories in The Netherlands
responded to our survey. LTA was used by 25 laboratories. A selection
of results of the survey is summarized (table):
Adherence to SSC guideline
(n = 25) (# is the recommendation)
Yes
No
Resting of the patient
Fasting of the patient (only light meal)
Use of discard
Sample resting before centrifugation
Centrifugal force 200 g
Centrifugal time 10 min
Reference values
Normal subjects
Adjustment
8#
3#
15#
18#
4#
12#
6#
11#
17
17
22
10
7
21
13
19
14
8#
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
12/06/2014
76
ABSTRACTS
The figure shows an example of one of the agonists used by the laboratories in different concentrations. De boxed results represent the SSC
recommended starting concentration for epinephrine. Differences in
results of healthy volunteers between laboratories were most prominent using low concentrations of agonists or when using collagen. At
the recommended starting concentrations, results of healthy volunteers
were within acceptable ranges of variance.
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PP19
Therapeutic platelet-rich plasma: characterization of
platelet activation and procoagulant markers, growth
factor release, and cell proliferation following pulse
electric field stimulation
Co
pi
Frelinger AL III1,2, Torres AS3, Caiafa A3, Morton C3, Berny-Lang
MA1, Gerrits AJ1,2, Carmichael SL1, Neculaes VB3 and Michelson
AD1,4
1
Hematology/Oncology, Center for Platelet Research Studies,
Boston Children’s Hospital; 2Pediatrics, Harvard Medical School,
Boston; 3GE Global Research Center, Niskayuna; 4Pediatrics and
Medicine, Harvard Medical School, Boston, USA
Objectives: To compare the ability of pulsed electric field (PEF),
bovine thrombin (BT), and thrombin receptor activating peptide
(TRAP) to activate clinically relevant preparations of human plateletrich plasma (PRP), release growth factors and induce cell proliferation. Therapeutic use of activated PRP has yielded mixed results in
clinical studies. PEF stimulation may provide more consistent platelet
activation and avoid complications associated with the addition of BT.
Methods: Blood from healthy aspirin- and NSAID-free subjects was
used to make PRP in the Harvest SmartPreP2 System. PRP treated
with vehicle, PEF, BT, TRAP, and Triton X-100 was analyzed by flow
cytometry for platelet surface P-selectin, glycoprotein (GP) Ib and
GPIIb, and, as markers of platelet procoagulant activity, platelet surface phosphatidylserine expression (by annexin-V) and platelet-derived
microparticles (PDMP). Platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), basic fibroblast growth factor, platelet factor 4 (PF-4), angiostatin, and endostatin were
measured by ELISA. Finally, supernatants were tested for their ability
to stimulate proliferation of cells in culture.
Results: Both PEF and BT significantly increased the% P-selectin positive platelets (BT more than PEF) and the number of annexin V-posi-
Conclusion: PEF treatment of fresh human PRP results in greater cell
proliferation, similar levels of procoagulant microparticles and growth
factor release, and lower% P-selectin positive platelets than BT. These
results, together with PEF’s inherent advantages, suggest PEF may be
a superior alternative to BT activation of PRP for therapeutic applications.
Disclosure of Interest: A. Frelinger III has grant/research support
from: GE Global Research Center, A. Torres is an employee of: GE
Global Research Center, A. Caiafa is an employee of: GE Global
Research Center, C. Morton is an employee of: GE Global Research
Center, M. Berny-Lang: none declared, A. Gerrits: none declared, S.
Carmichael: none declared, V. Neculaes is an employee of: GE Global
Research Center, A. Michelson has grant/research support from: GE
Global Research Center
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Conclusion: The LTA using laboratories in The Netherlands show discrepancies after comparing their current methods with the SSC guideline. Differences in results of healthy volunteers between 9 laboratories
with different methods are lowest at the recommended agonist concentrations. Adhering to the SSC guideline can minimize variation.
Disclosure of Interest: None Declared.
tive PDMP vs. vehicle. Platelet activation by PEF resulted in
significant release of PDGF, VEGF, and PF4, with levels similar to
those induced by BT, TRAP or Triton X-100. Supernatant from PEFtreated platelets, but not that from BT or TRAP-treated platelets, significantly increased cell proliferation compared to control plasma.
PP20
Systematic comparative analysis of bleeding phenotype in PT-VWD and type 2B VWD using an electronic
bleeding questionnaire
Kaur H1, Ozelo M2, Scovil S3, James P4 and Othman M1,5
1
Biomedical and Molecular Sciences, Queens University,
Kingston, Canada; 2University of Campinas, Campinas, Brazil;
3
New Atom Technologies Inc.; 4Medicine, Queens University;
5
Health Sciences, St Lawrence College, Kingston, Canada
Objectives: PT-VWD is a rare bleeding disorder that represents a commonly missed diagnosis among 2B VWD patients. Both diseases result
from gain-of-function mutations within GP1BA and VWF-A1 genes
respectively. Correct diagnosis’differentiation remains a challenge.
This study aims to investigate the utility of an electronic bleeding questionnaire (eBQ) in assessing the bleeding phenotype in PT-VWD and
type 2B VWD and to systematically compare the two disorders.
Methods: A call for participation in the study was made on ISTH website and participation of physicians and potential collaborators was
invited. A retrospective comparative analysis of the clinical bleeding
and laboratory phenotype of 25 patients was performed. 13 PT-VWD
[average age = 38 years, range: 4–67; 2M, 11F] representing 6 mutations together with 12 type 2B VWD patients [average age = 44 years,
range: 18–63; 6F, 6M] representing 4 mutations. An electronic version
of the condensed MCMDM-1VWD bleeding questionnaire that generated an automatically calculated bleeding score (BS) was used. BS,
platelet count and size and VWF levels were analyzed.
Results: Patients’ BS were variable within and between the two disorders. In PT-VWD, BS was (median = 4, range: 0–17) and in type 2B
VWD was (median = 12, range: 3–22). Variability was seen in patients
with similar mutations and appeared independent of patients’ age. BS
was significantly higher and platelet count was significantly lower in
type 2B VWD compared to that of PT-VWD. There was a significant
12/06/2014
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
ABSTRACTS
reduction in VWF:RCo/VWF:Ag in PT-VWD compared to type 2B
VWD. The sensitivity and specificity of the eBQ in diagnosing a bleeding condition were 76% and 100% respectively with a higher sensitivity in type 2B VWD.
Conclusion: Objective analysis of bleeding symptoms further the
understanding of the clinical phenotype of two closely similar bleeding
disorders, which may facilitate better management. Larger international prospective studies are warranted to further evaluate the utility
of the eBQ in other RBDs.
Disclosure of Interest: None Declared.
Methods: Using whole blood collected in ACD-B, platelet GPIa, Ib-a,
IIb, IIIa, IV, VI and IX expression were measured by FL-F, and platelet GPIa, IIIa and Ib-a copy numbers were measured by FL-C (BioCytex, Marseille, France). We first optimized pre-analytical, analytical
and post-analytical procedures including sample stability, antibody
titration and platelet counts. A total of 112 healthy donors (57% male,
age range 18–70 years) were recruited to establish upper 95% RR of
GP expression levels. Donors’ platelet indices were also recorded.
Results: Platelet GP measurements were stable in ACD-B whole blood
samples stored at room temperature for up to 3 days. The intra- and
inter-precision of both methods were approximately 10% when saturating conjugated antibodies and a fixed number of platelets were
used. The upper 95% RR of percentages of normal donor median GP
expression were as follows: GPIa, ≥ 62.7%; GPIb-a, ≥ 82.5%; GPIIb,
≥ 82.3%; GPIIIa, ≥ 81.9%; GPIV, ≥ 52.4%; GPVI (n = 70), ≥ 77.6%
and GPIX, ≥ 82.8%. The RRs for GP copy number/platelet are as follows: GPIa, ≥ 2,094; GPIb-a, ≥ 28,035; GPIIIa, ≥ 52,234; Among
GPs, only GPIIb and IIIa have a weak to fair association with MPV
and IPF (q between 0.24 and 0.45).
Conclusion: The performance characteristics of both platelet FL-F and
FL-C flow cytometry assays were acceptable.
Disclosure of Interest: None Declared.
PP21
Development of a laboratory model of thrombocytopenia in human whole blood
Bercovitz RS1, Brenner MK2 and Newman DK2
1
Medical Sciences Institute; 2Blood Research Institute,
BloodCenter of Wisconsin, Milwaukee, USA
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PP23
Analyzing platelet thrombus formation under arterial
blood flow conditions
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Ito T1, Nagasato T1,2, Oda Y1, Hosokawa K1,2 and Maruyama I1
1
Systems Biology in Thromboregulation, Kagoshima University
Graduate School of Medical And Dental Sciences, Kagoshima;
2
Research Institute, Fujimori Kogyo Co., Yokohama, Japan
Objectives: Thrombosis is a leading cause of death and disability
worldwide. Antiplatelet therapy is currently recommended to reduce
the risk of recurrent arterial thrombosis. However, the response to antiplatelet therapy is variable, and non-responders are associated with
an increased risk of adverse events. Several laboratory tests for platelet
function have recently become clinically available. However, it is still
controversial whether these tests are useful to optimize antiplatelet
therapy and to improve clinical outcomes. This indicates that classical
platelet function tests may not reflect thrombogenicity in vivo due in
part to the absence of blood flow and the addition of a single exogenous agonist at a supra-physiological concentration. In this study we
analyzed platelet functions under arterial blood flow conditions using
microchip-based total thrombus-formation analysis system (T-TAS).
Methods: Whole blood anticoagulated with hirudin was perfused over
a microchip coated with collagen, and processes of thrombus formation inside the microchip were analyzed by a video microscope and a
flow pressure sensor.
Results: Occlusion of microchip capillaries tends to be shortened in
cases of people with coronary risk factors. Occlusion was prevented in
about 75% patients treated with aspirin.
Conclusion: T-TAS may be useful in evaluating antiplatelet therapy. It
may also be useful in evaluating thrombotic tendency.
Disclosure of Interest: T. Ito has grant/research support from: This
study was supported in part by Fujimori Kogyo, the manufacturer of
the T-TAS., is an employee of: I hold an endowed faculty position in
thrombosis research and have received funds from Medipolis Medical
Research Institute, Shin Nippon Biomedical Laboratories, Asahi Kasei Pharma, and Asahi Kasei Medical., T. Nagasato is an employee of:
I am an employee of Fujimori Kogyo, the manufacturer of the TTAS., Y. Oda: none declared, K. Hosokawa is an employee of: I am
an employee of Fujimori Kogyo, the manufacturer of the T-TAS., I.
Maruyama is an employee of: I hold an endowed faculty position in
thrombosis research and have received funds from Medipolis Medical
Research Institute, Shin Nippon Biomedical Laboratories, Asahi Kasei Pharma, and Asahi Kasei Medical.
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Objectives: Thrombocytopenia (TP) can place patients at risk for lifethreatening bleeding, though it is difficult to identify thrombocytopenic patients who will experience bleeding. Current clinical tests cannot
assess platelet function, or the ability of different treatment interventions to correct hemostatic deficiency, in the setting of TP. Models of
TP that allow characterization of platelet function and evaluation of
treatment efficacy are therefore needed.
Methods: Whole blood (WB) collected from healthy volunteers was
subjected to a side-by-side comparison of two methods for preparation
of thrombocytopenic samples. For the Centrifugation Method (Larsen, et al., Ann Hematol 85:217, 2007) WB was centrifuged (15 m,
100 g) to separate red blood cells (RBCs) from platelet rich plasma
(PRP) and PRP was centrifuged (25 m, 3300 g) to produce platelet
poor plasma (PPP). PPP was mixed with RBCs to produce WB with a
reduced platelet count; the process was repeated up to 5 times until a
platelet count of < 20,000 lL–1 was achieved. For the Dilution
Method, WB was centrifuged (8 m, 300 g) to prepare RBCs and PRP
and PRP was centrifuged (10 m, 3800 g) to prepare PPP. WB, RBCs,
and PPP were mixed to achieve a platelet count < 20,000 lL–1.
Levels of expression of P-selectin (CD62P), GPIb, and GPVI in resting
and activated platelets were evaluated by flow cytometry.
The two methods were compared using the Wilcoxon-rank-sum test.
Results: The Dilution Method was superior to the Centrifugation
Method in that it took less time to achieve a platelet count < 20,000
lL–1, generated resting platelets with a lower level of CD62P and
higher level of GPVI expression, and produced platelets with a higher
fold change in CD62P upon activation with thrombin receptor-activating peptide (TRAP).
Conclusion: We will use the Dilution Method to compare the function
of platelets from healthy donors with those from thrombocytopenic
patients and to evaluate the efficacies of myriad interventions designed
to normalize hemostasis in thrombocytopenic samples.
Disclosure of Interest: None Declared.
PP22
Validation of two flow cytometric methods to measure
platelet surface glycoprotein proteins
Miller RS1, Lingineni R2, Bryant SC2, Heit JA1 and Chen D1
1
Hematopathology; 2Biomedical Statistics and Informatics, Mayo
Clinic, Rochester, USA
Objectives: While platelet glycoprotein (GP) expression can be measured by fluorescent-conjugated GP-specific antibodies (FL-F) or by a
copy number (FL-C) method, neither method has been validated and
normal ranges of GPs are unavailable. Our goals were to validate both
platelet GP flow cytometry methods and establish their reference
ranges (RR).
77
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
12/06/2014
78
ABSTRACTS
PP24
Rapid platelet concentrate delivery by pneumatic tube
system and pressured transfusion is a feasible option
Results: When platelets were stimulated with Cvx, a GPVI-specific
agonist, amp significantly decreased (P < 0.01). In addition, the
amount of generated intracellular ROS significantly increased
(P < 0.01). In the presence of the antioxidant N-acetyl-L-cysteine
(NAC, 10 mM), the generation of intracellular ROS was suppressed
and amp decreased, similarly to the case without NAC. When platelets
were irradiated with UV, amp decreased depending on the dose of UV
radiation. The amount of generated intracellular ROS increased with
the dose of UV radiation. However, when the generation of intracellular ROS was suppressed in the presence of NAC, the decrease in amp
was also suppressed.
Conclusion: The amount of intracellular ROS did not correlate with
the platelet shape change via GPVI signaling but correlated with the
platelet shape change induced by UV radiation. These findings suggest
that platelet shape change is controlled by at least two mechanisms,
namely, intracellular ROS-dependent and ROS-independent ones.
Disclosure of Interest: None Declared.
Kicken CH1, Lance MD1, van Egmond LT1, van Oerle R2 and
Henskens Y2
1
Anesthesiology and Pain Therapy; 2Central Diagnostic
Laboratory, Maastricht University Medical Centre, Maastricht,
Netherlands
Predictive Hemostatic Variables
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PHV01
D-dimer for the diagnosis of symptomatic upper
extremity deep vein thrombosis
or
CD
Sartori M1, Migliaccio L2, Favaretto E1, Legnani C1, Palareti G1
and Cosmi B1
1
Angiology and Blood Coagulation; 2Angiology and Bllood
Coagulation, S.Orsola-Malpighi University Hospital, Bologna, Italy
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PP25
Role of intracellular reactive oxygen species generation
in platelet shape change
Terada C, Satake M and Tadokoro K
Central Blood Institute, Japanese Red Cross Society, Tokyo,
Japan
Objectives: Recently, platelets have been found to generate intracellular reactive oxygen species (ROS), which control platelet activation
and thrombus formation. However, the effects of ROS generation on
platelet functions have not been fully clarified. In this study, we studied the role of intracellular ROS generation in platelet shape change.
Methods: An apheresis platelet concentrate was washed and suspended
in Tyrode’s HEPES buffer (pH 7.4) to be used as a sample. Changes in
platelet shape were monitored in real time during sample stimulation
with convulxin (Cvx, 0.1 lg mL–1) or sample irradiation with ultraviolet (UV) with a wavelength of 300 nm (0.1 J/cm2). Signal amplitude
(amp) obtained by frequency analysis of the change in the intensity of
90° scattered light was used as an index of platelet shape change and
thus indicated the percentage of discoid platelets. The amount of generated intracellular ROS was determined using CM-H2DCFDA fluorescence.
Objectives: To validate the diagnostic accuracy of D-dimer testing forsymptomatic upper extremity DVT (UEDVT)
Methods: A management study with data collection from 1 Jan 2012
to 30 Jun 2013, including 235 (female/male 149/86) consecutive
patients who were referred by the emergency department or by a primary care physician to our ultrasound laboratories. All patients (age
58.9 18.8 IQR 44.7–74.5 year) underwent an history-taking and a
physical examination, D-dimer testing (NV < 500 ng mL–1), and a
comprehensive real-time B-mode and color Doppler ultrasonography
examination of the upper limb by a vascular medicine physician. In
case of technical problems or anatomical barriers, the ultrasound was
repeated after 3–5 days. All the patients were followed up for
3 months to document the occurrence of symptomatic UEDVT or pulmonary embolism.
Results: In 24 (10.2%) and 36 (15.3%) patients a diagnosis of UEDVT
or superficial vein thrombosis was made, respectively. Patients with
superficial vein thrombosis were excluded from analysis. of the 175
patients with a normal diagnostic work-up, one developed UEDVT
during follow-up. The D-dimer had a sensitivity of 92% (CI 95%: 81–
98%), a specificity of 61% (CI 95%: 54–68%) with a negative predictive value of 98% (CI 95%: 95–99%).
Conclusion: D-dimer has a high negative predictive value for excluding
the UEDVT in symptomatic outpatients and can be used in the diagnostic work-up for suspected UEDVT.
Disclosure of Interest: None Declared.
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Objectives: Transfusion guidelines do not recommend rapid platelet
concentrate (PC) transport by pneumatic tube system (PTS) followed
by pressured or pressured and warmed transfusion. We investigated
whether this fast delivery route reduces platelet function.
Methods: After ethical board approval, 10 PCs underwent single PTS
transport and were subsequently pressured to 300 mmHg or pressured
to 300 mmHg and warmed in a RangerTM blood warmer. These conditions were tested on day 2 and 7 after donation. Platelet function was
measured with light transmission aggregometry (LTA) (agonists:
10 lmol L–1 ADP, 1 mmol L–1 Na-arachidonic acid, 4 lg mL–1 collagen and 30 lmol L–1 TRAP) and multiple electrode aggregometry
(MEA) (agonists: 6.5 lmol L–1 ADPtest, 1 mmol L–1 ASPItest, 3.2 lg
mL–1 COLtest and 32 lmol L–1 TRAPtest). Data are expressed as
median and were analyzed with one-sided Wilcoxon signed ranks test,
P < 0.05 was considered statistically significant.
Results: Single PTS transport reduces ADP response markedly in fresh
PCs (MEA 53U to 16U, P = 0.001; LTA 45.5% to 28.5%, P = 0.002).
Seven day storage has a greater impact on platelet response to ADP
(MEA 53U to 1U; LTA 45.5% to 20.1%, P = 0.001 for both) and collagen (MEA 98 to 43U; LTA 81.4% to 42.4%, P = 0.001 for both).
Additional pressure slightly decreased LTA ADP (20.1% to 18.9%,
P = 0.003) and LTA collagen (42.4% to 28.3%, P = 0.001) in stored
PCs. Additional pressure combined with warming slightly decreased
MEA ASPI (92U to 83U, P = 0.013) and MEA collagen (98U to 80U,
P = 0.003) in fresh PCs and MEA collagen (43U to 33U, P = 0.003) in
stored PCs. Other platelet responses were not affected.
Conclusion: PTS reduces ADP response, however storage affects ADP
and collagen response even more. Subsequent application of pressure
or pressure and warming does not relevantly reduce platelet function
in fresh and stored PCs. For patients who benefit from rapid PC delivery, e.g. in case of emergency, transport by PTS and pressured plus
warmed transfusion may be feasible.
Disclosure of Interest: None Declared.
12/06/2014
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
ABSTRACTS
PHV02
Hypoxia induces a prothrombotic state: results from
the ‘red meets white’ study
were: 1.incidents of BT, 2.the mean number of transfused RBCu, 3.BT
≥ 2 RBCu, 3.the mean age of transfused RBCu, 4.standard deviations
(SD) of the age of RBCu. The statistical analysis composed of chi2,
Cox regression. The age/SD of RBCu was defined as the number of
days between the expiratory and transfusion date and difference in the
age of RBCu transfused in each pt. BC was defined as VARC major
and life-threatening/disabling events ≥ 30 days after TAVI.
Results: We included 181 TAVIpts, age 55–91 years (80.19 7.36);
16pts were excluded (14 early deaths; 2 lost in follow-up). Follow-up
included 165 (91.16%)pts. We noted 27 (16.36%) late deaths. Analyzed parameters and their impact on prognosis are presented in
Table.
Ninivaggi M1, de Laat M1, Linssen-Thuis I1, Lanc
e M2, Konings
J1, Peters T1, Bloemen S1, Govers-Riemslag J3, Lindhout T1,
Krishnamoorthy G1, Hemker C1 and de Laat B1
1
Synapse bv, Maastricht University Medical Center;
2
Anesthesiology, Maastricht University Medical Center;
3
Biochemistry, Maastricht University Medical Center, Maastricht,
Netherlands
Late deaths (> 30 days)
Yes (n = 27)
No (n = 138)
P
HR [95% CI]; P
BT no.(%)
RBCu mean SD
(from-to)
BT ≥ 2RBCu no.(%)
Age of RBCu
(days) mean SD
(from-to)
SD of age RBCu
(days) mean SD
(from-to)
18 (66.66%)
3.076 4.33
(1–16)
16 (59.25%)
22.305 6.86
(4–31)
73 (52.89%)
1.514 1.88
(1–7)
59 (42.75%)
22.83 7.19
(4–35)
0.15
0.02
1.614 [0.681–3.825]; 0.27
1.156 [1.014–1.318]; 0.03
0.1
0.4
1.851 [0.805–4.257]; 0.15
1.022 [0.936–1.116]; 0.63
3.13 3.69
(0.57–13.67)
5.52 4.6
(0–16.46)
0.03
1.165 [1.00–1.356]; 0.048
R
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Conclusion: 1.Higher number of transfused RBCu after TAVI worsens
long-term prognosis. 2.Large discrepancy in the age of RBCu transfused early after TAVI is a risk factor of late mortality.
Disclosure of Interest: None Declared.
PHV04
Neutrophil extracellular trap formation (NETs) in
diabetes: correlation with inflammatory markers
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Objectives: Patients suffering from hypoxia are known for their prothrombotic phenotype, although the mechanism behind this phenomenon is not clear. Studies performed on altitude investigating the effect
of hypoxia related to differences in hypobaric pressure, reveal opposite
effects. We performed a study in the Swiss Alps in an attempt to get a
better comprehension of the coagulation state when suffering from
hypoxia.
Methods: Two groups of 15 consenting, healthy individuals were
formed that suffered from hypoxia due to an increase in altitude up to
3900 m. Group A ascended actively by climbing and group B was
transported passively to increasing altitudes. Both groups were
assayed for heart rate, oxygen saturation levels, blood pressure, factor
(F)VIII and Von Willebrand Factor (VWF) levels and thrombin generating (TG) capacity in plasma and whole blood. All TG parameters
had a coefficient of variation < 10%.
Results: Blood pressure was not affected by changes in altitude. Heart
rate increased and oxygen saturation levels decreased with increasing
altitudes for both groups. FVIII and VWF levels increased significantly only in the active group. TG in plasma showed an increase in
the active group, in contrast to the passive group. The increase in the
active group could be explained by the increase in VWF/FVIII levels.
TG in whole blood revealed that in both groups, hypoxia was associated with a faster and higher TG. This would indicate that the cellular
portion of the blood is at least partially responsible for the association
between hypoxia and venous thrombosis.
Conclusion: Whole blood TG has the advantage that it includes all the
blood cells and therefore is one step closer to physiology compared to
plasma. By applying TG in whole blood we found strong indications
that hypoxia induces a prothrombotic state. The results between whole
blood and plasma-based TG differ, suggesting that the cellular part of
the blood is involved in the prothrombotic phenotype.
Disclosure of Interest: None Declared.
