Acquired von Willebrand Disease Caused by an

From www.bloodjournal.org by guest on January 20, 2015. For personal use only.
Acquired von Willebrand Disease Caused by an Autoantibody Selectively
Inhibiting the Binding of von Willebrand Factor to Collagen
By Perry J.J. van Genderen, Tom Vink, Jan J. Michiels, Mars B. van ’t Veer, Jan J. Sixma,
and Huub H.D.M. van Vliet
An 82-year-old man with a low-grade malignant non-Hodgkin lymphoma and an IgG, X monoclonal gammopathy presented a recently acquired bleeding tendency, characterized
by recurrent epistaxis, easy bruising, and episodes of melena, requiring packed red blood cell transfusions. Coagulation studies showed a von Willebrand factor (vWF) defect
(Ivy bleeding time, > l 5 minutes; vWF antigen [vWFAg], 0.08
U/mL; ristocetin cofactor activity [vWFRCOF], ~ 0 . 0 5U/mL;
collagen binding activity [vWFCBA], 0.01 U/mL; absence of
the high molecular weight muitimers of vWF on multimeric
analysis). Mixing experiments suggested the presence of an
inhibitor directed against thevWFCBA activity of vWF
without significantly inhibiting the FVIIkC, vWFAg, and
V
ON WILLEBRAND factor (vWF) plays a crucial role
in the early phases of hemostasis. This complex,
multimeric glycoprotein (GP) mediates the shear-rate-dependent adhesion of platelets to the exposed subendothelium
at sites of vascular injury and platelet-to-platelet interactions.’.’ Likewise, a quantitative andor qualitative defect of
vWF, as observed in patients with congenital von Willebrand
disease (vWD), results in a bleeding diathesis. Acquired
cases of vWD, mimicking congenital vWD in their clinical
and laboratory features, have also been ~ b s e r v e d . ~ . ~
Acquired vWD has most frequently been reported in patients with lymphoproliferative or autoimmune disorders,’
suggesting an immunologic eti~logy.’~~
Two major mechanisms have been proposed to explain the pathogenesis of the
acquired deficiency of v W F in cases of lymphoproliferative
disorders or monoclonal gammopathies. In the first, antibodies inactivate immunologic or functional sites of VWF’.~or
form a c ~ r n p l e x , ~thereby
~ ’ ~ ~ inducing a rapid clearance of
vWF from the circulation. In the second mechanism, the
observed abnormal multimeric pattern of vWF results from
the selective absorption of large and intermediate multimers
of vWF by malignant cell^.^,^
In the present study, a patient with a low-grade malignant
B-cell non-Hodgkin lymphoma and a monoclonal IgG3 X
gammopathy is described in whom an IgM autoantibody
directed against the collagen binding domains of vWF is
implicated in the pathogenesis of the acquired deficiency of
vWF.
From the Department of Hematology, University Hospital Dijkzigt, Rotterdam;the Department of Hematology, UniversityHospital
Utrecht, Utrecht; and Dr Daniel den Hoed Cancer Centre, Rotterdam, The Netherlands.
Submitted November 22, 1993; accepted July 14, 1994.
Address reprint requests to PerryJ.J. van Genderen, MD, Department of Hematology, University Hospital Dijkzigt, Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands.
The publication costsof this article were defrayedin part by page
chargepayment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1994 by The American Society of Hematology.
0006-4971/94/8410-0002$3.00/0
3378
vWF:RCoF activities. The inhibitor was identified as an antibody of the IgM class by immunoabsorption of vWF and
inhibitor-vWF complexes from the plasma of the patient.
Subsequent immunoprecipitation experiments using recombinant fragments of vWF showed that the inhibitor reacted
with both the glycoprotein Ib binding domain (amino acids
[aal 422-826) and the A3 (aa 909-1112) domain of vWF, but
not with theA2 (aa 716-9081 or D4 (aa 1183-1535) domains.
We conclude thattheIgM
autoantibody inhibits the
vWF:CBA activity by reacting with an epitope present on
both the glycoprotein Ib and A3 domains of vWF.
0 1994 by The American Society of Hematology.