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PHV03
The impact of blood transfusions and the age of transfused red blood cells on long-term prognosis after
transcatheter aortic valve implantation (TAVI)
ska-Jelonkiewicz K1, Witkowski A2, Daz browski M3,
Czerwin
ska J1
Hryniewiecki T4, Demkow M5, Piotrowski W5 and Stez pin
1
2
Department of Intensive Cardiac Care; Department of
Interventional Cardiology and Angiology; 3Department of
Interventional Cardiology and Angiology; 4Department of
Acquired Valve Disease; 5Department of Coronary Artery Disease
and Structural Heart Diseases, Institute of Cardiology, Warsaw,
Poland
Objectives: TAVI is a rescue procedure dedicated to high risk, elderly
patients with severe aortic stenosis. Bleeding complications (BC) and
connected with them blood transfusions (BT) are the most frequent
early complications. The aim of the study was to assess the impact of
early BT and the age of transfused red blood cells units (RCBu) on
long-term prognosis after TAVI.
Methods: The single-center study conducted between 2009 and 2014.
Follow-up 1mth-5.8 year (21.97 14.62mths). Analyzed parameters
Carestia A1, Frechtel G2, Cerrone G2, Gonzalez C3, Schattner M1
and Casais P1
1
Laboratory of Experimental Thrombosis, Institute of
Experimental Medicine-CONICET-National Academy of
Medicine; 2Department of Microbiology, Immunology and
Biotechnology, School of Pharmacy and Biochemistry, University
of Buenos Aires (UBA); 3Department of Pharmacology, School of
Medicine, University of Buenos Aires (UBA), Buenos Aires,
Argentina
Objectives: The release of DNA-derived neutrophil extracellular
traps (NETs) is a mechanism by which these cells kill bacteria and
exert proinflammatory and prothrombotic activities. Diabetes is
characterized by a chronic inflammatory state, endothelial dysfunction, an increased risk of infections rates and early cardiovascular
disease.
The aim of this study was to determine whether the ability of neutrophils to form DNA traps differs in diabetic patients from healthy
controls, and the relationship between the presence of NETs with
inflammatory biomarkers.
Methods: Recently diagnosed diabetic patients (13) and healthy blood
donors (13) were included. NET formation was studied by microscopy
and using Syber Gold. Plasma levels of nucleosomes and von Willebrand factor (vWF) were measured by ELISA and P-selectin expression by cytometry.
Results: Microscopy studies showed that while neutrophils from
healthy donors did not form NETs without stimulation, 9 out of 13
neutrophil samples from diabetic patients exhibited spontaneous NET
formation. Also, analysis of free DNA revealed increased basal levels
in patient’s samples (control:0.3 0.02 vs. patients:0.5 0.1 lg mL–1,
P < 0.05). While TNF stimulation of control neutrophils resulted in
DNA release (0.5 0.1 lg mL–1), patient’s neutrophils were not
responsive (0.5 0.1 lg mL–1 vs. 0.5 0.06 lg mL–1).
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
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ABSTRACTS
PHV06
Can a multidisciplinary cardiac surgery blood conservation program succeed? A single center, 6 year, 687
patient retrospective blood/blood product utilization
review
Compared to healthy donors, nucleosome plasma levels were increased
in diabetic patients (control:0.06 0.09 vs. patients:0.44 0.2,
P < 0.05 vs. control). The expression of P-selectin (% of positive cells
control:11 2 vs. patients:8 2), and vWF levels (control:5.2 0.8
vs. patients:4.7 0.6;lg mL–1) were similar in both populations.
Conclusion: This pilot study shows that, compared to healthy controls,
patients with diabetes have spontaneous NET formation with statistically significantly increased nucleosomes and DNA plasma levels.
Other inflammation markers did not differ between the two populations. The clinical significance of these observations requires further
investigation.
Disclosure of Interest: None Declared.
Sharma AD, Behrend D, Seccombe JF, Al-Achi A, Nissen D,
Hummel R and Preston M
St Vincents Hospital Heart Center, Green Bay, USA
PHV05
Poor response to thienopyridines and thrombotic recurrent events in patients with ischemic heart disease
CD
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Objectives: Thienopyridines (clopidogrel and prasugrel) are prodrugs
that irreversibly bind to P2Y12 ADP receptor and inhibit platelet
aggregation. Poor response to thienopyridines is a multifactorial phenomenon characterized by platelet hyperactivity, identified by ADPinduced platelet aggregation. Thienopyridines poor response has been
related with thrombotic recurrent events.
The aim of this study was to identify the relationship between thienopyridines poor response and thrombotic recurrent events in patients
with ischemic heart disease.
Methods: A total of 400 patients who underwent a percutaneous coronary intervention were included. To establish the thienopyridines poor
responders we performed a 10 lmol L–1 ADP-induced platelet aggregation test. The patients were follow-up during 2 years to determinate
recurrent thrombotic events, considered as cardiac death, stroke, myocardial infarction, stent thrombosis and nuclear medicine imagingdefined silent ischemia.
Results: A hundred and one patients were lost during follow up. We
found that 25% of the patients showed thienopyridines poor response
and 13.4% developed a thrombotic recurrent event. The 20% of the
thrombotic events corresponded to silent ischemia which was directly
associated with clopidogrel poor response (Fisher exact test
P = 0.031). To provide a relative risk of a thrombotic recurrent event
we performed a Cox proportional-hazard model, results showed that
clopidogrel poor response and hypertension had a risk of 11 and 35
(P = 0.02 and P = 0.01) respectively.
Conclusion: Clopidogrel poor response is related to thrombotic events
especially to silent ischemia that points out to the extension of the
myocardial tissue damage, this clinical information can lead to an
appropriate forward treatment. We suggest that nuclear medicine
imagine-defined silent ischemia could be considered as an important
study variable for further studies.
Project supported by DGAPA, PAPIIT 221112.
Disclosure of Interest: None Declared.
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Calderon-Cruz B1, Elizalde-Hernandez PD2, Medina-Leyte DJ1,
~a-Duque MA3, Vargas-Alarcon G4,
Sobrevilla-Zarazua A1, Pen
4
~ a-Diaz A1,4
Fragoso JM , Martinez-Rios MA5 and de la Pen
1
2
Pharmacology, UNAM; General Hospital of Ticoman;
3
Hemodynamic; 4Molecular Biology; 5National Institute of
Cardiology ‘Ignacio Chavez’, Mexico, Mexico
Objectives: Cardiac surgery consumes 20% of the national blood supply. After implementing blood conservation measures in 2009, we
compared blood/blood product utilization in 2013–2009 to evaluate
the success of our program.
Methods: After IRB approval, we retrospectively collected blood/
blood product utilization for 657 patients that underwent CABG surgery from 2008–2013. Chi-square test for independent groups was used
to compare groups. The same test was used as a post-hoc test to compare groups pairwise along with Bonferroni correction to maintain
Type I error rate at 5% per comparison. A P value of < 5% was considered significance. Steps incorporated into the blood conservation
program included, A) Education of the clinical team about the adverse
aspects of blood transfusions. B) Perfusionists avoided hemodilution
associated hypo coagulation by utilizing micro circuits, priming the
CPB circuit with colloid, utilizing retrograde autologous priming
(RAP), modified ultrafiltration, and blood salvaging techniques. C)
Along with clinical judgement, utilization of post CPB TEGTM algorithm to anticipate and administer blood products, accepting of lower
platelet counts (< 100 9 109) provided function was adequate, and
correction of acidosis and hypothermia.
Results: In contrast to 2009, in 2013, A) Operative/post operative
patient percent blood/blood product use decreased from 47.5% to
22.7% (P < 0.05), and 55.4% to 42.7% (P > 0.05) respectively. B)
Total intra + post operative patient percent blood/blood product use
decreased from 63.1% to 50.4% (P > 0.05). C) Percent patients receiving 4+ RBC decreased from 25.4% to 13.6% (P < 0.05), 1+ FFP
decreased from 38.3% to 9.7% (P < 0.002), 1+ platelet transfusions
decreased from 37.5% to 20.4% (P < 0.05), and 1+ cryoprecipitate
decreased from 22.5% to 17.5% (P > 0.05).
Conclusion: After implementation, our multidisciplinary cardiac surgery blood conservation program succeeded in drastically reducing
percent of patients that received 4+ RBC, platelets, and FFP transfusions.
Disclosure of Interest: None Declared.
12/06/2014
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
ABSTRACTS
81
PHV07
A novel flow chamber system for modeling human
venous thrombosis
Sugita N1, Hirakata H2, Inoue K3, Tatsumi K4 and Murai T1
Psychiatry, Kyoto University/Japan; 2Anesthesiology;
3
Emergency Medicine, Kyoto Medical Center; 4Mechanical
Engineering and Science, Kyoto University/Japan, Kyoto City,
Japan
1
R
Conclusion: The process of venous thrombus formation looks complex
but we found there were some certain rules in it. Thrombi did not form
constantly but grew rapidly at a time trapping many red blood cells.
Thrombi did not always keep growing; some moved downstream
slowly and some were broken to drift away. Our flow chamber system
is useful for visualizing and quantifying thrombus formation, which
will be helpful for understanding the mechanism of venous thromboembolism.
Disclosure of Interest: None Declared.
or
CD
PHV08
Thrombin generation is a predictor of stroke in patients
with non valvular atrial fibrillation
da
p
Bagot C1, Leishman E1, Lowe G2 and Tait C1
Haematology, Glasgow Royal Infirmary; 2Institute of
Cardiovascular and Medical Sciences, University of Glasgow,
Glasgow, UK
1
Co
pi
aa
ut
or
iza
Objectives: Thromboembolism is a major risk for sudden death. Prevention of it is hard because the underlying mechanism is unknown.
So, we made a flow chamber system to visualize and quantify the process of venous thrombus formation. Some studies have reported rheological findings in artery models but there are few previous studies
modeling human veins.
Methods: We made polydimethylsiloxane (PDMS) into flow chambers
of rectangular-shaped cross section that is 20 lm in height and
500 lm in width (Fig. 1). The inner surface was fully siliconized and
then the downstream half was coated with collagen.
Human venous blood was obtained from healthy volunteers and
200 lg mL–1 of FITC-conjugated dextran was added. The sample was
flowed in the channel under a constant pressure (20 kPa) at 37 °C for
10 min. We recorded images of growing thrombi with a fluorescence
microscope and a high-speed camera at a fixed position. We also
recorded the time course of flow rate.
After experiment, we quartered a fixed part of the recorded images,
which contained a typical block of thrombi, into zones and named A,
B, C and D in order from upstream to downstream (Fig. 2). We measured area of thrombi in each zone.
Results: In the first 4 min, areas of thrombi in all zones were small and
grew slowly. In 6 min, the flow rate dropped and the every area began
to increase. Especially, the area of zone C grew sharply and dropped.
Then, the area of zone D grew sharply at the time the area of zone C
dropped. In 8 min, the areas of all zones decreased (Fig. 3).
Objectives: Atrial Fibrillation (AF) increases the risk of thromboembolic stroke 5-fold. Anticoagulation reduces this risk by 66% but with
an associated 1% annual risk of clinically significant bleeding. Clinical
prediction scores provide some guidance as to which patients will
derive most benefit from anticoagulation. We assessed the ability of
thrombin generation to improve risk stratification for stroke in these
patients.
Methods: Patients with AF were recruited to a prospective observational study in Greater Glasgow and Clyde Health Board and followed
up for stroke development. Thrombin generation was measured by
calibrated automated thrombography in platelet poor plasma, using
1 pmol L–1 tissue factor and thrombomodulin. Results were normalised to standard plasma and thrombin generation parameters compared between patients with and without stroke.
Results: 345 patients were followed up for an average of 2.7 years. 29
developed a thromboembolic stroke. Patients taking either aspirin or
no antiplatelet/anticoagulant agents, who developed a stroke (n = 12),
had a significantly shorter normalised thrombin generation lag time
[1.40 (95% CI 1.27–1.53) vs. 1.60 (1.52–1.68) P = 0.016] and time to
peak [1.02 (0.93–1.11) vs. 1.16 (1.09–1.21) P = 0.012] compared to
patients without stroke (n = 101). This difference was not seen in
patients using warfarin. The CHADS score was significantly higher in
patients with stroke [1.50 (1.26–1.74) vs. 2.50 (2.26–2.74), P = 0.02]
but there was no correlation between CHADS score and either lag
time or time to peak.
Conclusion: Lag time and time to peak are shortened in patients with
AF who are not anticoagulated and subsequently develop a stroke.
Neither parameter correlates with CHADS scores suggesting they are
independent variables. The use of normalised data implies that thrombin generation results could be standardised across laboratories. The
measurement of thrombin generation may improve risk prediction in
patients with AF and define more accurately who might benefit from
anticoagulation.
Disclosure of Interest: None Declared.
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
12/06/2014
82
ABSTRACTS
PHV09
Observation of clot formation and lysis in conjunction
with the thrombin generation assay
relationship between CFIB and PTDF. The analyses were divided into
two phases. Phase I included all 2,336 samples, while phase II targeted
on the 1,179 samples with abnormally low CFIB result (≤ 2.0 g/L).
Results: CFIB and PTDF values are significantly different (P < 0.001)
and PTDF values are on average 52% higher than CFIB values.
PTDF and CFIB values are highly correlated (correlation coefficient = 0.95). Polynomial relationship was established between CFIB
and PTDF values. Phase I study indicated that the equivalent critical
PTDF value is 1.6 g/L, which can identify 95% of patients (589 out of
619) with critically low CFIB (≤ 1.0 g/L). Phase II study found the
equivalent critical PTDF value is 1.5 g/L, which can identify 94% of
the patients (580 out of 619) with critically low CFIB.
Conclusion: To our knowledge, our study is the largest comparison of
PTDF and CFIB to date. PTDF and CFIB values are highly correlated. The equivalent critical PTDF value can be defined at 1.6 g/L,
which can identify 95% of patients with critically low CFIB. Bleeding
patients with PTDF ≤ 1.6 g/L may warrant prompt blood product
support.
Disclosure of Interest: None Declared.
Chang WC, Xin KZ, Lee TK and Ovanesov MV
Office of Blood Research and Review, US FDA, CBER, Rockville,
USA
CD
R
PHV11
The frequencies of thrombophilic alleles known from
GWAS studies in healthy population and in group of
patients with venous thromboembolism (VTE) in Czech
Republic
pi
aa
ut
Co
PHV10
A large-scale comparison study of plasma fibrinogen
by clauss and prothrombin time derived methods: an
attempt to define critically low value of prothrombin
time derived fibrinogen for bleeding patients when
clauss fibrinogen is not readily available
Sun P1,2, Wang Q3, Morales C1,2, Nasr M1,2, Turnbull G2 and
Musuka C1,2
1
Pathology, University of Manitoba; 2Hematopathology;
3
Quality, Diagnostic Services Manitoba, Winnipeg, Canada
Objectives: Plasma fibrinogen measurement is critical in managing
bleeding patients. Clauss fibrinogen (CFIB) method is widely accepted
as the ‘gold standard’; however unlike the quick, easily available prothrombin time derived fibrinogen (PTDF) method it is not frequently
performed at small laboratories. Manitoba has provincially run and
standardized coagulation testing. This study is aimed to define the
relationship between CFIB and PTDF and determine if PTDF can be
used as an alternative measurement of CFIB in bleeding patients.
Methods: We retrospectively reviewed all CFIB results from the urban
laboratories in 2012. 2336 samples had both CFIB and PTDF results.
Modified z-score method was used to check outliers in the datasets
and t-test was used to determine the significance between CFIB and
PTDF datasets. Regression analysis was employed to estimate the
or
Kvasnicka J1, Bobcikova P1, Hajkova J1, Kvasnicka T1, Malikova I2,
Brzezkova R1, Kudrnova Z1, Zenahlikova Z1 and Cverhova V1
1
Thrombotic Center; 2Central Haematology Lab, General
University Hospital, Prague, Czech Republic
da
p
or
iza
Objectives: Assaying thrombin generation (TG) in real time using a
fluorogenic substrate has been a popular approach for developing a
true global hemostasis assay. Benefits over other assay approaches
include sensitivity to moderate, not just severe, coagulation factor deficiencies. It is believed that reduced TG in factor deficient patients
results in weaker clots and reduced protection from fibrinolysis.
Methods: To expand the utility of the TG test, we are observing, concurrently with the fluorescent signal of the thrombin substrate, absorbance as a direct measurement of fibrin generation (FG) in vitro.
Tissue plasminogen activator (tPA) is added to our assay to induce
and allow observation of clot lysis. We run the same samples on
Thrombinoscope’s Calibrated Automated Thrombinography (CAT,
Stago USA) platform to assess the added value of our variations from
the current standard protocol.
Results: Increased tissue factor (TF) results in increased TG (higher
thrombin peak heights and lower thrombin lag time) and increased
fibrin generation (higher clot velocity and lower clot time), while clot
lysis does not appear to have a dose-dependent response to TF. Deficient plasma supplemented with 3% normal plasma showed more TG
than 100% normal plasma; thus it does not model moderately deficient
plasma. We restored TG in FV, FVII, FVIII, and FIX-deficient
plasma by supplementing with purified factors. Restoring FV and
FVII results in faster FG and clot lysis in addition to increased TG.
For FIX, TG and FG parameters correlate well: with increasing FIX
come decreasing thrombin lag time and clot time, and decreasing
thrombin peak time and lysis time.
Conclusion: Observation of clotting may add more value to the TG
assay giving us a more global assessment of hemostasis. Induction and
observation of clot lysis may reveal patterns not seen from FG alone.
Disclosure of Interest: W. Chang is an employee of: FDA. This is an
informal communication and it represents authors’ own best judgment. These comments do not bind or obligate FDA., K. Xin: none
declared, T. Lee: none declared, M. Ovanesov: none declared.
Objectives: The aim was to determine frequencies of mutations alleles
related to risk of VTE (FV Leiden, FII 20210G>A, 4/5G PAI-1,
SERPINC1 (IVS + 141G>A), GP6 13254T>C and CYP4V2
(Lys259Gln) in healthy Caucasians and in patients with VTE from the
area of Central Bohemia and to determine a level of VTE risk (odds
ratio, O.R).
Methods: DNA was isolated according to the MagNA Pure High-Performance DNA ExtractionTM protocol. The polymorphisms were
determined using PCR by FRET process with LightCyclerâ 480 System and LCâ480 Genotyping Master kits (all products supplied by
Roche). Specific primers and fluorescently labelled probes were
designed in cooperation with TIB MOLBIOL (Berlin, Germany). Chi
square test or Fisher extract test and logistic regression method (O.R.)
were determined both using SAS program, 9.2.
Results: Control group (n 1460): Frequencies of risk alleles A for the
mutations FV Leiden and FII 20210G>A were 4.5% and 1.3%,
respectively. Frequencies of the other risk alleles were: 4G PAI-1
55.5%, allele A of SERPINC1 (IVS + 141G>A) 11.3%, allele T of
GP6 13254T>C 87.7% and allele A of CYP4V2 (Lys259Gln) 65.2%.
Patients with VTE (n 2369): Risk alleles A frequencies for the FV Leiden and FII 20210G>A were 19.86% and 4.52%, respectively. Frequencies of the other risk alleles were: 4G PAI-1 55.89%, allele A of
SERPINC1 (IVS + 141G>A) 12.1%, allele T of GP6 13254T>C
87.9%, and allele A of CYP4V2 (Lys259Gln) 68.25%. Only frequencies of FV Leiden risk allele A(O.R. 5.19; CI 4.30–6.27) and FII
20210G>A risk allele A (O.R. 3.41; CI 2.42–4.79) were statistically significant (both P < 0.0001) for determining the risk of VTE in our control-case study. Frequency of risk allele A of CYP4V2 (Lys259Gln)
was also higher in pts with VTE (P = 0.007) bud with low O.R. (1.14;
CI 1.04–1.26).
Conclusion: We believe that from the observed mutations known from
GWAS represent a clinically relevant risk for VTE only FVL or FII
mutation in Czech Republic.
Disclosure of Interest: J. Kvasnicka has grant/research support from:
RVO-VFN64165, P. Bobcikova: none declared, J. Hajkova: none
declared, T. Kvasnicka: none declared, I. Malikova: none declared, R.
12/06/2014
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
ABSTRACTS
Brzezkova: none declared, Z. Kudrnova: none declared, Z. Zenahlikova: none declared, V. Cverhova: none declared.
are available on TG patterns in patients with transient ischemic
attacks (TIA).
Objective: To evaluate the association between TG patterns and carotid and intracranial (IC) atherosclerotic lesions in TIA patients.
Methods: 120 patients (male 52.5%), aged 69 12 years, with confirmed TIA diagnosis by a Neurologist were enrolled. Within 24 h
from diagnosis patients underwent: blood sampling for TG with or
without thrombomodulin (TM), D-dimer and C reactive protein
(CRP), CT scan, ECG and Carotid and intracranial (TCD) Duplex
ultrasound scan. Clinical follow-up was performed at 1, 3 and
6 months.
Results: 11 patients were on warfarin (VKA) at enrollment and had
significantly lower TG + TM and TG-TM than the others. Among the
patients without VKA, TG + TM and TG-TM did not differ according to TOAST classification etiology (large vessel vs. cardioembolic vs.
lacunar vs. idiopathic). TG + TM was higher in patients with intracranial stenoses than in those without (2049 105 vs. 1751 38,
P = 0.015), whereas it was similar in patients with carotid atherosclerosis and in those without.
Conclusion: TG will help give an etiopathogenetic hypotesis in cerebral
ischemia and it could represent the basis for new pharmacological trial
in patient with intracranial stenoses.
Disclosure of Interest: None Declared.
PHV12
Detection of a high rFVIIa binding subpopulation and
of donor variation in the interaction of activated
recombinant factor VII (rFVIIa) with human platelets by
flow cytometry
€hm E, Sedivy A, Dockal M and Scheiflinger F
Koehn J, Bo
Baxter Innovations Gmbh, Vienna, Austria
CD
R
Vascular Biology
da
p
or
VB01
Quantifying the impact of diet-induced atherosclerotic
plaque erosions on vessel hypercoagulability with
semipermeable nanoparticle beacons
Palekar RU1, Jallouk AP2, Goette MJ1, Myerson JW1, Allen JS2,
Akk A2, Chen J2, Yang L2, Tu Y2, Miller MJ2, Pham CT2, Wickline
SA1,2 and Pan H2
1
Biomedical Engineering; 2Medicine, Washington University in
St. Louis, St. Louis, USA
Co
pi
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iza
Objectives: Recombinant activated factor VII (rFVIIa) is a treatment
option for hemophilia patients requiring inhibitor bypassing agents.
rFVIIa is suggested to activate factor X (FX) on the platelet surface
independent of tissue factor (TF). However, treatment response varies,
and there is no standardized laboratory method to monitor or predict it.
Platelet characteristics and functions vary between individuals and
may influence rFVIIa platelet binding and treatment response. rFVIIa
has been shown to preferentially bind ‘coated’ platelets, characterized
by the exposure of fibrinogen and other procoagulant proteins. Variation in the ability to form coated platelets has been correlated with
bleeding frequency in hemophilia patients.
Methods: Fresh platelet concentrates were incubated with rFVIIa or
the corresponding vehicle for 7 min at 37 °C with or without platelet
activators thrombin and convulxin, a collagen receptor agonist. Platelet purity and activation status were analyzed by flow cytometry staining for CD61 and CD62P or fibrinogen and forward/side scatter
gating. rFVIIa bound to platelets was quantified using a fluorescentlabeled anti human FVIIa antibody.
Results: Quantifying binding of rFVIIa to platelets from healthy
donors, we frequently detected a high rFVIIa binding platelet subpopulation. rFVIIa binding to non-activated and dual agonist activated
platelets was concentration dependent and usually saturated at rFVIIa
concentrations of 200–1200 nmol L–1, with activated platelets showing
a higher binding capacity. rFVIIa binding to platelets showed considerable inter-individual variation, as did frequency, size, and binding
capacity of the high rFVIIa binding subpopulation. The rFVIIa high
binding subpopulation was not restricted to activated or ‘coated’
platelets.
Conclusion: Whether the occurrence of high rFVIIa binding subpopulations correlates with treatment response requires further populationbased functional and phenotypic characterization.