CASEREPORT
A 82-year-old man with an
IgG, A paraprotein (5 g/L) was referred
to our department in 1989 for further analysis of a prolongedpartial
thromboplastin time discovered before a liver biopsy in the staging
procedureofanon-Hodgkinlymphoma.Physicalexamination
showed a slight hepatosplenomegaly, without enlarged lymph nodes.
Hemoglobin levels and leukocyte, lymphocyte, and platelet counts
were normal, as were the serum values for total lgG, IgA, and IgM.
The bone marrow aspirate showed a nonnocellular hematopoiesis
A). Additionally,a
with 8%monoclonalplasmacells(cIgM+G
monoclonal B-cell population (slgM A) represented 5% and 2 1 % of
the total B-cell population in blood and bone marrow, respectively.
Skeletalx-raysshowednoosteolyticlesions.
The diagnosis was
made as low-grade malignant non-Hodgkin lymphoma stage IV
with
paraproteinemia IgG3 A.
Before 1989, the patient had no personal or family history
of a
bleeding tendency. Previous hemostatic challenges (appendectomy,
dentalextractions,andcholecystectomy)wereuneventful.
From
1989 on, the patient developed several episodes of easy bruising,
recurrent epistaxis, and severe gastrointestinal blood
loss, necessitating extensive replacement therapy with packed red blood cells and
factor VI11 (FVIII)/vWF concentrates. Gaatrointestinal radiographic
and endoscopic studies did not show a source of blood loss, except
for a mild gastritis at one occasion. A transurethral prostatectomy
was successfully performed with the administration of DDAVP and
frequent cryoprecipitate infusions. However,a trial of cyclophosphamide (2 mgikg body weight) for 6 weeks did not improve the coagulation defect.
MATERIALS AND METHODS
Coagulationassays.Bleedingtimesweremeasuredaccording
to Ivy et al.” Platelets were counted in EDTA blood samples using
the Platelet Analyzer 810 (Baker Instruments, Allentown,
PA). Platelet aggregation inducedby 0.63 and 1.75 mg/mL ristocetin (H. Lundbeck CO, Copenhagen, Denmark) was recorded with the use of a
serial aggregometer (Payton model 300B; Scarborough, Canada) in
platelet-rich plasma at a standard count 200
of X 1 0 9 LFVIII coaguof the Automatic Coaglant activity(FVI1I:C) was assayed by means
ulation Laboratory (ACL; Instrumental Laboratory, Ijsselstein, The
Netherlands) using FVIII-deficient plasma (Ortho Diagnostic Systems, Beerse, Belgium).
vWF assays. vWF antigen(vWF:Ag)wasassayed
by anenas described by
zyme-linked
immunosorbent
assay
(ELISA),
Cejka.” Ristocetin cofactor activity (vWF:RCoF) was assayed with
formalin-fixed platelets” using a PAP4 Model Aggregometer @ioData Corp. Hatboro, PA).
Blood, Vol 84, No 10 (November 15), 1994: pp 3378-3384
From www.bloodjournal.org by guest on January 20, 2015. For personal use only.
3379
ACQUIRED VON WILLEBRANDDISEASE
vWF binding to collagen. The collagen binding activity of vWF
(vWF:CBA) was measured according to the ELISA-based method
of Brown and Bosak” with slight modifications. Microtiter plate
wells (NS Nunc, Roskilde, Denmark) were coated overnight at 4°C
with 100 pL of a 0.2 mg/mL suspension of collagen (bovine achilles
tendon, type I; Sigma Chemicals, St Louis, MO) in 20 mmol/L acetic
acid. After washing with phosphate-buffered saline (PBS)-O.O5%
Tween 20 (PBS-Tween), 100 pL of 1/40 dilutions of plasmas in
PBS-Tween 20-1% albumin (PBS-Tween-Albumin) were added to
the wells and incubated for 2 hours at room temperature. After
rinsing, the wells were incubated with 100 pL of a 1/200 dilution
in PBS-Tween-Albumin of horseradish peroxidase-conjugated rabbit
polyclonal Igs to human vWF (Dakopatts N S , Glostrup, Denmark)
for another 2 hours at room temperature. After washing, 100 pL
ABTS substrate solution [27.4 mgABTS (2.2’-azino-di-[3-ethylbenzthiazolsulphonate(8)]) dissolved in 10 mL buffer, containing 10
mmol/L citric acid, 100 mmoVL NaH2PO4.H2O,and 10 pL 30%
H,OJ was added. After 15 minutes of incubation, the color development was stopped by the addition of 10 pL concentrated acetic acid
and the extinction was measured at 414 nm.
vWF multimeric analysis. The multimeric composition of vWF
was analyzed by sodium dodecyl sulphate (SDS) 0.9% agarose gel
electrophoresis according to Brosstad et al.I4
Inhibitor screening. Assay of an inhibitor against FV1II:Cwas
performed as described by Kasper et al.’5 Inhibitors of vWFAg,
vWF:RCoF, and vWF:CBA were sought by incubating 1 v01of
patient plasma (PP) with increasing volumes of pooled normal
plasma (NP) for 1 hour at 37°C. Equal volumes of normal pooled
plasma and PBS served as control samples.
Infusion studies. The commercial vWF concentrate Haemate P
(Behring, Marburg, Germany) was infused (3,000 IU) at the time of
a severe nasopharyngeal bleeding. Bleeding times and vWF parameters were measured before and at several times after infusion, after
obtaining full informed consent from the patient.
IdentGcation of the Ig class of the inhibitor with the use of antivWF immunobeads. To identify the Ig class of the inhibitor, rabbit
polyclonal Igs to human vWF (Dakopatts) were coupled to Immunobeads (BioRad, Hercules, CA) according to the instructions of the
manufacturer. Two hundred microliters of PP (vWF:Ag as estimated
by ELISA 0.25 ? 0.03 U/mL), 200 pL of NP (vWFAg 1.00 -C 0.04
U/mL), or, to allow complex formation between eventually free
inhibitor and vWF, 200 pL of a mixture of PP and NP (ratio, 76:l)
was diluted in 1,000 pL PBS-Tween-Albumin. After incubation
overnight at 4”C, 40 pL of anti-vWF immunobeads was added and
incubated for 3 hours at room temperature. It was previously established that the binding capacity of 40 pLof anti-vWF immunobeads
was in excess for 200 pL NP (data not shown). The immunobeads
were then pelleted (for 10 minutes at 1,500g at room temperature)
and washed three times with 10 mL PBS-Tween and resuspended in
500 pL of a 111,OOO dilution in PBS-Tween-Albumin of horseradish
peroxidase-conjugated rabbit polyclonal Igs to either human IgM,
IgG, or IgA (Dakopatts). After 3 hours of incubation at roomtemperature, the immunobeads were again pelleted and washed three times
with PBS-Tween and finally resuspended in 1,000 pL ofABTS
substrate solution. After 10 minutes, the color development was
stopped with the addition of 100 pL concentrated acetic acid. After
pelleting the immunobeads (for 10 minutes at 1,500g at room temperature), the extinction of the supernatant was measured at 414
nm. The background extinction was estimated by performing this
experiment with 1,OOO pL PBS-Tween-Albumin instead of plasma.
All experiments were performed in duplicate.
Expression of recombinant vWF fragments. A detailed description of the construction and expression of recombinant vWF (NWF)
and NWF fragments in baby hamster kidney (BHK) cells will be
published elsewhere (Vink et al, submitted). Briefly, the coding
20so
MI
I
W.
i
I
8P.
L
W.
,
,
.,
I
.