Disclosure of Interest: J. Koehn is an employee of: Baxter Innovations
GmbH, E. B€
ohm is an employee of: Baxter Innovations GmbH, A. Sedivy is an employee of: Baxter Innovations GmbH, M. Dockal is an
employee of: Baxter Innovations GmbH, F. Scheiflinger is an
employee of: Baxter Innovations GmbH
83
PHV13
Thrombin generation and blood coagulation activation
in patients with a transient ischemic attacks
Favaretto E1, Sartori M1, Migliaccio L1, Rondelli F2, Guarino M3,
Legnani C1, Palareti G1 and Cosmi B4
1
Angiology and Blood Coagulation, S.Orsola -Malpighi
University Hospital; 2Neurology, Angiology and Blood
Coagulation; 3Neurology, S.Orsola -Malpighi University
Hospital; 4Angiology and Blood Coagulation, S.Orsola-Malpighi
University Hospital, Bologna, Italy
Objectives: Late-stage atherosclerotic plaques presenting with eroded
endothelial barriers represent a subset of plaques at high risk for
thrombosis. We report that quantification of passive perfluorocarbon
nanoparticle (PFC-NP) accumulation in plaques with disrupted endothelium can be predictive of hypercoagulability, offering a functional
metric for measuring thrombosis risk.
Methods: ApoE-/- mice were treated as follows: normal chow or Western Diet for 2, 3, 4, 5, or 6 months. At 4 months on Western diet, a
subset of mice was switched to normal chow for 1 months or
2 months. A 1 mL/kg bolus of PFC-NP was delivered intravenously
and allowed to circulate in vivo for 2 h. A photochemically induced
carotid injury model was used to determine thrombosis risk, where
time to full occlusion is inversely related to hypercoagulability. Aortas
were removed for quantification of PFC-NP accumulation using 19F
magnetic resonance spectroscopy (19F-MRS) at 11.7T.
Results: ApoE-/- mice on Western diet exhibited accelerated time to
occlusion vs. mice on normal chow (P = 0.0016). Upon Western diet
removal, occlusion times gradually returned to control values after
2 months (Fig. A). Aortic PFC-NP accumulation increased with the
Western diet duration, and was elevated (P = 0.009) over controls by
6 months of feeding. Diet normalization resulted in decreased PFCNP accumulation, returning to normal after only 2 months, indicating
recovery of endothelial barrier function (Fig. B). An inverse correlation was observed between PFC-NP accumulation and carotid occlusion time (P = 0.02), indicating that PFC-NP accumulation is
predictive of thrombosis risk.
Objectives: Thrombin generation (TG) tests have been assessed in arterial diseases and showed a possible association with atherosclerotic
conditions and risk of vascular events. In stroke patients, limited data
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
12/06/2014
84
ABSTRACTS
A
Disclosure of Interest: R. Palekar: none declared, J. Chen: none
declared, C. Vemuri: none declared, J. Myerson: none declared, X.
Yang: none declared, H. Zhang: none declared, G. Lanza: none
declared, S. Wickline Shareholder of: AcuPlaq
B
VB03
Cyclosporine a prevents replicative senescence-related
endothelial dysfunction, pro-thrombotic and pro-coagulant responses and mp shedding in primary coronary
artery endothelial cells
Conclusion: Dietary normalization in ApoE-/- mice is highly effective
in delineating the recovery of endothelial barrier function and a natural antithrombotic state. Our results suggest that 19F-MRS quantification of PFC-NP accumulation could be used as a direct measurement
of thrombosis risk in subjects predisposed to atherosclerosis.
Disclosure of Interest: None Declared.
Abbas M1,2, Toti F3,4,5, Zobairi F2, Hamade E6, Merhi RA6,
Schini-Kerth V7 and Morel O8
1
University of Lebanon, Beyrouth, Lebanon; 2EA 7293, Facult
e de
e de
m
edecine, Universit
e de Strasbourg, Strasbourg; 3Universit
Strasbourg, Illkirch, France; 4Facult
e de Pharmacie, Universit
e de
Strasbourg; 5UMRCNRS 7213, Facult
e de Pharmacie, Illkirch,
France; 6Universite Libanaise, Beirut, Lebanon; 7UMRCNRS
7213, Facult
e de Pharmacie, Universit
e de Strasbourg, Illkirch;
8
^le d’activit
Po
e medico-chirurgicale cardio-vasculaire, Nouvel
^pital Civil,, Centre Hospitalier Universitaire, Strasbourg,
Ho
France
VB02
Anti-thrombin nanoparticles preserve renal function
after acute ischemic injury
Co
pi
aa
ut
A
B
or
CD
Objectives: Clinical studies have indicated that cyclosporine A (CsA)
limits myocardial infarction size. Aging is associated with vascular and
endothelial senescence, an irreversible cell cycle arrest characterizedby
increased activity of p53 and its downstream target p21. The possibility that CsA prevents endothelial senescence was evaluated in a senescent replicative model.
Methods: Replicative senescence was induced by sequential passaging
of primary cultures of endothelial cells (ECs) up to the fourth passage
(P4). Senescence associated b-galactosidase (SA-b-gal) activity and
mitochondrial membrane potential were evaluated using C12-FDG
and Dioc6 specific probes. NO formation by ECs cultured on beads
was indirectly assessed using washed human platelets. Procoagulant
microparticles in cell supernatants were measured by prothrombinase
assay following their capture onto annexin-5.
Results: ECs showed a progressive increase in microparticle shedding,
SA-b-gal, p53, p21 an p16 activities, expression of tissue factor,
NADPH oxidase subunits gp91 phox and p47 phox and p66 Shc from
P1 to P4. Conversely, eNOS and prohibitin were down-regulated and
Dioc6 signal reduced. ECs at P1 strongly inhibited platelet aggregation
in response to U46619 compared to cells at P3. CsA reduced SA-b-gal
activity at P3 (0.3–30 lg mL–1), changes in protein expression and in
the mitochondrial ΨD and microparticle shedding. In addition, CsA
restored the inhibitory activity of ECs at P3 on platelet aggregation.
Conclusion: Replicative senescence is associated with a prothrombotic
endothelial phenotype with increased expression of tissue factor and
microparticle shedding. CsA delays the senescence-related impairment
of the endothelial function, most likely by preventing the upregulation
of p66 Shc, NADPH oxidase, and by the down-regulation of eNOS
and prohibitin. These data suggest that CsA might thereby delay prothrombotic and pro-coaguant responses in the vessel.
Disclosure of Interest: None Declared.
da
p
or
iza
Objectives: Ischemic acute kidney injury (AKI) is a leading cause of
mortality in hospitalized patients. Ischemia and perfusion injury activates inflammation and coagulation pathways that involve thrombin
in both signaling events and microvascular thrombosis in the ‘extension phase’ of AKI. Herein, we demonstrate a novel therapeutic
approach to inhibit thrombin activity and intrarenal coagulation in
AKI through nanoparticle delivery of the direct thrombin inhibitor,
D-phenylalanyl-L-prolyl-L-arginyl chloromethylketone (PPACK-NP).
Methods: Male Sprague-Dawley rats underwent bilateral renal arterial
occlusion for 45 min followed by reperfusion. Rats were pre-treated
with either saline (N = 3) or PPACK-NP (0.5917 mg/kg PPACK,
N = 3) at 5–10 min prior to ischemic injury. At 24 h after AKI, rats
were euthanized and excised kidneys were prepared for histological
staining with H&E, anti-thrombin antibodies, and TUNEL assays to
evaluate renal damage. For all rats, blood samples were collected
before and at 24 h after AKI to analyze the serum creatinine level
([Cr]).
Results: The baseline [Cr] before AKI was 0.45+/0.13 mg/dL. At
24 h after AKI, [Cr] increased to 1.40+/0.45 mg/dL in saline treated
animals (P < 0.05 vs. baseline), which is higher than the 0.68+/
0.16 mg/dL [Cr] in PPACK-NP treated animals (P < 0.05 vs. saline
treated and vs. baseline). Immunofluorescent staining for thrombin
(green) revealed massive accumulation of thrombin in saline treated
kidneys (Fig. A) but was substantially reduced in PPACK-NP treated
kidneys (Fig. B).
R
Palekar RU1, Chen J2, Vemuri C2, Myerson JW1, Yang X2, Zhang
H2, Lanza GM1,2 and Wickline SA1,2
1
Biomedical Engineering; 2Medicine, Washington University in
St. Louis, St. Louis, USA
Conclusion: Our results demonstrate that PPACK-NPs protected renal
function as evidenced by lower serum [Cr] in PPACK-NP treated animals at 24 h after AKI.
12/06/2014
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
ABSTRACTS
85
VB04
Microparticles released by replicative senescence in
primary endothelial cells promote premature senescence associated with an impaired no formation and
oxidative stress
VB05
Utilization of elastic compression stockings (ECS) following first episode of deep venous thrombosis (DVT):
15 year experience at a single veterans affairs medical
center (VAMC)
Abbas M1,2, Toti F3,4, Zobairi F1, Yver B5, Schini-Kerth V6 and
Morel O7
1
EA7293, Faculte de M
edecine, universit
e de Strasbourg,
Strasbourg, France; 2University of Lebanon, Beyrouth, Lebanon;
3
Faculte de Pharmacie, Universit
e de Strasbourg; 4UMRCNRS
7213, Faculte de Pharmacie, Illkirch; 5EA 7293, Facult
e de
Medecine, universite de Strasbourg, Strasbourg; 6UMRCNRS
7213, Faculte de Pharmacie, Universit
e de Strasbourg, Illkirch;
7
^le d’activite medico-chirurgicale cardio-vasculaire, Nouvel
Po
^pital Civil,, Centre Hospitalier Universitaire, Strasbourg,
Ho
France
Bade NA1,2, Aggarwal A3, Amm JE1, Mobarek D1, O’Neill B4,
Faselis C5 and Rickles FR1
1
Hematology, Veteran’s Affairs Medical Center; 2Internal
Medicine, Georgetown University; 3Hematology, Veterans Affairs
Medical Center; 4Veteran’s Affairs Medical Center, Washington
DC; 5Internal Medicine, Veteran’s Affairs Medical Center,
Washington, DC, USA
R
CD
or
da
p
Co
pi
aa
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or
iza
Objectives: Circulating endothelial microparticles (EMPs) are
increased in coronary artery disease, peripheral vascular disease and
allograft rejection following heart transplantation. This in-vitro study
examined if the induction of endothelial senescence is associated with
EMPs shedding, and whether senescence-related EMPs promote endothelial senescence and prothrombotic changes.
Methods: Replicative senescence was induced by sequential passaging
of primary cultures of porcine coronary artery endothelial cells (ECs)
up to the third passage (P3). EMPs were harvested and isolated from
the supernatant of P3 and applied to cells at passage 1 (P1). The delivery of EMPs to target cells was evidenced by the fluorescent lipidic
probe PKH26. Cell cycle and senescence induced-b galactosidase activity (SA- b gal) were measured by flow cytometry. NO production was
inderectly assessed by incubating ECs with washed human platelets.
Results: ECs exhibited increased SA- b gal staining and% of cells in
the G0/G1 phase at P3. Exposure of P1 ECs to 10 nmol L–1
EMPs collected from the conditioned medium of ECs at P3 increased
the% of cells in G0/G1 phase and SA-b-Gal staining after 24 h. EMP
incubation led to increased expression of the senescence markers p53,
its downstream target p21, and the adaptor protein p66Shc and
enhanced AT2 expression. EMPs also induced oxidative stress in target P1 ECs as detected using dihydroethidine staining, and up-regulated TF expression. The ability of ECs to inhibit U46619-induced
platelet aggregation was reduced.
Conclusion: The findings indicate that endothelial senescence is associated with an increased shedding of EMPs, which, in turn, promote premature senescence and prothrombotic changes. The response to
senescence-related EMPs involves oxidative stress, induced the up-regulation of p53 and p21, AT-2 and a reduced formation of NO. They
further suggest that EMPs released by senescent endothelial cells in the
vessel may favour enhanced endothelial dysfunction and thereby
thrombogenicity.
Disclosure of Interest: None Declared.
Objectives: One of every 3–4 patients with symptomatic DVT of the
lower extremities (LE) will go on to develop post-thrombotic syndrome (PTS), which is burdensome and costly. The optimal management of PTS is prevention. While the efficacy of ECS is controversial,
several, well designed, randomized controlled trials (RCT) have demonstrated significant benefit in the prevention of PTS. Therefore, we
examined the rate of ECS utilization (prescription) in our institution
after a first episode of DVT.
Methods: In this IRB approved, retrospective observational study, the
electronic medical records for all patients diagnosed with LE DVT by
ICD-9 code over a 15-year period were reviewed. Patients with a history of prior DVT were excluded and only patients diagnosed with
first LE DVT by ultrasonography at VAMC were included for further
analysis. We collected demographic information including DVT site,
ECS prescription documentation of pain, cramps, heaviness, paresthesias, edema, erythema and pain on palpation.
Results: of the 510 patients who had first LE DVT, only 177 (35%)
were provided a prescription for ECS; however, 165/177 patients
(93%) already had signs and/or symptoms suggestive of PTS. Only 26
patients (5%) received an ECS prescription within 4 weeks of the initial event.
Conclusion: ECS prescription rate within 4 weeks after a first DVT
was low and ECS were rarely used in an attempt to prevent PTS.
While the results of a recently published RCT casts some doubt on the
efficacy of ECS in the prevention of PTS, this relatively inexpensive
and simple intervention may be useful in some patients. Therefore, we
plan to establish a quality improvement measure to ensure timelier
ECS prescription and provide education on its use.
Disclosure of Interest: None Declared.
VB06
Altered endothelium-dependent relaxation induced by
erythrocyte membrane from subjects with different
hemoglobin genotypes in isolated rabbit carotid arteries
Ajayi OI and Usunobun WO
Physiology, university of Benin, Benin city, Nigeria
Objectives: The obligatory roles of erythrocyte membrane interactions
with vascular endothelium have conflicting reports in the literature.
The goal of this study was to characterize the effect of constituents of
erythrocytes and their membranes from subjects with different Hb
genotypes on acetylcholine dependent vaso-relaxation
Methods: Isometric contractions of ring preparations (5 mm long) of
rabbit carotid artery suspended in 20 mL organ baths and bubbled
with 95% O2, 5% CO2 were studied pharmacologically using standard
in vitro techniques. Concentration-dependent contractile responses
induced by phenylephrine (PE) as well as relaxation responses induced
by acetylcholine (Ach) were examined. Acetylcholine (IC70M)induced relaxations of pre-contractions induced by EC70 mol L–1
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
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86
ABSTRACTS
phenylephrine were examined in control rings as well as in rings
exposed for 30 min to (a) intact washed erythrocytes (b) erythrocyte
ghosts and (c) hemoglobin – all obtained from subjects of different
hemoglobin genotypes (AA, AS and SS).
Results: Ach-induced relaxation was significantly (P < 0.05) enhanced
by AA erythrocytes (46.2 3.25 and 69.5 5.4% for control and
test, respectively). In contrast, AS and SS erythrocytes as well as exposure to hemoglobin (Hb) solution did not significantly alter Ach relaxation whereas AS and SS ghosts significantly (P < 0.01) attenuated
Ach relaxation. Compared with AA, erythrocytes but not Hb from AS
and SS subjects elicited significantly greater inhibition of Ach relaxation; futhermore, inhibition of Ach relaxation by erythrocyte ghosts
was significantly greater with AS than SS.
Conclusion: Our results show that a membrane-bound factor may
account for the genotype-dependent attenuated Ach-induced relaxation following interaction between erythrocytes and vascular endothelial cells.
Disclosure of Interest: None Declared.
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Objectives: Histones, major components of neutrophil extracellular
traps, are released at sites of infection, thrombosis, cancer and wound
healing. Considering that vascular repair and formation of new vessels
from preexisting ones (angiogenesis) and de novo (vasculogenesis) are
key events in these clinical situations, we aimed to analyze the effect of
histones on the angiogenic activity of endothelial cells involved in vasculogenesis such as late growth endothelial progenitor cells (EPC) and
angiogenesis including human umbilical vein endothelial cells (HUVEC) and dermal microvascular endothelial cells (HMEC).
Methods: Proliferation (pNPP), viability (fluorescence microscopy),
wound healing (scratch assay), tubule formation (matrigel) and intracellular signaling (western blot) were analyzed. Data are shown as%
of inhibition (%inh), n = 4–6, P < 0.05 vs. control.
Results: H1 and H2A (1–4 lM) had no significant effect on any of the
responses tested. However, H2B, H3 or H4 (4 lM) decreased proliferation of EPC (52 7, 47 5, 47 3%inh), HUVEC (74 8,
39 8, 44 4%inh) and HMEC (97 5, 26 5, 35 6% inh).
This effect was at least in part due to an increase in the% of necrosis
and apoptosis induced by this three histones (4 lM) (figure). When a
concentration that had no effect on survival or proliferation was used
(1 lM), H2B, H3 and H4 exerted a moderate but significant down-regulation of wound healing (EPC: 15 2, 18 2, 24 4, HUVEC:
20 1, 18 1, 15 2 and HMEC: 17 3, 20 2, 32 4%inh),
suggesting that histones might inhibit chemotaxis. Tubule formation
was also drastically inhibited by H2B, H3 and H4 (1 lM) in EPC
(38 4, 50 3, 56 5%inh) and HUVEC (15 2, 44 2,
56 3%inh), while in HMEC, only H3 showed a significant effect
(51 4%inh). These data correlated with the phosphorylation/activation levels of p38, an inhibitory pathway of tubule formation.
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n M2, Schattner M1 and Negrotto S1
Mena HA1, Centurio
1
Laboratory of Experimental Thrombosis, Institute of
Experimental Medicine, National Academy of MedicineCONICET; 2Service of Endocrine Gynaecology and Reproduction,
Hospital Bernardino Rivadavia, Buenos Aires, Argentina
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VB07
Effect of histones on angiogenic responses of progenitor and mature endothelial cells
Conclusion: Histones H2B, H3 and H4 exerted a suppressive effect on
vasculogenesis and angiogenesis as selectively inhibited angiogenic
responses of progenitor and mature endothelial cells.
Disclosure of Interest: None Declared.
VB08
Frequency of post thrombotic syndrome (PTS) in
patients with proximal deep venous thrombosis – a
prospective observational study from a tertiary care
hospital in North India
Suri V1, Sravan K1, Malhotra P1, Kalra N2, Ahluwalia J3, Prakash
G1, Khadwal A1, Kumari S1, Jain S1, Varma N3 and Varma S1
1
Internal Medicine; 2Radiodiagnosis; 3Hematology, PGIMER
Chandigarh, Chandigarh, India
Objectives: The post thrombotic syndrome (PTS) a chronic,potentially
disabling condition is estimated to develop within 1–2 years in 20% to
50% of patients of symptomatic deep vein thrombosis (DVT).
Methods: A prospective,observational study was undertaken at PGIMER Chandigarh,which is oneof the largest tertiary referral centers in
North India from Janauary 2005-April 2006.Patients with DVT of the
proximal veins of lower limbs (Iliac, femoral and popliteal veins)who
had been anticoagulated for at least 6 months were enrolled in the
study.PTS was defined as pain and swelling of a chronic duration
(occurring daily for at least 1 month) occurring 6 months or more
after an episode of DVT.
Patients were evaluated for symptoms and signs of post-thrombotic
syndrome by using CEAP clinical scale and Villalta’s scale at 0, 3 and
6 months.
Results: 100 patients (113 limbs) with PTS were enrolledin our study.
of these 100 patients,77 (88 limbs)&54 (63 limbs) patients were re-eval-
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ABSTRACTS
uated again at 3 and 6 months.The frequency of PTS at 0, 3 &
6 months was77.9%,71.6%&58.7%(CEAP clinical scale) and 46.9%,
20.5% &20.6% (Villalta’s scale) respectively.PTS. Severe PTS was seen
in 43.4%, 40.9% &38.1% (CEAP clinical scale) and 5.3%, 0% &1.6%
(Villalta’s scale) at 0, 3 and 6 months respectively.
Common signs and symptoms observed among patients with PTS were
pain (39.8%),cramps (24.8%), heaviness (62.8%), pruritus (24.8%),
edema (69%), induration (50.4%),hyperpigmentation (43.4%),telengectesias (44.2%).It was observed that the above signs and symptoms
ofPTS decreased with time with lower frequencies observed at the 3
and 6 months follow-up.
Previous history of recurrent ipsilateral DVT and a BMI OF > 25 kg/
m2 at enrollment were the only predisposing factors significantly associated with PTS (P < 0.0001 & P < 0.05).
87
503 mediates the effect of hypoxia upon eNOS expression. Taken
together, hypoxia-induced microRNA, miR-503, is involved in eNOS
alteration by hypoxia via its direct target RICTOR. Our work revealed
the impact of miR-503 on cellular function and the role of mTORC2
in endothelial cells under hypoxia.
Conclusion: Our data reveal a novel endothelial role for mTORC2, and
show that miR-503 regulates the mTOR pathway by targeting RICTOR.
Disclosure of Interest: None Declared.
VB10
Circulating endothelial cells and progenitors as prognosis factors during auto-immune thrombotic thrombocytopenic purpura: results of a prospective
multicenter french study
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Conclusion: The question is which of the above two tools is better for a
definitive diaagnosis of PTS will require a gold standard against which
both could be compared and for this more studies are needed.
Disclosure of Interest: None Declared.
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Widemann A1, Pasero C2, Arnaud L2, Poullin P3, Loundou A4,
Choukroun G5, Sanderson F6, Lacroix R2,7, Sabatier F7,8, Coppo
P9, Dignat-George F2,10, Kaplanski G11,12 and on behalf of ENDO13 study group
1
Aix-Marseille Universit
e, Vascular Research Center of Marseille,
Inserm UMR 1076; 2APHM, CHU de la Conception, Service
d’H
ematologie; 3APHM, CHU de la Conception, Service
d’H
emaph
er
ese et d’Autotransfusion; 4APHM, Unit
e d’aide
m
ethodologique a la recherche clinique, Marseille; 5CHU
D’Amiens, D
epartement de N
ephrologie, Amiens; 6Hopital
l’Archet, Service de M
edecine Interne, Nice; 7Aix-Marseille
Universit
e, Vascular Research Center of Marseille, Inserm UMR
1076; 8APHM, CHU de la Conception, Laboratoire de Culture et
de Th
erapie Cellulaire, Inserm, CIC-BT510; 9APHP, Hopital Saint
Antoine, D
epartement d’h
ematologie, Centre de r
ef
erence des
Microangiopathies Thrombotiques; 10Aix Marseille Universit
e,
Vascular Research Center of Marseille, Inserm, UMR 1076;
11
APHM, CHU de la Conception, Service de M
edecine Interne;
12
Aix Marseille Universit
e, Vascular Research Center of
Marseille, Inserm UMR 1076, Marseille, France
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miR-503 modulation of NOS3 signaling via RICTOR
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Yamakuchi M1, Hashiguchi T1 and Lowenstein CJ2
Laboratory and Vascular Medicine, Kagoshima University
Graduate School of Medical and Dental Sciences, Kagoshima,
Japan; 2Medicine, University of Rochester, Rochester, USA
1
Co
Objectives: Vascular cells adjust their metabolism, growth, and proliferation in response to environmental stress. The mechanistic target of
rapamycin (mTOR) pathway senses the availability of nutrients, oxygen, and growth factors; and regulates catabolic and anabolic pathways. We now identify microRNAs increased by hypoxia and
discovered that miR-503, one of hypoxia-induced microRNA, controls
mTOR signaling in endothelial cells under hypoxic conditions.
Methods: We performed microarray analysis for microRNA governed
by hypoxia in endothelial cells. We manipulated endogenous microRNA levels and performed Western blotting, qPCR, and immunostaining. We also measured nitric oxide (NO), NO synthase (NOS) activity,
and endothelial cellular functions.
Results: Using microarray analysis, we confirmed that hypoxia
increases expression of a set of microRNAs, such as miR-210, miR424, and miR-503 in different types of endothelial cells. We found that
miR-503 targets expression of rapamycin insensitive companion of
mTOR (RICTOR), a component of the mTOR complex mTORC2.
Over-expression of miR-503 decreases RICTOR levels, inhibiting
mTORC2 phosphorylation of AKT. In contrast, knockdown of
endogenous miR-503 increases RICTOR and activates AKT. In general, RICTOR regulates several downstream target of AKT, including
GSK3 and FOXO1. In endothelial cells, AKT controls NO synthesis
by phosphorylating NOS3 (eNOS); and we showed that miR-503 regulates NO production through a RICTOR-AKT-eNOS pathway by
Griess assay and NOS activity assay. Furthermore we found that miR-
Objectives: Autoimmune thrombotic thrombocytopenic purpura (AITTP) is characterized by excess circulating ultra-large von Willebrand
Factor (vWF) due to anti-ADAMTS-13 auto-antibodies, but animal
studies have shown that endothelial cell activation may also be an
important trigger of AI-TTP. We conducted a prospective muticenter
french study in order to evaluate the endothelial lesion/repair profile in
AI-TTP patients.
Methods: A prospective study of circulating biomarkers of endothelial
lesion (circulating endothelial cells CEC), activation (soluble (s)P-selectin and vWF) and repair (circulating progenitor cells [CPC], and
endothelial progenitor cells [EPC]) in 22 AI-TTP patients in correlation with disease severity and prognosis.
Results: CEC, vWF and sP-selectin were significantly increased during
crisis and returned to baseline during remission. CEC and sP-selectin
concentrations correlated with LDH levels and inversely correlated
with platelets counts. Both CEC and sP-selectin were significantly
higher in patients who died or developed sequelae. CPC counts were
highly increased during the acute phase of the disease, returned to
baseline during remission and correlated with platelet counts, LDH
concentrations as well as CEC and sP-selectin. Among CPC, EPC
were also increased during crisis and decreased during remission.
Patients who received < 16 plasma exchange therapy had significantly
higher initial levels of EPC than those who needed more numerous
plasma exchange to obtain remission, suggesting that initial EPC
counts may be associated with plasma exchange efficacy.
Conclusion: This profile of endothelial markers demonstrates important endothelial activation and repair/remodeling during AI-TTP, and
suggests that CEC and EPC may be promising prognosis factors of
disease outcome and response to treatment.
Disclosure of Interest: None Declared.
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
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ABSTRACTS
VB11
Retrospective database analysis of the prevalence of
cardiovascular comorbidities in a US patient population with hemophilia a: confirmation of findings
Pocoski J1, Kamalakar R2 and Humphries T1
Bayer HealthCare Pharmaceuticals, Whippany; 2Alpha
Consulting Corp, East Brunswick, USA
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Objectives: A previous retrospective study of the MarketScanâ claims
database reported increased prevalence and earlier onset of cardiovascular (CV) comorbidities in patients with hemophilia A compared with
patients without hemophilia. This study was designed to confirm these
findings in a second population of male patients with hemophilia A in
the United States.
Methods: Male patients with hemophilia A and continuous insurance
coverage were identified by ICD-9-CM code 286.0 using the PharMetrics LifeLink claims database (IMS Health) of patient records from
January 1, 2008 to December 31, 2011. Patients with hemophilia A
were matched 1:3 with controls for gender, age, plan type, geographical region, and eligibility months in the study period. The prevalence
of CV comorbidities (identified by ICD-9-CM codes) was compared
between matched cohorts. Statistical significance was calculated by
Fisher’s exact test.
Results: Overall, 1050 patients were included in the hemophilia A
cohort and 3150 in the control cohort (Table). The prevalence of hemorrhagic stroke (1.4% vs. 0.2%, P < 0.0001), ischemic stroke (4.1%
vs. 1.7%, P < 0.0001), coronary artery disease (8.9% vs. 5.2%,
P < 0.0001), myocardial infarction (1.4% vs. 0.6%, P = 0.013), hypertension (21.3% vs.13.2%, P < 0.0001), hyperlipidemia (15.0% vs.
12.0%, P = 0.016), arterial thrombosis (9.6% vs. 3.7%, P < 0.0001),
and venous thrombosis (4.9% vs.0.3%, P < 0.0001) was significantly
higher in the hemophilia A cohort. Increased prevalence of CV comorbidities was consistent across most age groups, and patients with
hemophilia A experienced CV comorbidities at an earlier age than
patients without hemophilia.
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Conclusion: This second retrospective study of claims databases confirms an increased prevalence and earlier onset of CV comorbidities in
patients with hemophilia A. These findings support increased screening in patients with hemophilia for CV comorbidities at an earlier age
than recommended for the general population.
Disclosure of Interest: J. Pocoski is an employee of: Bayer HealthCare,
R. Kamalakar: none declared, T. Humphries is an employee of: Bayer
HealthCare
VB12
Analysis of tissue factor pathway inhibitor isoforms on
macro- and microvascular endothelial cells
Pachlinger R, Baldin-Stoyanova A, Knofl F, Ullrich N, Scheiflinger F
and Dockal M
Baxter Innovations Gmbh, Vienna, Austria
Objectives: Tissue factor pathway inhibitor (TFPI) is a Kunitz-type
protease inhibitor that inhibits both FXa and TF-FVIIa and is an
important physiological inhibitor of the extrinsic coagulation path-
way. The main portion (~80%) of TFPI in humans is reportedly associated with endothelial cells (ECs). We aimed to identify possible
differences in the TFPI level of macro- and microvascular ECs of various tissues and to obtain further insight into the relevance of TFPI isoforms alpha and beta at the cellular level.
Methods: Primary macro- and microvascular ECs of various tissues
were treated with phosphatidylinositol phospholipase C (PI-PLC) to
remove the glycosylphosphatidylinositol (GPI) anchored TFPI isoform beta from the cell surface and subsequently used for fluorescence
activated cell sorting (FACS). TFPI alpha, the non-GPI anchored
TFPI isoform, is therefore unaffected by PI-PLC treatment. Measuring the fluorescence intensities of such treated cells by FACS allowed
us to determine the amount of cell surface TFPI beta. The remaining
supernatants and similarly treated cells were used to quantify TFPI by
applying an ELISA to quantify and another to discriminate between
TFPI alpha and TFPI beta.
Results: Four macrovascular (HUVEC, HAoEC, HPAEC and HSaVEC) and four microvascular (HDMEC, HDBEC, HCMEC and
HPMEC) cells from up to three individual donors were used for TFPI
analysis. FACS results revealed that ~80% of the cell surface TFPI of
all analyzed ECs represents TFPI beta. ELISA measurements of supernatants and cell lysates showed that TFPI alpha was not affected
by PI-PLC treatment whereas TFPI beta increases in the supernatant
and decreases in the total cell lysate after PI-PLC treatment.
Conclusion: In conclusion, we demonstrated that both TFPI isoforms
are present on micro- and macrovascular ECs and that ~80% of cell
surface TFPI represents TFPI beta.
Disclosure of Interest: R. Pachlinger is an employee of: Baxter Innovations GmbH, A. Baldin-Stoyanova is an employee of: Baxter Innovations GmbH, F. Knofl is an employee of: Baxter Innovations GmbH,
N. Ullrich is an employee of: Baxter Innovations GmbH, F. Scheiflinger is an employee of: Baxter Innovations GmbH, M. Dockal is an
employee of: Baxter Innovations GmbH
VB13
Microparticles inhibit angiogenic activities of microvascular endothelial cells through a CD36 dependent
signaling pathway involving reactive oxygen species
Ramakrishnan DP1,2,3 and Silverstein RL1,3,4
1
Department of Molecular Medicine, Cleveland Clinic Lerner
College of Medicine of Case Western Reserve University;
2
Graduate Program in Molecular Medicine, Case Western
Reserve University School of Medicine, Cleveland; 3Vascular
Pathobiology, Blood Research Institute; 4Department of
Medicine, Medical College of Wisconsin, Milwaukee, USA
Objectives: CD36, a scavenger receptor, inhibits angiogenesis in microvascular endothelial cells (MVEC) via its ligand thrombospondin-1.
Phosphatidylserine (PS) exposed on the surface of cell-derived microparticles (MP), ≤ 1 lm vesicles shed from vascular cells by activation
or apoptosis, is also a ligand for CD36. We hypothesized that CD36MP interaction would also inhibit angiogenesis.
Methods: MP were prepared in vitro from THP1 cells, RBCs, MVEC
and human umbilical vein endothelial cells (HUVEC). In vitro matrigel
tube formation and transwell migration assays were used to measure
angiogenic activities of endothelial cells. DCF dye based assay was
used to measure reactive oxygen species (ROS).
Results: THP1 MP dose dependently inhibited MVEC tube formation
by > 50% (P < 0.05) and migration by 80% (P < 0.001). HUVEC,
MVEC and RBC derived MP also inhibited MVEC tube formation
showing that the antiangiogenic effect of MP is independent of their
cell of origin. Specificity of CD36 in MP induced inhibition of migration was demonstrated by showing enhanced inhibition in high CD36
expressing early passage MVEC compared to late passage MVEC with
low or no CD36 expression (80% vs. 33%; P < 0.001). Similarly,
CD36 negative HUVEC showed low inhibitory response to MP, but
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© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
ABSTRACTS
VB15
Microparticle levels in patients undergoing coronary
revascularization surgery
responses were restored in murine CD36 cDNA transfected HUVEC
(82% inhibition vs. 44%; P < 0.001). Phospholipid binding protein
Annexin V reduced the inhibitory effect of MP on MVEC suggesting
that PS was required (35%; P < 0.001). Since ROS generated in
response to CD36 mediated signals inhibits cell migration in macrophages, we tested and found that MP induced ROS (2 fold; P < 0.05)
and that the NADPH oxidase inhibitor apocynin blocked MP induced
ROS (> 95%) and partially (33%) reversed inhibition of tube formation in MVEC by MP (P < 0.05).
Conclusion: Our studies identify a novel mechanistic pathway through
which MP via their exposed PS inhibit MVEC angiogenic activities via
activation of a CD36-mediated signaling pathway involving NADPH
oxidase-mediated ROS generation.
Disclosure of Interest: None Declared.
Jeske W1, Walenga JM1, Escalante V1, Schwartz J2 and Bakhos M2
Cardiovascular Research Institute; 2Thoracic and Cardiovascular
Surgery, Loyola University Chicago, Maywood, USA
1
VB14
Enhanced presence endothelial dysfunction biomarkers
in major depression disease and progressive down-regulation during antidepressant treatment
Objectives: Patients with assisted circulation (cardiac bypass surgery,
left ventricular assist devices) have altered blood rheology that may
lead to hemostatic abnormalites. Patients undergoing on-pump bypass
surgery have been shown to have higher levels of inflammatory cytokines and markers of activated platelets, leukocytes and fibrinolysis.
Cellular microparticles formed at sites of altered shear stress may be
mediators of hemostatic or inflammatory alterations.
Methods: In this study, blood samples were collected post-incision,
30 min after conduit harvest and following the last graft anastomosis
from patients undergoing on-pump and off-pump cardiac revascularization surgery. Blood samples were double centrifuged to produce
platelet-free plasma (PFP). PFP was subsequently ultracentrifuged
(20,000 g for 90 min) to pellet microparticles. Aliquots were labeled
with lactadherin (phosphatidylserine) and either CD41 (platelet) or
CD235 (red blood cell) and analyzed by flow cytometry. Microparticles were identified by their forward and side scatter profiles relative to
polystyrene beads of known size (0.5, 0.8 lm).
Results: Patients undergoing on-pump surgery had a higher fraction of
lactadherin (+) microparticles than those undergoing off-pump surgery
at each time point. In both surgical groups, the fraction of microparticles expressing phosphatidylserine increased with duration of surgery
(On-pump: 47.3 27.3% post-incision to 63.0 6.4% following
anastomosis; Off-pump: 1.5 1.0% post-incision to 12.0 8.9% following anastomosis).
Conclusion: Microparticle generation occurs in surgical patients and is
directly associated with the degree of intervention. Measurement of
microparticle levels from all hematologic and endothelial cell types
may provide insight into mechanisms relevant to outcomes and allow
for tailoring of antithrombotic and/or anti-inflammatory therapy.
Disclosure of Interest: None Declared.
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Molina P1, Lopez-Vilchez I1, Diaz-Ricart M1, Navarro V2, SerraMillas M3, Pino M1, Gasto C3, Galan AM1 and Escolar G1
1
Department of Hemotherapy-Hemostasis. Hospital Clinic, CDB,
IDIBAPS, Univ Barcelona; 2Clinic Psychiatry Institute. Hospital
Clinic; 3Clinic Psychiatry Institute. Hospital Clinic, Barcelona,
Spain
Objectives: Alterations of serotonergic mechanisms, involved in major
depression (MD), inflammation and cardiovascular risk could also
induce endothelial dysfunction (ED). We investigated presence of ED
biomarkers in MD-patients; and its modulation during antidepressant
treatment with escitalopram a selective serotonin reuptake inhibitor
(SSRI).
Methods: Studies were performed in samples from MD-patients and in
an experimental model using cultured endothelial cells (HUVEC). We
evaluated different biomarkers of ED and inflammation in 12 MDpatients at the time of diagnosis (MD-0), and after 8 (MD-8) and
24 weeks (MD-24) of treatment with SSRI; and compared with 12
healthy individuals (HI). ED was assessed through evaluation of circulating endothelial cells (CEC) and endothelial progenitor cells (EPC),
by flow cytometry, and assessment in plasma of vWF and VCAM-1,
by ELISA. We also investigated modifications in the expression of
ICAM-1, vWF and tissue factor (TF) on cultured HUVEC exposed to
sera pools form each study group, by immunofluorescence.
Results: CECs were higher at MD-0 than in HI (P < 0.01). SSRI-treatment gradually reduced CEC levels to values observed in HI. EPCs
were lower at MD-0 than in HI, and tended to increase during SSRItreatment. Soluble markers of endothelial damage, vWF and VCAM1, were statistically increased in MD-0 vs. HI, showing reductions at
MD-24. Similarly, studies in HUVEC revealed an elevated expression
of the inflammation marker ICAM-1 when exposed to MD-0 sera
pools (P < 0.01 vs. HI) that was fully normalized with sera at MD-24.
Presence of vWF and TF was also elevated on the extracellular matrix
generated by HUVECs exposed to sera at MD-0 vs. HI, and tended to
decrease at MD-24.
Conclusion: Our data demonstrate ED and a pro-inflammatory state in
MD-patients. These findings were experimentally reproduced in HUVECs exposed to patients’ sera. Treatment with SSRI significantly
down-regulated the different biomarkers of ED investigated.
Disclosure of Interest: None Declared.
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VB16
Protective effects of connexins in atherosclerotic plaques in patients with carotid artery stenosis
Nakase T1, Ishikawa T2 and Miyata H3
1
Department of Stroke Science; 2Department of Neurological
Surgery; 3Department of Neuropathology, Research Institute for
Brain & Blood Vessels, Akita, Japan
Objectives: Fragility of atherosclerotic plaques in the internal carotid
artery can be a risk of brain infarction. The activation of inflammatory
cells, the vulnerability of vascular endothelial cells and the thrombogenesis of plaques have been reported to influence on the fragility of
atherosclerotic plaques. In this context, gap junctional intercellular
communication has been remarked as a critical factor of worsening of
atherosclerotic plaques. Therefore, we aimed to reveal the effect of
connexins (Cx), which compose gap junction, on the stabilization of
atherosclerotic plaques immunohistochemically.
Methods: The samples were obtained from patients who were performed the carotid endoarterectomy on the stenosis lesion of cervical
internal carotid artery. Sections were taken from paraffin blocks with
paraformaldehyde fixation. Tyramide signal amplification method
(CSA, DAKO) were used. Cx37 and Cx43 as gap junction markers
and CD36 as oxidized low-density lipoprotein receptor marker were
used for the primary antibody. The expression of Cx was immunohistochemically observed in the atherosclerotic plaques under an optical
microscope.
Results: The expression of Cx37 and Cx43 was observed in 4 and 5 of
11 samples, respectively. The immunopositve forms for Cxs were local-
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
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ABSTRACTS
VWF02
Allosteric activation of ADAMTS13 involves conformational changes induced by its substrate von Willebrand
factor
ized in the cytosol of inflammatory cells in the surrounding area of
necrotic lesion. The Cx37 was mainly coexpressed with CD36 positive
macrophage. The Cx43 was mainly coexpressed with CD36 positive
spindle cell. The percentage of symptomatic brain infarctions was significantly lower in the Cxs positive plaques compared with the Cxs
negative plaques (P = 0.036: 20.0% and 83.3%, respectively).
Conclusion: The Cxs may regulate the reactivity of inflammatory cells
in the atherosclerotic plaques and enhance the stabilization of the plaque.
Disclosure of Interest: None Declared.
Muia J1, Zhu J1, Haberichter SL2, Friedman KD2, Feys HB3,
Vanhoorelbeke K4, Gupta G1, Westfield LA1, Tolia NH1 and Evan
Sadler J1
1
Washington University School of Medicine, St. Louis;
2
BloodCenter of Wisconsin, Milwaukee, USA; 3Belgian Red
Cross-Flanders, Ghent; 4IRC, KU Leuven Kulak, Kortrijk, Belgium
Von Willebrand Factor
VWF01
Misfolding of the von Willebrand factor A1 domain in
Type 2 von Willebrand disease
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Objectives: We have surveyed the effect of Type 2B and Type 2M von
Willebrand Disease (VWD) mutations the structure and rheological
function of the Von Willebrand factor (VWF) A1 domain. These
mutations have a dynamic range of clinical manifestations from a paucity of VWF-platelet interactions to severe thrombocytopenia.
Methods: To assess function, we have developed a real-time highspeed video microscopy analysis of platelet translocation dynamics
under shear flow in a parallel plate microfluidic flow chamber chelated with recombinant A1 domains harboring these mutations. To
assess structure, we have developed a number of solution biophysical and thermodynamic metrics that classify these mutational variants of the A1 domain as Native (with varying thermodynamic
stability), Native-Like (having reduced secondary structure but
retaining some thermodynamic stability), and Molten Globule (a
complete lack of tertiary structure with residual secondary structure
characterized by the absence of a urea and thermal unfolding transition).
Results: Our analysis of translocation dynamics results in statistical
distributions of pause (residence) times that are proportional to the
strength of the A1-GPIb interaction and quantitatively correlate to
reported VWD patient platelet counts and the severity of thrombocytopenia. Our biophysical analysis demonstrates that the majority of
these mutations cause the A1 domain to misfold to the Molten Globule conformation (11 of the 17 variants studied are not natively structured).
R
Auton M
Department of Internal Medicine, Division of Hematology and
Dept. Biochemistry and Molecular Biology, Mayo Clinic,
Rochester, USA
Objectives: ADAMTS13 cleaves a cryptic site in von Willebrand factor
(VWF) domain A2 that is exposed when VWF multimers are stretched
by fluid shear stress. Severe ADAMTS13 deficiency impairs this regulatory mechanism and causes thrombotic thrombocytopenic purpura.
ADAMTS13 is a multidomain protein with metalloprotease (M), disintegrin-like (D), thrombospondin-1 (T), Cys-rich (C) and spacer (S)
domains, followed by seven T and two CUB domains. The functions
of distal T and CUB domains are poorly understood. We now show
that distal domains inhibit ADAMTS13 and binding to VWF activates
ADAMTS13.
Methods: Proteins were purified from HEK293 media or plasma. ADAMTS13 was assayed with FRETS-rVWF71 or plasma VWF. Small
angle X-ray scattering (SAXS) data were collected at the SIBYLS
beamline (LBL).
Results: ADAMTS13 construct MDTCS was ~4-fold more active than
ADAMTS13 toward FRETS-rVWF71, suggesting that distal T-CUB
domains are inhibitory. ADAMTS13 construct M-T7 (truncated after
T7) was as active as MDTCS but construct M-T8 had the same markedly decreased activity as full length ADAMTS13, indicating that T8
is involved in autoinhibition. Monoclonal antibodies recognizing T67
(7C4), T8 (19H4), and CUB domains (12D4) additively activated ADAMTS13 ~4-fold, equaling the increased activity of MDTCS or M-T7.
VWF, recombinant VWF D4-CK or VWF D4 activated full-length
ADAMTS13 up to 4-fold, but did not change the activity of M-T8 or
MDTCS. SAXS data for M-T4, M-T5, M-T7, M-T8 and ADAMTS13
suggest that ADAMTS13 is folded roughly in half, placing distal
domains close to the active MDTCS domains.
Conclusion: ADAMTS13 adopts a folded conformation in which distal
T-CUB domains, particularly T8, inhibit ADAMTS13 activity. Autoinhibition is relieved by antibodies that bind distal domains or by
ADAMTS13 binding to VWF domain D4. Thus, VWF serves as both
an ADAMTS13 activator and substrate, and VWF induced allosteric
activation would concentrate ADAMTS13 activity at sites of thrombosis.
Disclosure of Interest: None Declared.
VWF03
Phenotype assignment of Type 2 von Willebrand disease (VWD) variants by a single ELISA-based assay of
von Willebrand factor (VWF) functions
Conclusion: Taken together, the effect of these mutations on the intrinsic conformational properties of the A1 domain sufficiently accounts
for the in vivo properties of multimeric VWF. Whether the misfolding
results in loss or gain of function depends on the local secondary structure elements involved.
Disclosure of Interest: None Declared.
Roberts JC, Morateck PA, Christopherson PA, Yan K, Hoffmann
RG, Gill JC, Montgomery RR and The Zimmerman Program
Investigators
BloodCenter of Wisconsin/Medical College of Wisconsin,
Milwaukee, USA
Objectives: Variant VWD is cumbersome to diagnose and involves
multiple laboratory tests done by few specialized laboratories. We
have developed an ELISA-based screening assay capable of rapid
determination of relative qualitative and quantitative VWF functionality to correctly assign variant VWD.
Methods: 165 Zimmerman PPG VWD plasma samples were analyzed
on a novel ELISA-based platform. Relative values of VWF:Ag (antigen), VWF:IbCo (Ib cofactor, no ristocetin), VWF:RCo (ristocetin co-
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© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
ABSTRACTS
factor), VWF:F8B (binding to FVIII), VWF:CB3 (binding to collagen
III), and VWFpp (propeptide) were measured and standardized to
30% normal control standard on a single ELISA strip. The study
included 21 type 1, 30 type 1C, 23 type 2A, 23 type 2B, 20 type 2M, 22
type 2N, 4 type 3 VWD, and 22 potential hemophilia A (HA) subjects.
Samples were run in single wells for each assay and optical densities
(OD) were compared to OD of 30% and 100% standard control
plasma. ELISA read time was 30 min and full assay can be done in
3 h. Analysis of VWF functional profiles was performed using the
Mann-Whitney test, ROC analysis, and linear discriminant analysis
(LDA) was used to assign VWD variant classification.
Results: LDA provided a unified diagnostic algorithm. Excluding type
3 VWD, LDA correctly assigned variant VWD in 145/161 subjects
(90.1%). Leave-one-out (jackknife) LDA correctly assigned 138/161
subjects (85.7%) overall and correctly assigned type 1 85.7%, 1C
83.3%, 2A 78.3%, 2B 100%, 2M 85%, 2N 86.4%, and HA 81.8% suggesting its utilization in a general population. ROC had P < 0.001 and
area under the curve was > 0.9 for separating 2B from 2A and 2B from
type 1 with VWF:IbCo/VWF:Ag; 2M from 2A and 1C from 2A with
VWF:CB3/VWF:Ag; HA from 2N with VWF:F8B/VWF:Ag; and 1C
from type 1 with VWFpp/VWF:Ag.
Conclusion: This VWF functional screening assay is able to discriminate variant VWD with desirable overall accuracy > 85%, and may
expedite variant VWD classification.
Disclosure of Interest: None Declared.
VWF05
Association between von Willebrand factor and factor
VIII levels and their changes during the aging process
nez S1, Ogiwara K1, Grabell J2, James P2 and Lillicrap D1
Alba
Department of Pathology and Molecular Medicine; 2Department
of Medicine, Queen’s University, Kingston, Canada
1
or
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Objectives: von Willebrand factor (VWF) plasma levels are influenced
by both genetic and environmental factors, while levels of Factor VIII
(FVIII) are believed to be largely dependent on VWF. Studies have
shown that the levels of these proteins increase with age, but the nature
of their relationship during this time has not been studied. Our aim
was to study how VWF and FVIII are associated during aging and to
determine the nature of their changes, by evaluating markers of VWF
secretion and clearance.