’
.‘l
Propeptide
Dl
D2
1
Mature subunit
HI
D’ A1D3
A2
AS
H
D4
H”
B C1 c2
A3
D4
Fig 1. Schematic representation of vWF and recombinant frag
menta. The upper drawing represents the vWF prepropeptide, divided into the signal peptide (sp),the propeptide, and the mature
subunit of 2,050 aa. The second drawing represents the typical vWF
domain structure. The lower four drawings representthe constructs
created to exprassthe four vWF fragmentsfor usein the irnmunoprecipitation experiments.
sequence for the GPIb binding domain of vWF (amino acids [aa]
422-826), the A2 domain (aa 716-908), the A3 domain (aa 9091112), and the D4 domain (aa1183-1535).were spliced to the coding
sequence of the signal peptide of vWF (Fig 1). This construct was
introduced in the mammalian expression vector pNUTI6 and stable
cell lines were obtained after transfection of BHK cells and selection
with methotrexate. The transient expression of rvWF and a mutant
lacking the A1 domain (AA1-rvWF) in Cos cells, has been described.” Both these constructs were introduced in the pNUT vector
and stable cell lines were established in BHK cells.
Metabolic labeling. Cell lines were washed twice with DulbecCO’smodified Eagle’s medium (DMEM; GIBCO, Paisley, UK) medium and starved for 1 hourinDMEM without methionine and
cysteine. Trans-S-label (ICN Biomedicals, Costa Mesa, CA) was
added at 500 pCi and the cells were incubated for 4 hours at 37°C.
Unlabeled methionine and cysteine (150 pg/mL) medium was then
added. The conditioned medium was harvested after 20 hours. Dead
cells were spun down and protease inhibitors were added (1 mmoV
L phenylmethylsulfonyl fluoride, 10 mmol/L EDTA, 10 mmol/L
benzamidine, and 5 m m o K N-ethylmaleimide [N-EM]).
Iodination of vWF. vWF was purified as described.” vWF was
labeled with NaIZ5Iusing the Iodogen method” to a specific activity
of 100,OOO c p d p g .
Immunoprecipitation. Conditioned medium (100 pL) was mixed
with 400 pL immunoprecipitation buffer (10 mmoVL Tris, 150
mmoVL NaCl, 1% Nonidet P-40, and 0.5% bovine serum albumin,
pH 7.4), mixed with 5 pL serum of the patient, and rotated for 2
hours at 4°C. Twenty microliters of rabbit polyclonal Igs to human
IgM (Dakopatts) was added and rotated for 1 hour at 4”C, after
which 40 pL protein A-sepharose (Pharmacia, Uppsala, Sweden)
was added and rotated for 1 hour at 4°C. The Sepharose beads were
pelleted (for 30 seconds at 14,OOOgat room temperature) and washed
four times with 1 mL of immunoprecipitation buffer. Bound protein
reducing
was eluted by incubation for 5 minutes at100°Cwith
sample buffer and analyzed by polyacrylamide gel electrophoresis
(PAGE) on a 5% 25% gel in the presence of SDS, performed according to Laemmli.” Competition experiments with unlabeled fragments were performed by incubating 100 pL conditioned medium,
with 100 pL 1OX immunoprecipitation buffer and 800 pL of BHK
From www.bloodjournal.org by guest on January 20, 2015. For personal use only.
VAN GENDEREN ET AL
3380
mediumor a mixture o f the GPlb and A3 domainfollowedby
immunoprecipitation, as described above. Immunoprecipitation o f
"'I-vWF was performed by incuhating I p g '"I-vWF ( I O pL) with
100 pL I0x immunoprecipitationbuffer.
10 p L of thepatient's
serum, and 900 pL o f the culture media. followedby an immunoprecipitation, a s described. The "'I-vWF precipitationswerescanned
with a Phospholmager (Molecular Dynamics, Sunnyvale, CA) and
analyzed using ImageQuant software (Molecular Dynamics).
RESULTS
Platelet function and vWF-related activities
in plmna.
The initial hemostatic data of our patient were characterized
by a prolonged Ivy bleeding time (> 15 minutes; normal,
c 4 minutes), absent ristocetin-induced platelet aggregation,
low levels of FVII1:C (0.10 U/mL: normal. 0.60 to 1.40 U/
mL) and vWF:Ag (0.08 U/mL; normal, 0.60 to I .40 U/mL),
and very low to undetectable levels of vWF:RCoF (<O.OS
U/mL: normal, 0.60 to I .40 U/mL) and vWFCBA (0.01 U/
mL: normal, 0.60 to 1.40 U/mL), confirming the diagnosis
of vWD. A selective absence of the high molecular weight
multimers of vWF, as seen in patients with type IIA congenital vWD. was noted (Fig 2, preinfusion value). The absence
of thehigh molecular weight multimers of vWF was not
caused by increased proteolysis. as shown by subunit analysis of vWF. or caused by the binding of vWF to malignant
lymphoma cells (results not shown).