Methods: VWF antigen (VWF:Ag), VWF propeptide (VWFpp),
VWFpp/VWF:Ag ratio and Factor VIII (FVIII:C) were measured in 3
normal populations comprised of 172 individuals: 52 young
(7 5 years), 42 middle-aged (41 6 years) and 78 old
(71 7 years), similar in gender and ABO blood type. Pearson correlations were performed and a P < 0.05 was considered significant.
Results: Distinct patterns of VWF and FVIII were observed with
aging. In the young, VWF levels were not determined by secretion
(P = 0.956, r2 = 0.00), but by clearance mechanisms (P < 0.0001,
r2 = 0.59). With aging, while VWF clearance continued to be a major
determinant, high VWF levels were also the result of increased secretion (P < 0.0001; middle-age: r2 = 0.45; old: r2 = 0.58). Interestingly,
FVIII:C was not correlated with VWF:Ag in the young population
(P = 0.334, r2 = 0.02), but as VWF:Ag increased with age, a positive
correlation between the two appeared (middle-age: P = 0.012,
r2 = 0.15; old: P < 0.0001, r2 = 0.29). This association was dependent
on higher VWF secretion, as shown in the old group where the correlation between FVIII and VWFpp was strongest (P < 0.0001, r2 = 0.24);
and less dependent on VWF clearance (old: P = 0.012, r2 = 0.09).
Conclusion: Our findings suggest that in contrast to young individuals, plasma VWF levels in the elderly are the result of both
increased secretion and reduced clearance of VWF. This study also
shows that FVIII levels are less dependent on VWF under normal/
young conditions, and that its increase with age depends on a higher
secretion of VWF.
Disclosure of Interest: None Declared.
or
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Casey L1, Bowman M1, Umana B2, Maurice D2 and James P3
1
Department of Pathology and Molecular Medicine; 2Department
of Pharmacology and Toxicology; 3Department of Medicine,
Queen’s University, Kingston, Canada
Conclusion: We demonstrate for the first time increased angiogenesis
in BOEC from a type 2B VWD patient through the increased tubule
length in 3D culture and an increased release of the angiogenic mediator Ang-2. These results may explain the angiodysplasia that occurs in
type 2B VWD patients, thus meriting the exploration of anti-angiogenic therapeutics for GI bleeding in type 2B VWD.
Disclosure of Interest: None Declared.
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VWF04
Patient-derived blood outgrowth endothelial cells from
a Type 2b von Willebrand disease patient exhibit
increased angiogenesis
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Objectives: In up to 20% of cases, von Willebrand disease (VWD) is
commonly associated with angiodysplasia, the vascular malformation
of the gastrointestinal (GI) tract. Recently, von Willebrand factor
(VWF) has been identified as a negative regulator of angiogenesis
through an intracellular pathway involving angiopoietin-2 (Ang-2)
storage and an extracellular pathway through VWF-integrin avb3
interaction. The current study aims to investigate the development of
angiodysplasia in type 2B VWD using patient-derived blood outgrowth endothelial cells (BOEC).
Methods: BOEC isolated from a type 2B VWD patient with the
p.Val1316Met VWF mutation were analyzed for VWF (n = 3) and
Ang-2 (n = 3) secretion into the supernatant using ELISA. To explore
angiogenic potential, total tubule length formed in 3D Matrigel culture
of type 2B BOEC (n = 1) was quantified by ImageJ analysis of images
of cells at 49 magnification. A student’s t-test with a cutoff P-value of
< 0.05 was used to determine significance.
Results: While type 2B VWD BOEC exhibited increased VWF secretion compared to normal BOEC, this result was not significant
(P = 0.07). However, Ang-2 secretion in type 2B VWD BOEC was
increased significantly (P = 0.002). Moreover, total tubule length
formed in 3D culture was increased in type 2B VWD BOEC
(P = 0.049) compared to normal BOEC.
Table 1. Angiogenesis in type 2B VWD BOEC
Normal
Type 2B
P-value
Normal VWF
Secretion (%)
Normal Ang-2
Secretion (%)
Total tubule
length (lm)
100
171
0.07
100
248
0.002
18927
21759
0.049
VWF06
Diurnal variation of von Willebrand factor in plasma:
the Bispebjerg study of diurnal variations
Timm A1, Fahrenkrug J2, Jørgensen HL2, Sennels HP3 and Gøtze JP1
1
Clinical Biochemistry, Rigshospitalet, Copenhagen University
Hospital; 2Clinical Biochemistry, Bispebjerg Hospital, University
Hospital, Copenhagen; 3Clinical Biochemistry, Glostrup Hospital,
Copenhagen, Denmark
Objectives: Quantitation of von Willebrand factor (VWF) in plasma is
a central element in assessing von Willebrand disease (VWD). VWF
activity is known to vary, which has partly been ascribed to biological
and preanalytical variation. However, a possible diurnal expression of
VWF has not been thoroughly tested. We examined whether VWF
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
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92
ABSTRACTS
antigen and VWF activity in plasma display a diurnal profile in
healthy young males, and whether such variation is related to changes
in release or elimination.
Methods: Plasma from 20 healthy young males was collected at 9 timepoints over 24 h (15 h of light and 9 h of darkness); the plasma concentration of melatonin was used as an internal control to confirm the
normal 24-hour rhythms of the individual participants. VWF activity
was measured optically by ristocetin cofactor activity methodology.
Measurement of VWF antigen, coagulation factor VIII (FVIII) and
VWF propeptide (VWFpp) were performed by ELISA analyses. Time
related data for all 20 subjects were analyzed under the assumption of
24-h periods, using the methods for cosinor-rhythmometry as
described by Nelson W et al, 1979. To examine whether a diurnal pattern was related to changes in elimination or secretion, the ratio
between a) coagulation factor VIII and VWF and b) VWFpp and
VWF was determined.
Results: The data revealed a significant diurnal variation (P = 0.02)
with a total amplitude of 22.6% in VWF antigen concentrations. A
pronounced variation in VWF activity was also observed, although
not significant according to the 24-h statistical model. A prominent
diurnal variation of the ratio FVIII/VWF in addition to an almost
constant ratio between VWFpp to VWF suggests changes in release
and not in clearance.
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Methods: We obtained samples from 26 patients with IBD receiving
anti-inflammatory treatment. Fifteen patients had Crohn disease, 10
patients had ulcerative colitis, and 1 patient had unclassified IBD. Age
ranged from 3 to 15 years old. aVWF was measured using an antibody
against von Willebrand factor (VWF) A112 that recognizes sequence
Glu1238–Ser1253, which has previously shown to regulate the binding of
VWF to platelet glycoprotein 1ba. VWF antigen (VWF Ag) and ristocetin cofactor activity (RCoF) assays were performed using established automated methods.
Results: There was a median of 2.5 times higher reactivity with antiA112 antibody in IBD patients compared to normal subjects, with interquartile range of 1.3–3.8. The median for VWF Ag and RCoF were
126.0% and 98.5%, with interquartile ranges of 90.0–192.5% and
73.3–153.3%, respectively. As expected, VWF Ag showed a strong
direct correlation with RCoF (Pearson’s correlation coefficient
R = 0.95, P = 0.00001). aVWF on the other hand, showed no correlation with RCoF (R = 0.31, P = 0.13) and VWF Ag (R = 0.27,
P = 0.19).
Conclusion: As expected, vWF Ag and RCoF were elevated among
patients with IBD. aVWF, measured using anti-A112 antibody, also
appeared to be elevated among patients with IBD compared to normal
subjects. The weak inverse correlation between aVWF and both VWF
Ag and RCoF was unexpected. This observation may suggest a unique
physiological regulation of aVWF under chronic inflammatory state
like IBD as opposed to other more acute inflammatory conditions.
Disclosure of Interest: None Declared.
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VWF08
Molecular characteristics and recombinant protein
expression of type 2 and type 3 von Willebrand disease: studies of a Chinese cohort of 25 patients
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Conclusion: Diurnal variation in von Willebrand antigen and activity
in plasma represents an important aspect of the biological variation.
Standardized time-of-day plasma sampling for quantitation of VWF
in VWD patients seems warranted.
Disclosure of Interest: None Declared.
VWF07
Increased active von Willebrand factor among patients
with inflammatory bowel disease
Teruya M1, Cruz MA1, Teruya J1,2,3,4, Nguyen TC5,6, Pelkey GR3,4
and Hui S-KR3,4
1
Department of Medicine; 2Department of Pediatrics;
3
Department of Pathology & Immunology, Baylor College of
Medicine; 4Department of Pathology, Texas Children’s Hospital;
5
Department of Pediatrics-Critical Care, Baylor College of
Medicine; 6Department of Pediatrics-Critical Care, Texas
Children’s Hospital, Houston, USA
Objectives: Increased active von Willebrand factor (aVWF) has been
found in von Willebrand disease (VWD) type 2B, acquired or congenital thrombotic thrombocytopenic purpura, and malaria. It has also
been associated with increased morbidity in systemic inflammatory
response syndrome and sickle cell anemia. Purpose of our study is to
investigate the status of aVWF among patients with inflammatory
bowel disease (IBD).
Yin J, Su J, Ma Z, Zhao X, Wang Z, Yu Z, Ouyang W, Bai X and
Ruan C
Jiangsu Institute of Hematology, The First Affiliated Hospital of
Soochow University, Key Laboratory of Thrombosis and
Hemostasis of Ministry of Health, Suzhou, China
Objectives: Gene mutations are the common events in the pathogenesis
VWD patients. Jiangsu Institute of Hematology is one of main centers
focusing on the treatment of VWD patients in China. More than 200
VWD patients were enrolled in our center. In this study, we evaluated
the VWF gene in 25 type 2 and type 3 patients, and restructured the
mutant VWF, further explored the pathogenesis of VWD in Chinese.
Methods: Among 25 VWD patients, 12 were type 3 and 13 were type 2
(8 type 2A, 1 type 2M, 4 unclassified). All 52 exons and intron–exon
boundaries of the VWF gene were amplified by PCR and were DNA
sequenced. Mutant VWF was reconstructed by site-directed mutagenesis, and rVWF antigens were analyzed by ELISA.
Results: Sequence analysis detected 17 mutations in VWD patents, 19
(p.V9L, p.E251G, p.Q469X, p.C827Y, p.W856X, p.E1015X, p.C1165R,
p.S1442G, p.Y1974X, p.Y2043X, p.Q895R, p.D1184N, p.S1731P,
c.4990delC, c.5170G>A, c.6678_6679insC, p.F1299LfsX4, p. D1472PfsX39,
p.C2227LfsX14) of which were novel. Of all mutations, seven were nonsense
(p.R34X, p.Q469X, p.W856X, p.E1015X, p.R1779X, p.Y1974X and
p.Y2043X), 12 were missense (p.D141N, p.V9L, p.E251G, p.C827Y,
p.Q895R, p.T1122M, p.C1149Y, p.C1165R, p.D1184N, p.S1442G,
p.S1731P, p.L2142F). Three splice-site muations (c.3379 + 1G>A,
c.5170G>A, c.1730-2A>G), one insertion (c.6678_6679insC) and 4 deletion
(C.4990delC, c.3895delT, c.4414delGinsCC, p. D1529_V1530del) were also
found. As expected, most termination codon mutations were identified in
type 3 except two in type 2. Mutation c.6678_6679insC produced frame-shift
translation and a new stop codon in 2241 acid amine, which were detected in
two unrelated VWD patients. Furthermore, we transfected HEK293 cell
with VWF mutations (p.Q895R and p.S1731P). The levels of rVWF in medium were lower than that of wide type VWF.
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ABSTRACTS
Conclusion: This study widened the mutational spectrum of VWD in
Chinese patients. Gene analysis and recombinant protein expression
are helpful tools to elucidate the pathogenesis of VWD.
Disclosure of Interest: None Declared.
VWF09
Effect of factor von Willebrand on fibrin structure and
lysis
Marchi R1, Rojas H2 and De Agrela M1
Medicina Experimental, Instituto Venezolano de Investigaciones
Cientıficas; 2Inmunologıa, UCV, Caracas, Venezuela, Republic of
Bolivarian
0
0.51 0.23
216 51
0
1.47 0.76*
455 149
8.3 1.1
CD
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16732 1566*
0.185 0.082*
pi
22208 6727
0.047 0.018
VWF11
Identification of a novel polymorphism in P.N258 of
the GP1BA gene
iza
With FvW
Co
Polymerization
Lag phase (sec)
Slope (mOD/sec)
MaxAbs (mOD)
External Fibrinolysis
T50%
LR (sec)
Permeation
Ks (cm2) 9 109
Without FvW
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Objectives: Factor von Willebrand (FvW) is constitutively secreted by
the endothelium and incorporated to the fibrin clots under slow clotting conditions. In the present work we have studied the effect of FvW
on clot structure and fibrinolysis.
Methods: 100 lg of purified fibrinogen was mixed with 0.6 lg FvW or
Tris-buffered saline (control clot formed without FvW). Then 5 mM
of CaCl2 (final) was added and incubated during 1 min. Clotting was
triggered by adding a mixture of thrombin – activated factor XIII
(1 unit mL–1 and 48 ng, respectively). The optical density (OD)
changes were recorded at 350 nm during 2 h and a half. After this
time, on the top of the clots 12 lg mL–1 of plasmin were added, and
OD was read until baseline values. Permeation coefficient was calculated with and without FvW, using the same clotting conditions as for
fibrin polymerization. Finally, clot structure was visualized and analyzed by laser scanning confocal microscopy (LSCM).
Results: FvW dramatically increased fibrin polymerization, decreased
fibrin lysis rate, and increased Ks (Table). The analysis of LSCM
images showed that FvW increased fibrin fibers diameter and networks0 pores size.
Objectives: The aim of study is to characterize the genotype of Von
Willebrand type III patients in Pakistani population.
Methods: Blood samples of 48 known index cases of type III VWD
were collected in this cohort from throughout Pakistan including
NIBD & BMT Karachi, Chughtais lab, Children’s Hospital Lahore,
PAEC Islamabad and HMC Peshawar.Genomic DNA was extracted
from peripheral blood by QIAamp DNA Blood mini kit (Qiagen).
Sequencing of VWF and interpretation was done by medical technologist from NIBD,Pakistan undersupervision of experts in university
clinics Bonn, Germany. All 52 exons were amplified with 56 pairs
primers. Direct sequencing of VWF was done for identification of
mutation using BigDye termination Cycle sequencing v3.1 and ready
reaction kit by automated ABI-3130 DNA sequencer (Applied Biosystems, Foster city, CA, USA). DNA Sequences were analyzed on SeqScape (Applied Biosystems). Variation in VWF sequences were
checked on ISTH-SSC VWD homepage, VWFdb hemobase and biobase biological database. Simple descriptive was applied for frequencies.
Results: We found mutations in 46 (95.83%) out of 48 cases and distributed as 28 cases (60.8%) homozygous and 18 (39.1%) were compound heterozygous. Total 58 mutations were identified comprising 37
missense (52%), 20 nonsense (35%), 2 small deletion (3%), splice site
and insertion 3(5%). Twenty-one of these were novel in this cohort
and most of them detected as missense mutations. Nonsense
c.3931C>T, p. Gln (CAG) 1311 X (TAG) was found most common
mutation in Pakistani patients.
Conclusion: Two patients required multiplex ligation dependent probe
amplification (MLPA) for detection of large deletions. Most of the
mutations identified in this cohort were homozygous due to Consanguinity in the family of the patients.
Disclosure of Interest: None Declared.
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1
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20.4 1.6*
*P < 0.05
Conclusion: FvW covalently crosslinked to fibrin modified fibrin structure (increasing fibrin diameter and the pores filling space of the meshwork) that favors the fibrin lysis rate.
Disclosure of Interest: None Declared.
VWF10
Genotype characteristics of von Willebrand disease
type III in Pakistani patients
Naz A1, Yadegari H2, Ahmed S3, Driesen J2, Oldenburg J2, Ahmed
N4, Tariq S4, Amanat S5, Raziq F6, Nadeem M3 and Shamsi TS7
1
Thrombosis and Haemostasis, National Institute of Blood
Disease & Bone Marrow Transplantation, Karachi, Pakistan;
2
Institute of Experimental Haematology and Transfusion
medicine, University Clinics Bonn, Bonn, Germany; 3National
Institute of Blood Disease & Bone Marrow Transplantation,
Karachi; 4Children Hospital Lahore, Lahore; 5Pakistan Atomic
Energy Commission Islamabad, Islamabad; 6Hayatabad Medical
Complex Peshawar, Peshawar; 7Hayatabad Medical Complex
Peshawar, Karachi, Pakistan
Woods A1, Sanchez-Luceros A1, Paiva J2, Romero ML2, Kempfer
AC1 and Lazzari MA1
1
Hemostasis and Thrombosis, Imex-Conicet, National Academy
of Medicine, Buenos Aires, Argentina; 2Hemostasis and
Thrombosis, National Academy of Medicine, Buenos Aires,
Argentina, C.A.B.A., Argentina
Objectives: Platelet-type von Willebrand disease (PT-VWD) and type
2B von Willebrand disease (2B-VWD) are rare bleeding disorders. The
differential diagnosis between the two entities is especially challenging
as evidenced by high levels of misdiagnosis of both conditions, particularly PT-VWD. GPIba is the largest component of the GPIb/IX/V
platelet receptor complex and carries the VWF-binding site. Five
mutations and 6 polymorphisms (SNP) in the GP1BA gene were
reported worldwide (www.pt-vwd.org). Recently, we have reported the
6th mutation, the p.W246L.
Methods: Genotypic analysis for the GP1BA gene was performed on
DNA from 100 controls and 12 2B-VWD patients (pts). It was amplified by PCR, and sequenced (ABI PRISM 310 Genetic Analyzer PE
Applied Biosystems). We have adopted the new nomenclature for
numbering amino acids of GPIba as recommended by M Othman.
Allelic frequencies were determined by gene counting. Chi-square was
used for statistical analysis.
Results: We identified a novel single synonymous substitution (C/T)
located at nucleotide 3842 in the leucine rich repeat C-terminal domain
of the GP1BA gene. This transition C/T is located at residue N258,
and resulted in no change of amino acid. Six controls and 3 2B-VWD
pts were heterozygous; therefore, the allelic frequencies were: 0.97 and
0.875 for the C allele, and 0.03 and 0.125 for the T allele in controls
and 2B-VWD pts, respectively. No statistical difference was found
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
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ABSTRACTS
between the frequencies of T allele between controls and 2B-VWD pts
(P = 0.091).
Conclusion: According to our results, we consider this substitution C/T
at residue N258 as a novel SNP in GB1BA gene, which does not affect
the function of GPIba. This new description adds more genotypic
characterization of the GP1BA gene.
Disclosure of Interest: None Declared.
with laboratory and clinical data of patients with various types of
VWD.
Methods: Complete VWF gene loci (exons, introns and flanking
regions) were sequenced using long-range PCRs combined with Sanger/next generation sequencing. Laboratory investigations included
Factor VIII:C, VWF antigen, VWF gp Ib-binding, PFA-100 closure
time, VWF collagen binding, multimeric analysis, and blood group. In
addition, CRP and BMI were assessed as environmental confounders.
Results: Molecular analysis of whole VWF gene in 92 unrelated
patients with VWD has led to identification of 281 missense sequence
variations, 182 heterozygous and 99 homozygous. Furthermore, we
found 277 sense sequence variations (193 heterozygous and 84 homozygous). Numerous intronic variants were detected, in addition. The
highest allele frequency of sequence variation (mutation) per nucleotide was detected in exon 13, 18, 20 and 28. Several mutations are
newly described. Genetic results are presented and analyzed together
with a comprehensive panel of tests used for the primary diagnosis of
VWD, and a standardized bleeding score.
Conclusion: Even when a comprehensive panel of assays including
genetic analysis is used both diagnosis as well as classification of VWD
remain uncertain in some cases. Especially classification of VWD
based on the relation between level and function and reflecting simplicity and clinical utility remains unreliable in a significant proportion of
patients.
Disclosure of Interest: None Declared.
VWF12
Contribution of defective von Willebrand factor (VWF)
multimerization, regulated storage, and secretion to
type 1C von Willebrand disease (VWD)
Jakab DA1, Jacobi PM1 and Haberichter SL1,2
BloodCenter of Wisconsin; 2Pediatrics, Medical College of
Wisconsin, Milwaukee, USA
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VWF13
Diagnosis of von Willebrand disease using a comprehensive panel of assays including gene analysis by
next generation sequencing
von Depka M, Detering C, Martensen H, Halves C and EkhlasiHundrieser M
Werlhof-Institute, Hannover, Germany
Objectives: Von Willebrand disease (VWD) is the commonest inherited
bleeding disorder and results from diminished or dysfunctional von
Willebrand factor (VWF). However, diagnosis of VWD is difficult
because the relationship between laboratory assays and clinical presentation remains uncertain. Here, we present genetic results combined
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VWF14
Overlap of collagen 4 and platelet binding sites in
VWF A1 domain
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Objectives: One mechanism causing type 1 VWD is the reduced survival of VWF in plasma (type 1C VWD). Little is known about the
mechanisms causing type 1C VWD, but it is assumed that VWF
undergoes normal intracellular processing/secretion with rapid plasma
clearance. We hypothesized that defective intracellular processing may
contribute to the type 1C phenotype.
Methods: We studied 12 VWF sequence variations (C1130Y, R1315C,
R1315L, V1411E, N2041S, Y2160C, W1144G, V2141I, R1205H,
N1231S, R1527W, S2179F) identified in patients enrolled in the
Zimmerman Program for the Molecular and Clinical Biology of VWD
meeting the criteria for type 1C VWD (VWFpp/VWF:Ag ≥ 3 & VWF:
Ag ≤ 30 IU dL–1). Variants were expressed homozygously or heterozygously (with varying ratios of wild-type (WT) to variant VWF) and
VWF secretion, multimer structure, and pseudo-Weibel-Palade body
(pWPB) formation assessed. Collagen-, GPIba-, and FVIII-binding
were analyzed.
Results: Six variants had severely decreased secretion with multimer
and binding function defects when homozygously expressed. These
variants did not form pWPB and co-localized with the endoplasmic
reticulum, consistent with severely impaired secretion. Two variants
had moderately reduced secretion (one with multimer and functional
defects). The remaining variants had normal processing and function.
Co-expression with WT VWF mostly corrected processing and functional defects, but some variants still had moderately reduced secretion.
Conclusion: When variants were homozygously expressed, we observed
many processing and functional defects. Only 4 variants demonstrated
normal processing and function. Heterozygous expression (mimicking
patients) corrected most of the defects, although reduced secretion persisted for a subset of variants. While reduced plasma survival of VWF
is a major determinant of the type 1C phenotype, additional processing defects may contribute to the severity of the overall VWD phenotype.
Disclosure of Interest: None Declared.
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Schlauderaff AC1, Haberichter SL1,2, Slobodianuk TL2, Morateck
PA2, Montgomery RR1,2, Flood VH1 and on behalf of Zimmerman
Program for the Molecular and Clinical Biology of VWD
1
Pediatrics, Medical College of Wisconsin; 2Blood Research
Institute, BloodCenter of Wisconsin, Milwaukee, USA
Objectives: Von Willebrand factor (VWF) plays a critical role in coagulation by tethering platelets to injured subendothelium. VWF binds
platelet glycoprotein Iba (GPIba) via the VWF A1 domain. Collagens
1 and 3 bind to the VWF A3 domain, while collagen 6 binds to the
VWF A1 domain. We recently localized the binding site for type 4 collagen to the VWF A1 domain and now investigate its relationship to
the platelet-binding domain.
Methods: Scanning alanine mutagenesis was performed for the VWF
A1 domain region encompassing residues 1387–1412. Full length
VWF DNA was expressed in HEK293T cells, and secretion measured
by VWF antigen. Multimer distribution was assessed by gel electrophoresis. Collagen binding ELISAs were performed with types 1, 3, 4
and 6 collagen. Platelet GPIba-binding ELISAs were performed using
recombinant mutant GPIba. All results were normalized to VWF antigen to control for total protein present.
Results: All alanine constructs had a normal expression profile compared to wild type VWF with normal multimer distribution. No difference in binding to types 1 and 3 collagen was observed. However,
several alanine mutants had decreased binding to types 4 and 6 collagen and/or platelet GPIba. Variants 1392A, 1395A, 1399A, 1405A
and 1406A had significantly decreased collagen 4 and 6 binding. Variants 1389A, 1396A, 1397A, 1400A, and 1404A had significantly
decreased platelet GPIba binding, while variants 1402A and 1407A
had decreased binding to both platelet GPIba and collagens 4 and 6.
A construct with a novel 1402P mutation also showed absent binding
to collagen 4, 6, and platelet GPIba.
Conclusion: Although platelet GPIba and collagens 4 and 6 bind to the
VWF A1 domain, these ligands for the most part interact with different regions of VWF. VWF mutations in the regions that affect both
ligands may confer increased bleeding due to disruption of multiple
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© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
ABSTRACTS
VWF16
Update on bleeding scores in the Zimmerman program:
comparison of ISTH BAT, PBQ, and MCMDM-1VWD
scoring systems
ligand binding sites as compared to mutations that disrupt exclusively
collagen or exclusively platelet interactions.
Disclosure of Interest: None Declared.
Flood VH1, Gill JC1,2, Christopherson PA2, Bellissimo DB2,
Friedman KD2, Haberichter SL1,2, Udani R2, Montgomery RR1,2
and on behalf of Zimmerman Program for Molecular and Clinical
Biology of VWD
1
Pediatrics, Medical College of Wisconsin; 2Blood Research
Institute, BloodCenter of Wisconsin, Milwaukee, USA
VWF15
Evaluation of six commercial von Willebrand factor
collagen binding assay kits
Objectives: Diagnosis of von Willebrand disease (VWD) is generally
considered to require a personal and family history of bleeding as well
as laboratory findings consistent with the diagnosis. While von Willebrand factor (VWF) levels are easily quantified, bleeding symptoms
are less amenable to standardization. Recent attempts at calculating
bleeding scores (BS) have utilized several different methodologies.
Methods: We analyzed 440 healthy control subjects and 627 index
cases with all types of VWD enrolled in the Zimmerman Program for
the Molecular and Clinical Biology of VWD using 3 different bleeding
assessment tools (ISTH BAT, PBQ, and MCMDM-1VWD). Laboratory testing, including VWF antigen and VWF ristocetin cofactor
activity, was performed in a central laboratory. DNA sequencing of
the VWF gene included all coding sequence.
Results: Bleeding scores were highest with the ISTH BAT, while the
PBQ and MCMDM-1 VWD scores were similar across groups. For
the healthy controls, mean ISTH BAT was 1.1 and mean PBQ and
MCMDM-1 VWD were 0.23. For all index cases, mean ISTH BAT
was 7.3, mean PBQ 6.8 and mean MCMDM-1 VWD 6.7. Older age
was significantly associated with BS (P < 0.001). Female gender was
significant using the ISTH BAT (P < 0.02) and MCMDM-1VWD
(P < 0.05) but not the PBQ. Presence of a pathogenic sequence variation in VWF was also statistically significant (P < 0.02) for all scoring
systems. As expected, VWD diagnosis correlated with BS, with a mean
ISTH BAT of 6.5 for type 1, 8.6 for type 2, and 16 for type 3 VWD
subjects. However, neither VWF antigen nor ristocetin cofactor activity displayed a significant correlation with BS regardless of the bleeding assessment tool used or the type of VWD.
Conclusion: VWF levels are best correlated with BS in more severe
forms of VWD. Although the ISTH BAT yields higher overall scores,
it performs similarly to other bleeding assessment tools. Lack of correlation with laboratory values, however, may limit the relevance of
bleeding score data for diagnostic purposes.
Disclosure of Interest: None Declared.
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D
Mean Precision
(CV%) (n = 18)
14.1%
16.2%
18.1%
7.3%
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3.6%
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Rachel LR, Warad DM, Cayou JG, Pruthi RK and Chen D
Hematopathology, Mayo Clinic, Rochester, USA
Objectives: Von Willebrand factor (VWF) collagen binding assay
(CBA) has been shown to be the most sensitive method for detecting
loss of VWF high molecular weight multimers (HMWM) in both type
2 VWD and AVWS. However, its wide application has been hindered
by the low precision and divergent laboratory characteristics of various kits. The study objective was to compare the laboratory performance of 6 commercial CBA kits.
Methods: Six CBA kits (A-F) from 5 manufacturers were obtained. Citrated plasma samples from 1 normal donor, 4 VWD patients (one
each of type 1, 2, 3 and AVWS), and 3 commercial controls were tested
repetitively for 3 days. Kits with low inter-precision (CV > 10%) were
excluded. Remaining kits were further tested using a larger subset of
patient samples (see Table) to evaluate their sensitivity to loss of
HMWM. The VWF:CBA/Ag ratios were normalized using the average ratio of the normal samples as a divisor.
Results: Of the 6 CBA kits, only 3 kits (D, E and F) had adequate precision with CV < 10% (see Table). Due to the lack of a common certified CBA calibrator, the absolute VWF:CBA activities of the
individual samples are slightly different between kits. Therefore, the
VWF:CBA/Ag ratios need to be normalized for the purpose of comparison. The average and range of the normalized VWF:CBA/Ag
ratios of the normal and type 1 VWD samples were all close to 1.0,
while VWF:CBA/Ag ratios were markedly decreased in samples from
type 2 VWD and AVWS patients.
95
Not Applicable
1.00
1.00
0.89
0.90
0.83
0.17
0.19
0.18
0.48
0.52
0.49
1.00
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Normal donor
(n = 7)
Type 1 VWD
(n = 4)
Type 2 VWD
(n = 4)
AVWS (n = 5)
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Mean Normalized VWF:CBA/Ag Ratios
Conclusion: There are wide ranges of precision of the commercially
available CBA kits. Normalization of the VWF:CBA/Ag ratios is necessary due to the lack of a certified CBA calibrator. Three of the 6 kits
appear to have desirable precision and sensitivity to VWD or AVWS
due to the loss of HMWM.
Disclosure of Interest: None Declared.
VWF17
Improved diagnosis of VWD in affected family members using the isth bleeding score
Christopherson PA1, Gill JC1,2, Flood VH2, Friedman KD1,
Haberichter SL1,2, Montgomery RR1,2 and on behalf of The
Zimmerman Program Investigators
1
Blood Research Institute, BloodCenter of Wisconsin; 2Pediatrics,
Medical College of Wisconsin, Milwaukee, USA
Objectives: Use of bleeding assessment tools (BAT) to quantify bleeding symptoms is of growing interest for the evaluation and potential
diagnosis of von Willebrand Disease (VWD). To explore this further,
we assessed the correlation of bleeding score (BS) with von Willebrand
Factor (VWF) levels in Index Cases (IC) who were clinically diagnosed
with VWD and their Affected Family Members (AFM).
Methods: We analyzed the quantitative BS using the ISTH Bleeding
Assessment Tool (ISTH- BAT) and current VWF antigen (VWF:Ag)
or VWF ristocetin cofactor activity (VWF:RCo) levels in subjects
enrolled in the Zimmerman Program for the Molecular and Clinical
Biology of VWD. This included 232 type 1 (VWF:Ag or VWF:RCo
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
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ABSTRACTS
≤ 40) and 93 low-VWF (LVWF, VWF:Ag or VWF:RCo between
40 IU dL–1 and the lower end of the normal range) Index Cases as well
as 276 type 1 and 209 LVWF AFM. A similar approach was taken to
compare the ISTH BS to VWF levels in 168 type 1H (historical levels
below the normal range but not substantiated in current testing) and
203 type 2 VWD IC and AFM.
Results: No correlation was found between BS and VWF:Ag level in
type 1 Index Cases, however type 1 AFM did show an increase in BS
with decreasing VWF:Ag level. Similarly, for IC with type 2 VWD,
VWF:RCo levels did not correlate with BS, while it did for their
AFM. For the type 1H cohort, there was no association of current
VWF levels with BS in the IC or the AFM, adding further uncertainty
to the diagnosis of VWD in these patients.
Conclusion: In summary, correlation of BS and VWF levels was found
for AFM but not for IC with type 1 or type 2 VWD. Quantitative BS
may be useful in diagnosis of AFM, but appears less useful in IC, particularly when obtained after the initial diagnosis.
Disclosure of Interest: None Declared.
VWF19
Effect of recombinant von Willebrand factor and fractions thereof on the procoagulant activity of factor VIII
Till S, Knappe S, Scheiflinger F and Dockal M
Baxter Innovations Gmbh, Vienna, Austria
VWF18
Classification of patients with von Willebrand disease
after systematic genotypic analysis
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Objectives: Von Willebrand disease (vWD) is a bleeding disorder
caused by inherited defects in the concentration, structure, or function
of Von Willebrand factor. VWD can be divided in three different subclasses, but classification remains difficult if based on well-standardized laboratory assays. Since classification is still a challenge we
initiated a national reference centre for von Willebrand factor genomic
analysis. The aim of this center is to improve vWF classification. To
do so, genetic abnormalities in the vWF gene in patient suspected to
suffer from vWD will be correlated to the other vWF parameters.
Methods: DNA of patients suffering from vWD was analyzed by Sanger sequencing the coding regions including the intron-exon boundaries of the vWF gene. Large deletions and duplications in the vWF
gene were detected by MLPA.
Results: Up to February 2014, 287 patients were analyzed. 135 patients
had a genetic abnormality. The mutations found were 10 nonsense
mutations, 129 missense mutations, 11 small deletions, 4 duplications,
and 7 large deletions detected by MLPA. In 152 patients no mutations
were found. Of all mutation detected 125 were reported previously and
29 were novel. 20 of the novel mutations were missense mutations, 3
nonsense and 6 small deletions. 37 patients were carrying the D1472H
polymorphism affecting the von Willebrand Factor Ristocetin cofactor activity as reported by Flood et al, Blood, 2010. The de novo mutations were further analyzed by in silico analysis.
Conclusion: Our systematic genotypic approach resulted in 47% of the
cases in a confirmation of von Willebrand disease. Furthermore, we
were able to confirm in 82% of the cases a vWD classification. Finally,
we expect that our approach will result in the identification of specific
haplotypes since the rate of polymorphisms in von Willebrand factor
is high. The results of our analyses will be included in ISTH/WFHrelated registries.
Disclosure of Interest: None Declared.
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Schoormans S1, Duren CV1, Krouwel S1, Pennings M2, Diekstra
A2, Simons A2, Hoefsloot L2, Laros B3, Brons P4 and Heerde WV1
1
Department of Laboratory Medicine, Laboratory of Hematology,
Unit Thrombosis Hemostasis; 2Department of Human Genetics;
3
Department of Hematology; 4Department of Pediatrics,
Radboud University Medical Centre, Nijmegen, Netherlands
Objectives: Von Willebrand factor (VWF) is a multimeric plasma glycoprotein with multiple physiological functions. VWF mediates
between platelet surface receptors and the extracellular matrix component collagen during primary hemostasis. In addition, it binds and stabilizes procoagulant factor VIII (FVIII) in plasma. FVIII/VWF
complex formation prevents FVIII from interaction with lower affinity
binding partners such as FIXa, phospholipids and clearance receptors.
FVIII is also protected from enzymatic (in)activation when bound to
VWF. Thus, FVIII survival in the circulation depends on its ‘chaperone’ VWF.
Methods: In this study we investigated the effect of a recombinant
VWF (rVWF; BAX 111) and fractions thereof on FVIII in the absence
of platelet binding and activation. Therefore, activity of a recombinant
FVIII (rFVIII, Advate) was determined by thrombin generation
assays (calibrated automated thrombography, CAT) with different
coagulation triggers as well as by a chromogenic assay in FVIII/VWFdouble deficient platelet-poor plasma.
Results: Thrombin generation in FVIII/VWF-deficient plasma was
only partially restored by supplementation with 1 IU mL–1 FVIII.
Addition of rVWF further increased thrombin formation reaching a
normal plasma control. The activity increase was dependent on rVWF
concentration, reaching saturation at about 0.4 IU mL–1 rVWF,
which corresponds to a 20-fold molar excess of rVWF monomers.
rVWF fractions with reduced molecular weight showed a lower effect
on FVIII procoagulant activity. Thus, rVWF size-dependently protects or stabilizes FVIII in plasma in the absence of VWF’s plateletmediated effects. Similar results were obtained by the chromogenic
assay.
Conclusion: In conclusion, we established thrombin generation as a
new tool to characterize FVIII/VWF complexes. We showed that
FVIII activity is influenced by rVWF depending on its size and concentration, and that standard clinical assays may be affected by VWF
plasma levels.
Disclosure of Interest: S. Till Employee of: Baxter Innovations GmbH,
S. Knappe Employee of: Baxter Innovations GmbH, F. Scheiflinger
Employee of: Baxter Innovations GmbH, M. Dockal Employee of:
Baxter Innovations GmbH
VWF20
In vitro characterisation of missense mutations located
in the von Willebrand factor (VWF) D1 domain associated with quantitative VWF deficiency
Dsouza MM1, Cartwright A1, Budde U2, Goodeve AC1 and
Hampshire DJ1
1
Department of Cardiovascular Science, University of Sheffield,
Sheffield, UK; 2Hemostaseology Department, Medilys Hamburg,
Hamburg, Germany
Objectives: Quantitative deficiency of von Willebrand factor (VWF)
manifests as either type 1 (VWD1; mild-moderate reduction) or type 3
(VWD3; severe reduction) von Willebrand disease (VWD). VWD1 is
associated primarily with missense mutations that alter VWF protein
sequence, while VWD3 is primarily associated with mutations that
result in a null allele. Missense mutations located in the D1 domain of
VWF have however been shown to cause both VWD1 and VWD3.
This study therefore aimed to investigate the cellular expression of
VWD1 and VWD3 missense mutations in the D1 domain to elucidate
the disease mechanism(s) involved and determine whether these differ
dependent on VWD type.
12/06/2014
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
ABSTRACTS
VWF22
New insights into type 3 von Willebrand disease: the
3Winters-Ips project update
Methods: Bacterial plasmid pcDNA3.1 expressing either wild-type
VWF cDNA (WT) or a D1 missense mutation (p.R34G, p.D47H,
p.S49R, p.L60P, p.R81G, p.L129M) were transfected into HEK293T
cells to mimic homozygous (Hom) inheritance, with co-transfections
performed to mimic heterozygous (Het) inheritance. The quantity of
mutant VWF expressed relative to WT only was assessed using
ELISA. Multimer analysis of secreted VWF was performed via electrophoresis on 1.6% (w/v) SDS-agarose gels.
Results: All mutants displayed a significant reduction in VWF secretion compared to WT. VWD1 mutant p.S49R showed ~50% reduction
in Hom and Het secretion, whereas VWD3 mutants p.R34G, p.D47H
and p.R81G showed > 90% reduction (Hom) and ≥ 75% reduction
(Het) in secretion. Expression of varying WT:mutant ratios (25:75,
75:25) confirmed that p.R34G and p.R81G had a dominant-negative
effect. p.L129M (reported in VWD1 and VWD3) and p.L60P when
expressed alone (Hom) showed a > 90% reduction in secretion, but a
milder ~50% reduction when co-expressed with WT (Het). Notably,
none of the mutants showed increased intracellular retention.
Conclusion: Both VWD1 and VWD3 D1 missense mutations have
been shown to cause reduced VWF secretion with varying severity.
Lack of intracellular retention suggests abnormal cellular processing
of VWF as a possible disease mechanism.
Disclosure of Interest: None Declared.
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Objectives: Von Willebrand disease type 3 (VWD3) is still of major
interest because of: severe clinical presentation; need for replacement
therapy with VWF/FVIII concentrates; risk of occurrence of antiVWF inhibitors after concentrates. To evaluate: 1) role of VWF phenotypic data measured with standardized clinical and laboratory
markers on the bleeding tendency; 2) frequency of bleeding and the
requirement for VWF/FVIII concentrates in VWD3; 3) correlation
between clinical or molecular markers and bleeding tendency, response
to therapy with VWF/FVIII concentrates and risk of anti-VWF inhibitors
Methods: The type 3 von Willebrand disease International Registries
and Inhibitor Prospective Study (3WINTERS-IPS) is a no-profit,
investigators initiated, multicenter, European and Iranian observational, retrospective and prospective study on 250 patients with
VWD3. Patients meeting the enrolment criteria will be consecutively
enrolled at each participating centre and data entered into the website
database available for Investigators. The work planned to achieve the
objectives of the project will take place over 5-year period (2011–
2016).
Conclusion: A total of 173/250 (69.2%) VWD3 cases are included into
database by February 28 with gender of 76/97 (M/F); median age
(range) = 26 (1–74) years; median VWF:Ag levels (range) = 1 (< 1–3)
IU/dL; FVIII levels (range) = 2.6 (1–22) IU/dL; anti-VWF inhibitors
in 11/173 (6.4%). 124/173 (71.7%) VWD3 previously exposed to
VWF/FVIII concentrates: other blood products and/or derivatives in
the other cases. Only 21/173 (12%) VWD3 included into secondary
long-term prophylaxis. Enrolment of the 250 VWD3 cases to be completed by 2014 and diagnosis confirmed using centralized phenotypic
and genotypic assessments by 2015. The prospective 24-month observational study on efficacy and safety of the VWF/FVIII concentrates
to be started only in VWD3 patients centrally confirmed.
Disclosure of Interest: None Declared.
iza
Nardi MA1,2
1
Pediatrics; 2Pathology, NYU School of Medicine, NY, NY, USA
Federici AB and on behalf of For the European and Iranian
Investigators of 3Winters-Ips
AB Bonomi Foundation, University of Milan, Milan, Italy
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VWF21
Performance of an automated latex particle-enhanced
immunoturbidimetric von Willebrand factor (VWF)
activity assay on samples from patients receiving
DDAVP or VWF concentrates
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Objectives: Accurate measurement of von Willebrand factor (VWF)
activity is essential for the diagnosis of von Willebrand disease
(VWD). Recently, a validation study of an automated latex particleenhanced immunoturbidimetric method reagent (VWF:Lx) for VWF
activity (Instrumentation Laboratory) was published by others (J
Thromb Haemost 2011;9:1993–2002) demonstrating its excellent laboratory characteristics and diagnostic accuracy compared to the reference method, von Willebrand ristocetin cofactor (VWF:RCo) by
manual light-transmission aggregometry. The report did not, however,
evaluate its accuracy in determining VWF activity in patients receiving
desmopressin (DDAVP) or VWF concentrates (VWFC).
Methods: We report here our findings for the performance of VWF:Lx
vs. VWF:RCo in patients receiving DDAVP (n = 14) or VWFC
(n = 6). DDAVP samples (n = 50) were obtained from patients undergoing challenges (n = 10) or treatment (n = 4). VWFC samples
(n = 34) were obtained from patients treated with Humate-P (n = 4)
or Willate (n = 2). Only samples obtained post-DDAVP or postVWFC treatment are included in this report.
Results: The patient population had a median age of 33.5 years (range
5–90 years), 2:3 M:F ratio, Type 1 VWD (n = 17), Type 2A (n = 2)
and acquired VWD (n = 1). Linear regression for VWF:Lx vs. VWF:
RCo for the DDAVP, VWFC and Total populations were
r2 = 0.86553, P < 0.001; r2 = 0.91327, P < 0.001; r2 = 0.92069,
P < 0.001, respectively. The mean% difference for VWF:Lx vs. VWF:
RCo for the DDAVP, VWFC and Total populations were 0.13%,
3.99% and 1.70%, without significant bias (< 0.001) using BlandAltman bias analysis.
Conclusion: These results demonstrate the VWF:Lx performs equal to
the VWF:RCo for the assessment of patient samples receiving
DDAVP or VWFC. Its automation and performance time (6 min)
allow rapid reporting of accurate results to assist in the monitoring
and expeditious medical care of patients receiving such modes of treatment.
Disclosure of Interest: None Declared.
VWF23
Determination of the VWF activity with the ristocetin
independent gain of function glycoprotein 1b innovance von Willebrand activity assay
Van Duren C1, Schoormans S1, Brons P2, Laros B3 and Heerde
WLV1
1
Laboratory for Hematology; 2Department of Pediatrics;
3
Department of Hematology, Radboudumc, Nijmegen,
Netherlands
Objectives: Von Willebrand disease (vWD) is a bleeding disorder
caused by abnormalities in Willebrand factor (vWF) concentration
and/or function. The prevalence is 1: 100, but only 1:10,000 cases have
a clinical significant bleeding tendency. The vWF ristocetin assay
(VWF:RICOF) is a cumbersome assay and affected by polymorphisms
present in the ristocetin binding site of vWF resulting in in vitro
decreased vWF activity (Flood et al, Blood, 2010). The gain of function Glycoprotein 1b (GP1b) assay is an automated assay that does
not require ristocetin. In this study we compared both assays in a phenotypic and genotypic well defined patient cohort.
Methods: The GP1b assay uses polystyrene particles coated with an
antibody directed against the platelet receptor GPIb. After addition of
plasma the two gain-of-function mutation containing recombinant
GP1b, is added. vWF induces GPIb-based particle agglutination
which can be measured by turbidimetry. 44 VWD patients (genotypically confirmed Type 1: N = 9, type 2A: N = 9, type 2B: N = 6, 2M:
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
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N = 9, type 2N: N = 6, type 1/2N: N = 4, type 3: N = 1, acquired
VWD: N = 1) and 12 non VWD patients were analyzed with both
assays.
Results: By studying the total group no clinical significant differences
were observed between the two assays. The overall slope was
0.958 0.077. The positive predictable value (PPV) of the VWF:RICOF and the GPIb assay for type 1 and 2N patients (cut off ratio
≥ 0.7) is 85% and 94% respectively with a sensitivity of 90% and
79%. For type 2 patients (cut off ratio < 0.7) the PPV is 91% and 85%
respectively with a sensitivity of 87% and 96%. The negative predictable value (NPV) of the VWF:RICOF and the GPIb assay for type 1
and 2N patients is 91% and 85% respectively with a specificity of 87%
and 96%. For type 2 patients the NPV is 85% and 94% respectively
with a specificity of 90% and 79%.
Conclusion: The GP1b assay is able to distinguish between the different
types of VWD. The assay is a good alternative for the Von Willebrand
ristocetin cofactor activity assay.
Disclosure of Interest: None Declared.
ratios for VWD Type 2A and 2B (Mean = 0.41 and 0.72 respectively)
were lower than the ones obtained for VWD Type 2M (Mean = 1.29).
Results with characterized samples are equivalent to those obtained
with the on market assay Asserachrom VWF:CB.
Conclusion: The new VWF:CB assay, in conjunction with HemosIL
AcuStar VWF:Ag and VWF:RCo assays, could be useful for VWD
diagnosis and to discriminate between VWD types 2A and 2B from
2M. The use of these three assays could avoid performing multimer
analysis. However thorough studies with more characterized samples
are required.
Disclosure of Interest: J. Puig Employee of: Biokit R&D, D. Mane
Employee of: Biokit R&D, F. Stufano: none declared, S. Morcillo
Employee of: Biokit R&D, G. Cozzi: none declared, J. Serra Employee
of: Biokit R&D, L. Baronciani: none declared, F. Peyvandi: none
declared.
VWF25
Secondary prophylaxis in von Willebrand disease with
highly purified VWF/FVIII concentrates: interim results
of the PRO.WILL study
VWF24
Fully automated chemiluminescent assay for the detection of von Willebrand factor collagen binding (VWF:
CB)
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Objectives: VWF:CB determination is used as a supplemental assay to
FVIII activity, VWF:Ag and VWF:RCo assays in typing of VWD.
Optimized VWF:CB assays show preferential sensitivity for high
molecular multimers (HMWM) and can help to distinguish between
VWD types 2A and 2B from 2M. The analytical and clinical performance of the new HemosIL AcuStar VWF: CB assay (IL) was evaluated.
Methods: The new VWF:CB assay is a 2-step sandwich assay using
recombinant Type III collagen-coated magnetic particles, and a chemiluminescent detection system based on an isoluminol labeled antiVWF polyclonal antibody. Typed according to ISTH-SSC guidelines
VWD patient plasma from Angelo Bianchi Bonomi Hemophilia and
Thrombosis Center of Milano (Italy) and Blood Bank samples were
tested with the new VWF:CB assay and with the HemosIL AcuStar
VWF:Ag and VWF:RCo assays. The ratios VWF:CB/Ag and VWF:
CB/RCo were calculated.