Infirsion studies. Infusion of 3,000 U of the vWF concentrate Haemate P (Fig 2) resulted in a partial correction
BT (min)
>lo'
FVIII:C (U/ml)
0.09 0.40
vWF:Ag (U/ml)
0.16
1.78
0.78
7'45" 7'15" >IO'
0.22
0.16
>lo'
0.14
0.50 0.36
vWF:RCoF (U/ml) <0.05 0.86 0.23 <0.05 c0.05
vWF:CBA (Ulml)
<0.01 0.38 0.04 ~0.01<0.01
NP before
0'
60'
120'
240'
Fig 2. The effect of infusion of 3,000 IU Haemate P on patient's
I v y bleeding time (ET), FVlll/vWF activities, and mukimeric patternof
plasma vWF before and at different times after infusion. NP, normal
pooled plasma.
of the bleeding time. In addition, a rapid clearance to preinfusion levels of the FVlWvWF-related activities and the high
molecular weight multimers of vWF was observed. Interestingly, the vWF:CBA activity remained decreased immediately after Haemate P infusion, despite normal levels of ristocetin cofactor activity and vWF antigen.
Inhibitor of FVIII/vWF. The patientplasma contained
no inhibitory activity against FVI1I:C (data not shown). Incubation of different volumes of normal pooled plasma with 1
v01 of PPdidnotinduce
significant inactivation of the
vWF:Agand
vWF:RCoF activities (results not shown).
However, a significant reduction of the vWFCBA activity
was observed in themixture of 1 v01 of PPwith I v01 of
normalpooledplasma
(0.29 t 0.02 U/mL) as compared
with controls (0.49 5 0.02, P < ,001). Incubation of I v01
of PP with increasing volumes of normalpooledplasma
showed that the inhibitory effect of the PP on the vWF:CBA
activity was lost after incubating I v01 of PP with 9 v01 of
normal pooled plasma (1.02 2 0.03 U/mL v controls 1.04
2 0.06; P > .OS). No significant changes of the vWFAg,
vWF:RCoF, and vWF:CBA activities were measured after
prolonged incubation (data not shown). The mixture of normal and patient plasma was found to retain the high molecular weight multimers of vWF. indicating no specific in vitro
proteolysis of vWF (data not shown).
Identification of the Ig class of the inhibitor with the use
of anti-vWF immunohead.~. The results of a representative
experiment of immunoabsorption of vWF or vWF-Ig complexes are shown in Fig 3. Two times more IgM, bound to
vWF isolated by immunoabsorption of plasma from the patient, was observed in comparison to the results found when
using 200 yL of normal pooled plasma. This indicates the
presence of IgM-vWF complexes in the plasma of the patient. Preincubation of normal pooled plasma with an excess
of PP (ratio 1:76) resulted in a sevenfold increase in the
binding of IgM to vWF isolated from the mixture of normal
pooled and patient plasma as compared with normal pooled
plasma. The binding of IgG and IgA was comparable. Furthermore, after the plasma of the patient was depleted of
IgM, no inhibition of the vWF:CBA activity was observed
in mixing experiments with normal pooled plasma (data not
shown).
Immunoprecipitation of n ~ W Ffragments I>? /he inhibitor,
To determine which part of vWF interacted with the inhibitor, recombinant fragments of vWF expressed in mammalian
cells (Fig 1 ) were immunoprecipated. As shown in Fig 4A,
the inhibitor reacted both with the GPIb binding domain and
the A3 domain of vWF. butnot with the A2 or theD4
domains of vWF. This indicates that either the inhibitor has
an epitope present on aa 422-715 and aa 909- 1 I 12 or that
two anti-vWF antibodies are involved. To further show that
the inhibitor reacted exclusively with the vWF GPlb and A3
domains, we performed additional inhibition experiments.