Results: The new VWF:CB assay exhibited good precision (< 5.4%
CV) along the working range (0.3–4000 IU dL–1) with the re-run
enabled. A reference range study yielded a range of 54.9–227.7 IU
dL–1. The VWF:CB/Ag ratio distribution considering a cut-off of 0.6
was:
VWF:CB/Ag distribution
Sample
N
> 0.7
0.5–0.7
< 0.5
Normal
VWD Type 1
VWD Type 2A
VWD Type 2B
VWD Type 2M
262
14
8
13
5
253
11
0
0
0
7
3
1
1
5
2
0
7
12
0
Objectives: Desmopressin (DDAVP) is not always effective to manage
bleeding episodes of all patients with von Willebrand Disease (VWD).
For severe forms of VWD, the main treatment for bleeding is the
administration of FVIII/VWF concentrates. The objective of this
study was to investigate whether long-term prophylaxis with FVIII/
VWF concentrates (Fanhdiâ and Alphanateâ) is more effective than
the treatment on demand to stop recurrent bleedings in patients with
severe VWD.
Methods: PRO.WILL is an international, prospective, open-label, randomized clinical trial. Clinical data are to be obtained in 24 severe
VWD patients presenting frequent spontaneous bleeding and unresponsive/contraindication to DDAVP. Efficacy, safety and pharmacoeconomic results are assessed. Dose for recurrent haemarthroses/haematomas is 60 IU RiCof kg 3 days–1, while for mucosal bleeding it is
60 IU RiCof kg 2 days–1. Dosage in the on demand arm follows standard recommendations for the treatment of bleeding events.
Results: In the first half of 2013, 7 patients treated on demand and 5
on prophylaxis met the interim analysis criteria. The total number of
bleeding episodes were 58 and 20, respectively, of which 53 (91%) and
9 (45%) required FVIII/VWF concentrate. Mean number of bleeding
episodes per patient were, respectively, 8.3 5.8 (mainly reported as
epistaxis; 6.7 6.8) and 6.7 4.0 (mainly reported as gastrointestinal
bleeding in 2 patients). Two patients in the prophylaxis arm had experienced joint bleeding prior to the study. No study drug related adverse
events and no thrombotic events occurred. Bleeding, especially joint
bleeds, could be prevented in patients receiving prophylaxis: gastrointestinal bleeding was not reduced probably because of other co-morbidities.
Conclusion: Based on these preliminary data, the beneficial effect of
secondary prophylaxis in severe VWD using highly purified FVIII/
VWF can be proven, particularly for joint bleeds. The role of prophylaxis in gastrointestinal bleeding remains to be evaluated.
Disclosure of Interest: F. Peyvandi has grant/tesearch support from:
Grifols, A. Federici has grant/research support from: Grifols
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Puig J1, Mane D1, Stufano F2, Morcillo S1, Cozzi G2, Serra J1,
Baronciani L2 and Peyvandi F3
1
R&D, Biokit R&D, Llica d’Amunt, Spain; 2Fondazione IRCCS Ca’
Granda Ospedale Maggiore Policlinico Milano, Angelo Bianchi
Bonomi Hemophilia and Thrombosis Center; 3Department of
Pathophysiology and Transplantation, Universit
a degli Studi di
Milano, Milan,, Milano, Italy
Peyvandi F1 and Federici AB2
Angelo Bianchi Bonomi, Hemophilia and Thrombosis centre,
University of Milan; 2L. Sacco University Hospital & School of
Medicine, Milan, Italy
1
The low detection limit and the good precision of all AcuStar VWF
assays allowed a more precise VWF:CB/RCo ratio calculation. The
12/06/2014
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
ABSTRACTS
VWF26
Acquired von Willebrand disease syndrome associated
with Klebsiella pneumoniae infection
tions, concentrate was used 5–7 days. In minor procedures as dental
extractions only one dose of concentrate was used together with tranexamic acide. FVIII levels monitored perioperatively. However RiCOF level was also checked in patients with type-3 vwD. The FVIII
target level was 100% perioperatively and maintained above 50% until
healing. Hemostasis was obtained in all cases and no need for platelet
infusions. Thrombo-embolic complications were not occured during
and after surgery. No inhibitor to FVIII was found prior to or after
operations.
Conclusion: One-fifth of our patients had to operated. Plasma-derived
FVIII-vWF concentrate was evaluated as safe and efficient product in
all type of patients with vWD whether minor or major operations.
Careful monitoring of FVIII levels is necessary for preventing thrombosis.
Disclosure of Interest: None Declared.
Narendran V1, Nardi MA2,3, Jacobson J2 and Nierodzik ML1
1
Medicine, 2Pathology; 3Pediatrics, New York University School
of Medicine, New York, USA
Women’s Health Issues in
Thrombosis and Hemostasis
R
WH01
Association between newborn birth weight and
maternal venous thromboembolism in the postpartum
period: a population-based case-control study
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Blondon M1,2, Quon B3, Harrington L2, Bounameaux H1 and
Smith N2
1
Angiology and Haemostasis, Geneva University Hospitals,
Geneva, Switzerland; 2Epidemiology, University of Washington,
Seattle, USA; 3Division of Respirology, St. Paul’s Hospital,
Vancouver, Canada
Objectives: A precise understanding of the risk factors for postpartum
venous thromboembolism (PPVTE) allows for more targeted use of
thromboprophylaxis post delivery. We hypothesized that the birth of a
newborn at term with low or high birthweight would be associated
with a greater risk of maternal PPVTE than the birth of a normal
weight newborn.
Methods: We conducted a population-based case-control study in
Washington State. Cases comprised all women with selected ICD-9
codes for hospitalized VTE within the 3 months post-delivery from
1987 through 2011. Controls were randomly selected postpartum
women without VTE, matched by newborn birth year to cases. Characteristics of women and their deliveries were abstracted from birth
certificates. Using logistic regression models, and restricting to White
women with a singleton birth after 36 weeks of gestation, we compared the risk of maternal PPVTE for mothers of newborns with low
birthweights (< 2500 g) and with large birthweights (> 4000 g, macrosomia) with mothers of newborns with normal birthweights (2500–
4000 g). Models were adjusted for maternal age, BMI, parity, education, smoking, gestational diabetes, gestational hypertension, preeclampsia, delivery method and gestational length.
Results: Our study comprised 442 case and 6927 control women who
had given birth to a singleton at term. Among controls, the prevalence
of low-birthweight and macrosomic newborns was 1.3% and 14.1%,
respectively. Compared with mothers of newborns with normal
weights, mothers of newborns < 2500 g had a 4-fold increased risk of
PPVTE, which persisted after multiple adjustment (OR 3.6, 95%CI
2.1–6.3). Mothers of macrosomic newborns (> 4000 g) had a slightly
increased risk of PPVTE, which was somewhat attenuated after multiple adjustment (OR 1.4, 95%CI 1.1–1.8).
Conclusion: Mothers of newborns born at term with low and high
birthweights may carry an increased risk of PPVTE. This increased
risk appears to be independent of common causes of growth restriction
or macrosomia.
Disclosure of Interest: None Declared.
Co
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Objectives: Acquired von Willebrand disease (aVWD) syndrome is a
rare disorder usually associated with autoimmune or lymphoproliferative disease. Here we describe a patient with aVWD associated with
bacterial infection.
Methods: A 55 year old male, post-surgical for uneventful repair of left
shoulder rotator cuff, was admitted to the hospital with severe left
shoulder pain non-responsive to intra-articular injections. He was
found to have septic arthritis requiring surgical intervention with complications due to hemarthrosis.
Results: Coagulation studies revealed Factor VIII (FVIII) = 3%,
VWF antigen (VWF:Ag) = 11% and VWF:ristocetin cofactor (VWF:
RCo) < 10%. PT, Thrombin Time, Fibrinogen, Factor IX and Factor
XI were normal. Measurement of PTT, FVIII, VWF:Ag and VWF:
RCo in mixing study samples without and with incubation
(37 °C 9 1 h) demonstrated a mild immediate-acting PTT inhibitor
and lack of a neutralizing inhibitor to FVIII, VWF:Ag and VWF:
RCo. Platelet aggregation was absent in response to ristocetin. VWF
multimeric composition showed lack of all multimers. Joint fluid culture subsequently was positive for Klebsiella pneumoniae. The patient
was treated with cefepime and vancomycin. Initially, the patient was
unresponsive to Humate-Pâ. On day 17 FVIII, VWG:Ag and VWF:
RCo increased to 59%, 27% and 15%, respectively, with only antibiotic therapy. VWF multimers analysis showed normal pattern with
decreased amount of all multimer bands. The patient responded to
DDAVP therapy with a sustained increase in FVIII, VWF:Ag and
VWF:RCo (113%, 62% and 53%, respectively). The patient continued
to improve. On day 29 FVIII, VWF:Ag and VWF:RCo were 254%,
269% and 233%, respectively, with normal multimers.
Conclusion: We describe here a patient with a transient VWF non-neutralizing autoantibody with a strong causal relationship between the
bacterial infection and aVWD. A 482 AA homology between VWF
domain A and Klebsiella pneumoniae has been reported. Studies are
on-going to characterize the antibody.
Disclosure of Interest: None Declared.
VWF27
Surgical experience in patients with von Willebrand
disease: one center report
Kavakli K1, Balkan C2 and Karapinar DY2
Hematology, Ege University Children Hospital; 2Hematology,
Children’s Hospital, Izmir, Turkey
1
Objectives: Patients with von Willebrand disease (vWD) who undergo
surgical interventions need regular clinical and laboratory evaluation
and monitoring. In this paper, surgical experience was evaluated in
respect of efficacy and safety. Totally 140 patients is being followed up
for vWD. Type-1 patients (n = 106) (mean age: 12; range 2–30 year)
and 48 of them were male. Fifteen patients were Type-2 and 6 of them
male. Nighteen patients were type-3 and 12 of them were girl.
Methods: For last 10 years, all surgical operations were collected retrospectively. Six major operations were made (hidronephrosis correction, artrodesis, ovary operation and sinus pilonidal cyst) in 6 patients.
25 minor surgeries were done in 25 patients; radioisotope synovectomy
(n = 5), dental extractions (n = 7), circumcision (n = 8), adenoectomy
(n = 3), harelip correction and inguinal hernia repair (n = 1). All operations were elective except over surgeries related acute abdomen.
Results: For hemostasis, FVIII-vWF concentrate (Haemate-P) was
used for all patients included DDAVP unresponsive patients. Bolus
infusions were used in every 12 h in the first day. For major opera-
99
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
12/06/2014
100
ABSTRACTS
WH02
Pregnancy complications
thrombocytopenic purpura
in
acquired
USA; 5Division of Angiology and Haemostasis, Geneva University
Hospitals, Geneva, Switzerland; 6Department of Health Services;
7
School of Nursing, University of Washington; 8Seattle
Epidemiologic Research and Information Center, Department of
Veterans Affairs Office of Research and Development, Seattle,
USA
thrombotic
CD
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Objectives: Pregnancy is considered an important risk factor for
relapse of acquired thrombotic thrombocytopenic purpura (TTP). The
risk of miscarriage could also be increased in these women, similar to
other autoimmune disorders. However, the exact entity and causes of
these risks are unknown. The aim of this study was to evaluate risk
factors associated with gravidic TTP relapse and miscarriage in
women with a history of acquired TTP.
Methods: We conducted a nested case-control study in women with a
history of acquired TTP enrolled in the Milan TTP registry from 1994
to Octobe 2012. Sixteen out of 254 women had a pregnancy after diagnosis of acquired TTP and inclusion in our registry. We contrasted
women with a complicated pregnancy (i.e., cases of either gravidic
TTP or miscarriage) with women with uncomplicated pregnancy (i.e.,
controls). Clinical variables (age at pregnancy, gravidity, time from
the last TTP episode, TTP recurrence) and laboratory features (ADAMTS13 activity, anti-ADAMTS13 antibody) were studied. We used
odds ratios as an approximation of relative risks for these variables.
Results: According to pregnancy outcome, 4 cases with gravidic TTP,
5 with miscarriage and 7 controls with uncomplicated pregnancy were
included. ADAMTS13 activity levels in the first trimester were reduced
in the cases, severely (median < 3%) in gravidic TTP and moderately
(20%, range 14–40%) in miscarriage; in the controls median ADAMTS13 activity level was 77% in the first trimester (range 40–129%) and
remained above 39% until delivery, in the absence of detectable antiADAMTS13 antibodies. The presence of anti-ADAMTS13 antibodies
during pregnancy was associated with an over 5-fold increase in the
risk for both gravidic TTP and miscarriage (lower boundary of the
confidence interval of the odds ratio).
Conclusion: ADAMTS13 activity levels and anti-ADAMTS13 antibody assays may help to predict the risk of complications in pregnant
women with a history of acquired TTP.
Disclosure of Interest: B. Ferrari: none declared, L. Lotta: none
declared, A. Maino: none declared, A. Artoni: none declared, S. Pontiggia: none declared, S. Trisolini: none declared, A. Malato: none
declared, F. Rosendaal: none declared, F. Peyvandi has grant/research
support from: NovoNordisk, Kedrion
Objectives: Bilateral salpingo-oophorectomy (BSO) is associated with
endogenous hormonal changes, yet the relation between hysterectomy
with BSO and incident venous thrombosis (VT) is incompletely characterized. We aimed: 1) to test whether hysterectomy with BSO is associated with the risk of incident VT, compared with hysterectomy with
ovarian conservation; and, 2) to evaluate this association by age at
hysterectomy.
Methods: In the Heart and Vascular Health Study, a population-based
case-control study, we identified postmenopausal female cases of incident VT, aged 50–89 years, occurring from 1995 to 2010, and their
matched controls. Index date was defined as the VT event date for
cases, and for controls, as a randomly chosen date. Hysterectomy and
oophorectomy status and date were abstracted from medical records.
Eligible participants had undergone hysterectomy and had no cancer
history. We excluded participants with an unknown hysterectomy or
oophorectomy status or date, or current progestogen (P)-only or estrogen (E)+P hormone therapy use, resulting in 331 eligible cases and
1,317 controls. Women were categorized as having ≥ 1 ovary remaining (ovarian conservation) vs. BSO. Using multiple logistic regression,
we estimated the risk of VT associated with BSO compared with ovarian conservation; we then evaluated this association within categories
of age at hysterectomy.
Results: In adjusted analyses (Table), hysterectomy with BSO was not
significantly associated with the risk of VT compared with hysterectomy with ovarian conservation. Among women with hysterectomy
at < 40 years of age, BSO was associated with an increased risk of
VT compared with ovarian conservation (OR: 1.75; 95% CI: 1.07–
2.85).
R
Ferrari B1, Lotta LA1, Maino A1, Artoni A1, Pontiggia S1, Trisolini
SM2, Malato A3, Rosendaal FR4 and Peyvandi F5
1
Angelo Bianchi Bonomi Hemophilia and Thrombosis Centre,
Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico,
Milan; 2Cellular Biotechnologies and Hematology, Sapienza
University, Rome; 3UOC di Ematologia con UTMO, Ospedali
Riuniti Villa Sofia-Cervello, Palermo, Italy; 4Department of
Clinical Epidemiology and Department of Thrombosis and
Haemostasis, Leiden University Medical Center, Leiden,
Netherlands; 5Angelo Bianchi Bonomi Hemophilia and
Thrombosis Centre, Fondazione IRCCS Ca’ Granda Ospedale
Maggiore Policlinico, Universit
a degli Studi di Milano, Milan,
Italy
WH03
The association between bilateral salpingo-oophorectomy and incident venous thrombosis among postmenopausal women with hysterectomy
Harrington LB1, McKnight B2, Heckbert SR1,3, Wiggins KL4,
Blondon M1,5, Psaty BM1,3,4,6, Woods NF7, LaCroix AZ1 and
Smith NL1,3,8
1
Department of Epidemiology, 2Department of Biostatistics,
University of Washington; 3Group Health Research Institute, ;
4
Department of Medicine, University of Washington, Seattle,
Conclusion: In this population of women with hysterectomy but without cancer, BSO was not associated with the risk of incident VT. However, among women undergoing hysterectomy at a young age, BSO
may be associated with an increased risk of VT compared with ovarian
conservation.
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© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
ABSTRACTS
Disclosure of Interest: L. Harrington: none declared, B. McKnight:
none declared, S. Heckbert: none declared, K. Wiggins: none declared,
M. Blondon: none declared, B. Psaty Consultant for: Psaty serves on a
DSMB for a clinical trial of a device funded by the manufacturer (Zoll
LifeCor) and is on the steering committee of the Yale Open Data
Access Project funded by Johnson & Johnson., N. Woods: none
declared, A. LaCroix: none declared, N. Smith: none declared.
101
ICH, and ECH was 2.5% and 3.7%, respectively. The incidence of
asymptomatic head bleeding was 15% from one prospective study.
The RR of ICH in newborns with haemophilia was significantly higher
with vaginal delivery (61; 95% CI 25.8–146 for assisted vaginal and 51;
95% CI 33.2–79.1 for spontaneous vaginal delivery) and lowest with
caesarean delivery (7.3; 95% CI 1.81–29.3) (P = < 0.001).
WH04
Clinical utility of antithrombotic prophylaxis in IVF/
ICSI
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Conclusion: The risk of cranial bleeding is significantly higher in newborns with haemophilia compared to the general population
(P ≤ 0.001). MOD is an important determinant of risk and neurological morbidity.
Disclosure of Interest: None Declared.
WH06
Association of factor V Leiden G1691A and prothrombin gene G20210A mutation with adverse pregnancy
outcomes
pi
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Objectives: We examined the contribution of an antithrombotic prophylaxis in influencing clinical pregnancy and live-birth in an unselected cohort of women approaching ART.
Methods: 1107 women with fertility problems and a valid indication
for ART were recruited. Baseline and follow-up information of obstetric outcomes and antithrombotic treatment were collected.
Results: Median follow-up time was 34.5 months (range: 2–143). During the follow-up period, 595 (53.8%) women underwent ART (total
1234 cycles); 202 (33.9%) women achieved a pregnancy for a total of
255 clinical pregnancies.
The concomitant use of LMWH and aspirin was significantly associated with a higher rate of clinical pregnancies (P: 0.001, OR: 5.3, 95%
CI: 1.9–14.6). The pregnancy rate was also significantly increased by
the use of LMWH alone (P: 0.03, OR: 1.9, 95% CI: 1.1–3.3). Carriership of inherited or acquired thrombophilia did not affect clinical outcomes of the ART. The efficacy of antithrombotic treatment was
confirmed when the outcome ‘live-birth’ was considered.
Conclusion: Present data suggest a potential benefit of antithrombotic
prophylaxis during ART and do not support the clinical utility of universal thrombophilia screening in improving the number of live-births.
Disclosure of Interest: None Declared.
R
Villani M1, Tiscia GL1, Dentali F2, Colaizzo D1, Cappucci F1,
Fischetti L1, Ageno W2, Margaglione M3 and Grandone E1
1
IRCCS Casa Sollievo Della Sofferenza, San Giovanni Rontondo,
FG; 2Clinical Medicine, University of Insubria, Varese; 3Mediacal
Genetics, University of Foggia, Foggia, Italy
Co
WH05
Mode of delivery and cranial bleeding in newborns
with haemophilia, a systematic review of the literature
Davies J and Kadir RA
Obstetrics and Gynaecology, Royal Free Hospital, London, UK
Objectives: Cranial bleeding at birth can result in significant neurological morbidity in newborns with haemophilia. The optimum mode of
delivery (MOD) of a potentially affected fetus remains controversial
largely due to the lack of prospective data in this cohort.
The aim of this review is to ascertain the cumulative incidence of cranial bleeding and its neurological sequelae in newborns with haemophilia in comparison to the general population. The impact of MOD
on rates of cranial bleeding is also determined.
Methods: An EMBASE/MEDLINE search using key terms revealed
the relevant studies. Studies were included if the newborn period and a
denominator population was defined. The proportion of newborns
with cranial bleeding in both populations was compared using Chisquared or fisher’s exact test and relative risk (RR) with 95% confidence intervals (CI) were calculated.
Results: In the general population the cumulative incidence of symptomatic intracranial haemorrhage (ICH) and extracranial haemorrhage (ECH) was 5.8 and 47 per 10,000 deliveries, respectively. The
incidence of asymptomatic head bleeding was 24% (range 8–46%). In
newborns with haemophilia the cumulative incidence of symptomatic
Asad S and Moiz B
Pathology and Microbiology, Aga Khan University Hospital,
Karachi, Pakistan
Objectives: Determine the association of factor V Leiden G1691A and
prothrombin gene G20210A mutation with adverse pregnancy outcomes.
Methods: It was a case control study, conducted at clinical laboratory,
section of haematology, and PCR-RFLP technique is used at multidisciplinary laboratory, Aga Khan University Hospital. Females with
adverse pregnancy outcomes who came to obstetrical clinic were
included in the study as cases. Adverse pregnancy outcomes included
recurrent pregnancy loss (defined as > 2 first trimester miscarriages or
one or more second trimester miscarriage), severe pre-eclampsia, placental abruption, intrauterine growth restriction and still birth.
Control samples are selected from females with ≥ 2 consecutive normal
pregnancies. Calculated sample size is 172 which comprise of 86 cases
and 86 controls.
Results: Overall mean age of all subjects was 28.5 years (4.9). Mean
age of cases was 29.3 (5.17) years and of controls was 27.6 years
(4.5). 73 (84.8%) cases had recurrent pregnancy loss, 12 (13.9%) had
pre-eclampsia, 8 (9.3%) had IUGR while placental abruption and still
birth was present in 2 (2.3%) cases each. 10 (11.6%) cases had more
than one adverse pregnancy outcomes. 19 (22.09%) cases had > 4
pregnancy losses. Among cases, 40 (46.5%) females had previous live
births and 9 (10.4%) were pregnant at the time of sample collection.
Two cases with recurrent pregnancy loss (P = 0.155 OR = 0.49) showed
heterozygous mutation of factor V Leiden G1691A and while no mutation identified in the control arm. Heterozygous prothrombin gene mutation was identified in one case with recurrent pregnancy loss (P = 0.316
OR = 0.497) while none of the control exhibited this mutation.
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
12/06/2014
102
ABSTRACTS
women with Type 1 DM (24.31 3.68 years) (who had a diabetes
duration < 5 years), 24 healthy pregnant women (HPW)
(23.96 5.18 years) and 10 healthy non-pregnant women (HNPW)
(23.82 4.58 years) of the control groups. Adiponectin was measured
using commercially available kits. Platelet disaggregation was determined by light transmission using an AP 2110 computerized analyzer
of platelet aggregation (SOLAR, Belarus).
Results: Adiponectin serum levels were lower in women with gestational diabetes mellitus (19.3 1.4 ng mL–1) when compared with
HNPW (24.3 2.4 ng mL–1), HPW (20.4 1.6 ng mL–1) and were
significantly higher in women with Type 1 DM (35.7 4.0 ng mL–1).
Addition of sodium nitroprusside (SNP) (400 lM) to platelets preaggregated with ADP (1.5 lM) induces platelet disaggregation. The
velocity of induced disaggregation at the pregnant women with Type 1
DM was higher than in control group in 3, 4 times. Adiponectin serum
levels were correlated positively with platelet disaggregation, induced
by SNP.
Conclusion: The present study reveals a new role of adiponectin as an
endogenous antithrombotic factor. As studies related to platelet functions in gestational diabetes and Type 1 DM increase will be improvements in prenatal and postnatal observation and treatment, and thus a
decrease in the complications for both the fetus and the mother.
Disclosure of Interest: None Declared.
Conclusion: This is a small sample sized study which does not support
a significant association between inherited thrombophilia mutations
and adverse pregnancy outcomes. The apparent lack of association
may be reconciled by the low numbers of subjects recruited.
Disclosure of Interest: None Declared.
WH07
Obstetric patients in the intensive care unit (ICU):
hematologic aspects
Co
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WH08
Effects of serum adiponectin levels on platelet
disaggregation in pregnant women with gestational
diabetes mellitus and type 1 diabetes mellitus
Bichan V1 and Zabarovskaya Z2
1
Belarusian State University; 2Belarusian State Medical
University, Minsk, Belarus
Objectives: Adiponectin is an adipocyte-specific protein that has been
found to be associated with insulin sensitivity and obesity. The physiological role of adiponectin has not yet been fully elucidated, but it is
believed that it has the ability to reduce glucose, triglycerides, and free
fatty acids and that it plays a major role in the pathogenesis of metabolic syndrome. Because gestational diabetes mellitus (GDM) is associated with obesity and decreased insulin sensitivity, we have analyzed
plasma adiponectin levels in women with gestational diabetes mellitus.
Methods: We compared 36 pregnant women with GDM
(24.68 5.12 years) and gestational age (24–26 weeks), 15 pregnant
or
CD
WH09
Outcomes of 339 pregnancies in 181 women suffering
from 13 different forms of inherited thrombocytopenia
enrolled in a retrospective and multicentric study (on
behalf of EHA-SWG on thrombocytopenias and platelet
function disorders)
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Objectives: The challenge of caring for critically ill obstetric patients
requires urgent attention; pregnant women are at risk to develop complications due to illness related to pregnancy or due to complications
of preexisting disease. We have an obstetric intensive unit. Only in special conditions obstetric patients are admitted to general ICU. Objectives: to describe hematologic aspects of obstetric admissions to ICU.
Methods: Retrospective observational study of obstetric patients
admitted to ICU (3 years). We identified 20 admissions (obstetrichematology databases).
Results: Reasons for admission: 1-Post-partum hemorrhage (PPH)
(n = 10); PPH was classified as:1a-PPH without disseminated intravascular coagulation (DIC) (n = 7); 1b-PPH with DIC (n = 2) and 1cPPH in Jehovah Witness. 2-Hipertensive disorders (n = 3): HELLP
Syndrome (n = 2), eclampsia with intracerebral hemorrhage (n = 1); 3Sepsis (n = 2); 4- venous thromboembolic disease (VTE, n = 3):postpartum pulmonary embolism (TEP) (n = 1);catastrophic antiphospholipid syndrome (CAPS) at 12 weeks of gestation (n = 1) and an
antenatal TEP at 22 weeks in a women with sickle cell anemia; 5Other (n = 2): leukemia and FX deficit. Associated morbidities and
complications: von Willebrand disease (n = 1) and gonadal venous
thrombosis in a PPH (n = 1). Therapeutic hematologic management
included: blood derivates, tranexamic acid, intravenous gammaglobulin and corticoids (CAPS), anticoagulation with heparin, high doses of
erythropoietin and intravenous iron.
Conclusion: Critical illness in pregnancy is uncommon, but it is essential to adopt an early multidisciplinary management. PPH is the first
cause of ICU admission. Hematology as a specialty field forms part of
this approach either during the acute intervention or previously during
the antenatal or pre-pregnant period by counseling pregnant women
and the obstetric team how to manage high risk thrombotic and/or
hemorrhagic situations. This approach can prevent complications and
avoid late admissions with advanced medical problems.
Disclosure of Interest: None Declared.
R
Grand B, Alcantara MG, Orti J, Lapidus A and Voto LS
Department of Maternal and Perinatal Medicine, Hospital Juan A
Fernandez, Buenos Aires, Argentina
Noris P1, Schlegel N2, Klersy C3, Heller PG4, Civaschi E1, PujolMoix N5, Fabris F6, Favier R7, Gresele P8, Latger-Cannard V9,
Cuker A10, Nurden P11, Greinacher A12, Cattaneo M13, De
Candia E14, Pecci A1, Hurtaud-Roux M-F2, Glembotsky AC4,
~ iz-Diaz E15, Randi ML6, Trillot N16, Bury L8, Lecompte T17,
Mun
Marconi C18, Savoia A19 and Balduini CL1
1
Department of Internal Medicine, University of Pavia-IRCCS
Policlinico San Matteo Foundation, Pavia, Italy; 2National
Reference Centre on Inherited Platelet Disorders and Service
d’H
ematologie Biologique, CHU Robert Debr
e and Paris 7 Denis
Diderot University, Paris, France; 3Service of Biometry &
Statistics, IRCCS Policlinico San Matteo Foundation, Pavia, Italy;
4
Hematology Research, Institute of Medical Research Alfredo
Lanari, University of Buenos Aires, Buenos Aires, Argentina;
5
noma de Barcelona, Institut de Recerca
Universitat Auto
Biom
edica Sant Pau, Barcelona, Spain; 6Department of
Medicine-DIMED, University of Padova Medical School, Padova,
Italy; 7AP-HP, Armand Trousseau children Hospital,
Haematological Laboratory, French reference center for inherited
platelet disorders, Paris, France; 8Department of Internal
Medicine, University of Perugia, Perugia, Italy; 9Centre de
Comp
etence Nord-Est des Pathologies Plaquettaires from the
frame of the Reference French Centre, Service d’H
ematologie
Biologique, Centre Hospitalo-Universitaire, Nancy, France;
10
Department of Medicine and Department of Pathology &
Laboratory Medicine, Perelman School of Medicine, University of
Pennsylvania, Philadelphia, USA; 11Plateforme Technologique et
^pital Xavier Arnozan, Pessac,
d’Innovation Biom
edicale, Ho
€r Immunologie und Transfusionsmedizin,
France; 12Institut fu
Greifswald, Germany; 13Medicina III, Ospedale San Paolo,
Dipartimento di Scienze della Salute, Universit
a degli Studi di
Milano, Milano; 14Servizio Malattie Emorragiche e Trombotiche,
Istituto di Medicina Interna e Geriatria, Policlinico Agostino
12/06/2014
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
ABSTRACTS
tered: lipoprotein-A in 29.8%, total cholesterol > 250 mg dL–1 (5.4%)
and triglycerides > 200 mg dL–1 (3%). In 54(18%) pregnancies were
positive for more than one marker. Of the 257 pregnancies that had
deep vein thrombosis, 73 (28%) had a thrombotic event in the current
pregnancy and 184 (71%) had a history of venous thrombosis. In this
last group 33/184 (18%) the event had occurred in previous pregnancy,
9(4.8%) on contraceptive use, 16 (8.6%) in the postpartum period and
126(68%) had no extrinsic risk factor associated. Thus, of the 298
patients with thromboembolic event, 122 (41%) was related to pregnancy and postpartum
Conclusion: The period of pregnancy and childbirth have an increased
risk for thromboembolic events. Antiphospholipid syndrome and antiphospholipid antibodies were the main findings in these events.
Disclosure of Interest: None Declared.
Gemelli, Universita Cattolica del Sacro Cuore, Roma, Italy;
15
Immunohematology Department, Banc de Sang i Teixits de
Catalunya, Barcelona, Spain; 16Institut d’H
ematologie^le Biologie Pathologie G
Transfusion, Po
en
etique, CHRU Lille,
ecialit
es de M
edecine, Service
Lille, France; 17Departement des Sp
^pitaux Universitaires de Gen
d’Hematologie, Ho
eve, Universit
e de
Geneve, Faculte de Medecine, Gen
eve, Switzerland; 18Genetica
Medica, Dipartimento di Scienze Mediche Chirurgiche,
Policlinico Sant’Orsola-Malpighi, University of Bologna,
Bologna; 19Department of Medical Sciences, University of
Trieste, Institute for Maternal and Child Health – IRCCS Burlo
Garofolo, Trieste, Italy
WH11
Descriptive analysis of pregnant women with recurrent
fetal death and thrombophilia
Andres MDP, Igai AMK, Barros V, Francisco RP and Zugaib M
Obstetrics and Gynecology, Clinics Hospital, Sao Paulo, Brazil
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Objectives: Evaluate pregnancy outcome and thrombophilia frequency
in women with recurrent fetal death.
Methods: Analyses of obstetric outcomes in a retrospective cohort of
pregnant women with recurrent stillbirth after the 20th week, accompanied at Thrombosis and Pregnancy Department, Clinics Hospital, Faculty
of Medicine, University of S~ao Paulo, from 2001 to 2013. Antithrombin
activity, protein C and S activity, Factor V Leiden, G20210A Prothrombin mutation and antiphospholipid syndrome were tested.
Results: 527 patients and 585 pregnancies followed in the thrombophilia and pregnancy ambulatory of HCFMUSP had had previous stillbirth. Included in this group were 20 patients who had recurrent fetal
death. The incidence of recurrent fetal death was 3.79%. Thrombophilia were found in 11(60%), seven diagnosed as antiphospholipid
syndrome (63%), three as protein S deficiency (27%) and one as prothrombin gene mutation (9%). All of them used subcutaneous heparin
(unfractionated or enoxaparin) and 14 of them acetylsalicylic acid
(AAS) during pregnancy. Obstetric complications occurred in 15
patients and included: intrauterine fetal growth restriction (25%), placenta previa (15%), amniotic fluid decreased index (25%), severe preeclampsia (10%), fetal distress (5%) and stillbirth (1–5%). The mean
gestational age at delivery was 35.8 3.7 weeks and newborn weight
averaged 2417.3 666.2 g.
Conclusion: Recurrent stillbirth can be associated with the presence of
acquired and inherited thrombophilias, especially protein S deficiency
and antiphospholipid syndrome. The use of antithrombotic therapy in
these cases appears to result in better obstetric outcomes and increased
live birth rate.
Disclosure of Interest: None Declared.
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Objectives: Pregnancy in inherited thrombocytopenia (IT) women is a
main cause of concern because both mothers and infants are at risk of
bleeding. Since evidence-based medical treatments are not available
due to the paucity of literature, we collected data on maternal and neonatal bleeding risk in different forms of IT.
Methods: 181 women with 13 different forms of IT confirmed by
genetic analysis were enrolled in this study: data from 339 pregnancies
and 156 IT newborns were collected from 45 institutions worldwide.
Results: Thrombocytopenia and bleeding tendency in the mothers did
not worsen during pregnancy. Gestations were uneventful in 304 cases
while miscarriages and preterm births had the same frequency than in
healthy women (10.1% and 9.9%, respectively). IT newborns had
platelet counts similar to those of their mothers; only 5 IT newborns
had minor bleeding, while 2 died for cerebral hemorrhage. Prophylactic platelet transfusions were given in preparation for delivery in 46 of
301 evaluable cases; 38% of births were by cesarean section. Deliveryrelated maternal bleeding was higher than in general population (6.8%
to 14.2% depending on blood loss entity) but no women required hysterectomy to stop bleeding. Bleeding frequency was similar in vaginal
and caesarean deliveries. Excessive bleeding at delivery requiring
transfusion correlated significantly with a history of grade 3 or 4 (OR
5.32) and grade 4 (OR 24.50) of WHO bleeding scale. ROC analysis
identified the value of 50 9 109 platelets L–1 as the optimal cut-off of
platelet count for the identification of patients with a higher risk for
bleeding requiring transfusion (OR 7.61, CI 1.55–37.60).
Conclusion: Delivery-related bleeding risk was higher in subjects with
ITs than in healthy population for both the mothers and the affected
newborns. The degree of thrombocytopenia and a history of severe
bleeding tendency in the mother have been identified as useful parameters to predict the risk of delivery-related bleedings.
Disclosure of Interest: None Declared.
103
WH10
Incidence of thrombophilia testing in pregnancies complicated by current or prior thromboembolism
Barros V, Torres I, Baptista FS, Francisco RP and Zugaib M
Obstetrics and Gynecology, Clinics Hospital, Sao Paulo, Brazil
Objectives: To determine the incidence of thrombophilia markers in
pregnancies with thromboembolic events.
Methods: Between December 2001 and February 2013, 851 pregnancies were followed in the Clinic of Thrombophilia and Thrombosis in
Pregnancy. Of these, 298 had thromboembolic events and underwent a
routine investigation for the presence or absence of markers for thrombophilia. The thromboembolic events considered were: deep vein
thrombosis, pulmonary embolism and stroke.
Results: Of the 298 pregnancies, 257 cases had deep vein thrombosis
(86.2%), 5 had pulmonary embolism (2%) and 3 (14%) stroke. Recurrent thrombosis was identified in 46(15.4%). Thrombophilia markers
were detected in 237(79.5%) pregnancies. The findings were: APS in
19.1%, presence of antiphospholipid antibodies (11%), Protein S Deficiency (9.4%), Factor V Leiden (9.7%), Prothrombin Mutant (3%),
hyperhomocysteinemia (> 15) in 1.7%. Other markers were encoun-
Working Group on Genomics in
Hemostasis
GH01
Use of human phenome ontology (HPO) is an effective
approach to cluster 519 cases of inherited bleeding and
platelet disorders enrolled by 12 referral centres in an
exome sequencing study
Kelly A1,2 on behalf of on behalf of the BRIDGE consortium
1
Department of Haematology, University of Cambridge; 2NHS
Blood and Transplant, Cambridge, UK
Objectives: Clinicians use prior knowledge of patterns of phenotypes
in the diagnosis of disease and classical patterns of bleeding or associ-
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
12/06/2014
104
ABSTRACTS
ated clinical features can be easily recognised in bleeding or platelet
disorders (BPDs) of known molecular aetiology or in small cohorts of
novel BPD cases.
High-throughput whole exome sequencing (WES) allows the rapid parallel genotyping of large numbers of BPD cases with relatively heterogeneous pathobiologies for whom detailed phenotypic information is
known. Automated clustering of cases into smaller groups with similar
clinical phenotypes is essential to maintain power of gene discovery.
Methods: The BRIDGE-BPD study is an international collaborative
study. So far 519 BPD cases have been recruited at 12 enrolment centres from 8 countries and both detailed clinical phenotyping and WES
has been performed and data entered into a shared study database.
The HPO1, a curated body systems based strategy for clinical phenotype capture by ontologically related terms, has been expanded and
used to cluster cases.
Results: Affected members of pedigrees and cases with classical syndromes (e.g. Hermansky-Pudlak Syndrome) or deleterious mutations
in well-described BPD genes (e.g. MYH-9) but with more obscure presentation can be seen to cluster (see Fig. 1. Clustering of cases of
known BPDs, shown with association P-values).
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subsequent confirmation. Patients were grouped by testing status and
groups compared on patient and thrombophilia risk characteristics.
Results: A total of 1314 patients were included. Patients were mixed
evenly by sex with a mean age of 65 years (Table). Thrombophilia testing was ordered in 24% of patients with 9% of these positive for at
least one thrombophilia type. Prothrombin mutation (4%) and factor
V Leiden (3%) were most commonly detected. Strongly thrombophilic
patients (i.e., no surgery within prior 30 days plus family history of
VTE, recurrent VTE, or age < 50 years) comprised only 45% of those
tested. Thrombophilia test timing was within 7 days of acute VTE in
35% of patients. Family history of VTE, age < 50, hormone therapy,
and pregnancy were more common in the tested group (all P < 0.001).
Upper extremity deep vein thrombosis, cancer, an indwelling catheter,
and hospitalization in the prior 30 days were more common in the
non-tested group (all P < 0.01).
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Conclusion: Recruitment of large numbers of BPD cases for sequencing studies requires automated phenotype comparison between cases.
We have demonstrated proof of principle in the use of the HPO coding
system as BPD cases with the same or related causative sequence variants tend to cluster closely. It is postulated that this HPO-driven clustering approach will maintain power of gene discovery in a large
collection of relatively heterogeneous BPD cases of unknown molecular aetiology.
References: K€
ohler, S. et al. The Human Phenotype Ontology project:
linking molecular biology and disease through phenotype data.
Nucleic Acids Research (2013).
Disclosure of Interest: None Declared.
GH02
Thrombophilia testing among patients with venous
thromboembolism
Meyer MR, Delate T, Johnson SG and Witt DM
Kaiser Permanente Colorado, Aurora, USA
Objectives: The utility of thrombophilia testing in patients with acute
venous thromboembolism (VTE) is limited as results rarely impact
therapy (choice of anticoagulant, intensity/duration of therapy). This
study sought to describe the proportions of patients who received
thrombophilia testing, had a positive test result, and their thrombophilia types and thrombophilia testing patterns in our organization.
Methods: This was a cross-sectional study of patients with validated,
acute VTE between January 2000 and December 2010. Data were collected via electronic queries and verified via chart review. For nongenetic assays thrombophilia diagnosis required a positive test and
Conclusion: Thrombophilia testing was widespread in patients with
acute VTE; although, few tests confirmed thrombophilia. Not testing
during the acute VTE period and limiting later testing to strongly
thrombophilic patients may improve appropriateness of thrombophilia testing and reduce healthcare costs.
Disclosure of Interest: None Declared.
GH03
Identification of coding variants in susceptibility genes
through genome-wide association and next generation
sequencing implicated in pediatric venous thrombosis
Stoll M1, Barysenka A1, Arning A1, Witten A1
€ttl U2
and Nowak-Go
1
Genetic Epidemiology, Lifa At the University of Muenster,
€nster; 2Hemostasiology, University Clinics SchleswigMu
Holstein, Kiel, Germany
Objectives: Recent genome-wide association studies (GWAS) for
venous thromboembolism (VTE) have implicated novel susceptibility
loci in adults. Studies in families with a first onset of VTE in childhood
12/06/2014
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
ABSTRACTS
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GH04
The prevalence of gene polymorfisms of thrombocytes
in patients with evidential venous thromboembolism
(VTE)
Conclusion: Our results did not show any significant differecies
between patients with identified VTE and controls in prevalence of
gene polymorfism of thrombocytes, no statistical analysis have shown
any association with either allele or genotype frequencies. Prevalence
of inherited thrombophilic mutations (FV Leiden and prothrombin)
were statistical higher in thromboembolic patients.
Disclosure of Interest: T. Kvasnicka has grant/research support from:
NT11176-5, RVO-VFN64165/2012, J. Hajkova: none declared, P.
Bobcikova: none declared, V. Cverhova: none declared, P. Kvasnickova: none declared, J. Ulrych: none declared, J. Briza: none declared,
I. Malikova: none declared, J. Kvasnicka: none declared.
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are lacking. To identify coding variants contributing to VTE risk in
children using GWAS in 212 families followed by next generation
sequencing (NGS) in 24 discordant siblings.
Methods: GWAS was assessed using the Transmission Disequilibrium
Test and corrected for multiple testing using permutation testing. Subsequently, we performed NGS in 24 discordant siblings.The target
regions (~11 Mb) comprise 30 genes from 16 chromosomes. DNA
libraries were paired-end sequenced on an Illumina HiScanSQ yielding
in 142.5 Gb sequence with a QScore > 30.Sequence reads were
mapped using the BWA algorithm and analyzed by GATK yielding
coverage of 196X. Variant annotation was done using SNPEff and
Annovar softwares.
Results: For two SNPs exceeding the threshold for genome-wide association (P > 105) replication was conducted in 201 trios with thromboembolic stroke (TS). Among these, rs1304029 and a SNP curtly
missing the threshold for permutated P-value (rs2748331) reside in a
region comprising the gene for beta-1,3-glucoronyltransferase 2, and
are associated with pediatric VTE (rs1304029; P = 1.42 9 106,
rs2748331; P = 6.11 9 106), and was replicated (P = 0.00719) in
pediatric TS (combined P = 7.88 9 107) and a second cohort for
adult VTE (P = 0.015). 27 additional SNPs are associated at confident
P-values (P < 1 9 104).For NGS analysis, a sibling disequilibrium
test was applied on 41,478 variants, 4,062 of which were novel. 23 significant (P < 0.05) coding non-synonymous or UTR SNPs in 10 genes
were identified and subsequently validated and genotyping within the
full cohort (257 families).
Conclusion: Our data support the presence of novel susceptibility genes
and coding variants, which are not directly linked to the coagulation
system. Future functional studies are warranted to further characterize
their role in the pathogenesis of VTE.
Disclosure of Interest: None Declared.
105
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Kvasnicka T1, Hajkova J1, Bobcikova P1, Cverhova V1,
Kvasnickova P1, Ulrych J2, Briza J2, Malikova I1 and Kvasnicka J1
1
Thrombotic Center; 21st Surgical Department, General Faculty
Hospital, Prague, Czech Republic
Objectives: The aim of our study was to determine prevalence of gene
polymorfism of thrombocytes [(GPVI (13254T/C), P2Y12 (H1/H2
haplotype i742T), P2Y12 (32C/T), PAR-1 (IVSn-14A/T), COX-1
(-842a/G), GPIa (807C/T), GPIIIa (PlA1/PlA2)] joined with atherosclerotic cardiovascular disease (CVD) and known thrombophilic
mutations [FV Leiden (Arg534Gln, rs6025), prothrombin F2
(20210G > a, rs1799963) and SERPINE1 (4G/5G, rs1799889)] in subjects with evidential VTE (n = 2360) compared with a control group
(n = 1460) of healthy blood donors.
Methods: Genome DNA was extracted from their leukocytes in
peripheral blood and isolated using the MagNA Pure LC Nucleic Acid
Extraction systemTM. DNA was isolated according to the MagNA Pure
High-Performance DNA ExtractionTM protocol. Mutations were determined using PCR in a process called FRET (Fluorescence Resonance
Energy Transfer). Tests were performed using the LightCyclerâ 480
System with LCâ 480 Genotyping Master kits.
Results: Determined genotypes of the alleles FVL, FII 20210G>A,
PAI-1 4G/5G, GP6 (Ser219Pro, rs1613662), SERPINC1
(IVS + 141G>A, rs2227589), CYP4V2 (Lys259Gln, rs13146272) and
gene polymorfism of thrombocytes (GPVI [13254T/C], P2Y12 [H1/H2
haplotype i742T] P2Y12 [32C/T], PAR-1 [IVSn-14A/T], COX-1
[-842a/G] GPIa [807C/T], GPIIIa [PlA1/PlA2]) in subjects with identified VTE and a control group of healthy blood donors are presented in
Table. All results met the criteria of Hardy-Weinberg equilibrium
(HWE)
GH05
The EAHAD coagulation factor variant databases
Hampshire DJ1, Gomez K2, Goodeve AC1, Kemball-Cook G2,
Ludlam CA3, McVey JH4, Oldenburg J5, Perkins SJ6, Peyvandi F7
and Rallapalli PM6
1
Department of Cardiovascular Science, University of Sheffield,
Sheffield; 2The Katherine Dormandy Haemophilia Centre and
Thrombosis Unit, Royal Free Hampstead NHS Trust, London;
3
University of Edinburgh, Edinburgh; 4Department of
Biochemistry and Physiology, University of Surrey, Guildford,
UK; 5Institute of Experimental Hematology and Transfusion
Medicine, University of Bonn, Bonn, Germany; 6Research
Department of Structural and Molecular Biology, UCL, London,
UK; 7A. Bianchi Bonomi Hemophilia and Thrombosis Center,
Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico,
Milan, Italy
Objectives: Locus-specific databases available for coagulation factors
(CoagDB) are a highly valued clinical/scientific resource; providing
searchable lists of sequence variants and information on how these
variants relate to phenotype in patients with inherited bleeding disorders. CoagDB receive little or no administrative/financial support,
putting their long-term viability at risk. In addition, for several coagulation factors multiple CoagDB exist, each containing different (and
sometimes conflicting) information causing confusion amongst users.
The European Association for Haemophilia and Allied Disorders
(EAHAD) aimed to create a combined CoagDB portal to provide centralised administrative/financial support to CoagDB curators and consistent information to CoagDB users.
Methods: Initially focusing on CoagDB for coagulation factor VIII
(F8), factor IX (F9) and von Willebrand factor (VWF), the current
information, data submission and data curation processes were evalu-
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106
12/06/2014
106
ABSTRACTS
ated and data protection and ethical issues associated with all three
addressed.
Results: Access to all three CoagDB has been combined under a central EAHAD portal (www.eahad-db.org) allowing a common data
protection policy and variant submission process to be adopted. Basic
versions of the databases providing information on known variants
and associated phenotypic data have been established in a joint Leiden
Open Variation Database (LOVD) v.2.0 installation. In addition,
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enhanced F8 and F9 databases provide additional information, e.g.
regarding protein structure.
Conclusion: EAHAD have established a common CoagDB front-end,
initially incorporating F8, F9 and VWF with plans for incorporation
of further genes. This portal will serve as a worldwide repository providing accurate and consistent information for all coagulation factors
relevant to all those working in the clinical and scientific sectors, while
also ensuring long-term administrative/financial support.
Disclosure of Interest: None Declared.
12/06/2014
© 2014 International Society on Thrombosis and Haemostasis 12 (Suppl. 1) (2014) 1–106

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