Immunoprecipitation studies (Fig 4B) showed that an excess
of mixed unlabeled GPlb and A3 domain could compete for
the immunoprecipitation of metabolically labeled rvWF and
AAl-rvWF (missing aa 478-717) by the inhibitor. These
domains seem to be the only parts of vWF interacting with
the inhibitor(s). To investigate the monoclonality of the in-
From www.bloodjournal.org by guest on January 20, 2015. For personal use only.
3381
ACQUIRED VON WILLEBRANDDISEASE
CA
C
1000
.
I
U
S
.m
n
Q)
>
ICI
(R
I
Q)
a
800
600
400
200
0
IgM
100
IgA
Fig 3. Relative bindingof IgM, IgG, and IgA Igs to vWF or vWF-lg
complexes isolated by immunoabsorption from
(W) 200 pL ofpatient’s plasma (PP), (01200 pL of normal pooled plasma (NP), or(W)
200 pL of a mixture of PPandNP(PP + NP). Note the increased
of patient, particbinding of l@ Igs to vWF isolated from plasmathe
ularly after preincubation of normal pooled plasma with an excess
plasma oftha patiant (ratio, 1:76). The relative binding
of either IgM,
of the patient, normal pooled
IgG, or IgA to vWF isolated from plasma
plasma, or a mixture is expressed as the extinction obtained after
incubation of the sample with a horseradish peroxidaseconjugated
Ig to IgM, IgG, or IgA, respectively, after correction for background
activity IPBS-Tween-Albumin). The relative amounts
of IgM, total
IgG, and IgAin the plasma of the patient and normal pooled plasma
were comparable.
hibitor, we performed additional immunoprecipitations of
‘251-vWF with serum of the patient. As shown in Fig 5 , an
excess of either unlabeled recombinant GPIb fragments or
unlabeled recombinant A3 fragments was able to compete
for binding of the inhibitor to %
’‘WF
to the same extent
as unlabeled vWF, thereby excluding the presence of two
anti-vWF antibodies. The culture medium, unlabeled recombinant A2 domain, and unlabeled recombinant D4 domain
were unable to compete for binding of the inhibitor to Iz5IVWF.
DISCUSSION
The adhesion of platelets to the exposed subendothelium
or perivascular collagen at sites of vascular injury requires
the binding of v W F to these tissues.’l The high molecular
weight multimers of vWF are known to play an important
role in this initial phase of hemostasis by expressing numerous binding sites for collagen” and other subendothelial cons t i t u e n t ~ .In~ ~the described patient, an IgM autoantibody
was found to be involved in the pathogenesis of acquired
vWD that had severe consequences in vivo.
In the last 3 years, the patient developed a progressive
bleeding tendency consisting of recurrent epistaxis, easy
bruising, and severe gastrointestinal blood loss, requiring
almost weekly hospitalization. To achieve adequate hemostasis at times of bleeding, intensive replacement therapy
with DDAVP and FVIIYvWF concentrates was instituted
with only minor improvement. This finding suggested the
presence of a circulating inhibitor to the EVIIuvWF complex.
Inhibitors of vWF in plasma are normally shown in mixing
experiments with normal pooled plasma by their ability to
inhibit an immunologic (vWF:Ag)or functional activity
(vWF:RCoF) of v W F . In this patient, the mixing experiments did not indicate the presence of an inhibitor. However,
the vWF:CBA activity, which reflects the in vitro ability of
vWF to bind to collagen type I, was selectively inhibited.
This suggests that the inhibitor was directed against an epitope involved in the binding of vWF to collagen type I or
located near these collagen binding domains, causing steric
hindrance of the vWF-collagen interaction.
The results of the infusion study are in agreement with
this hypothesis. After Haemate P infusion, a discrepancy
between the functional activities of vWF, the vWF:RCoF,
andvWF:CBA activity, was noted. The vWF:RCoF and
vWF:Ag activities normalized after infusion, indicating the
presence of sufficient amounts of functional vWF. Nevertheless, the bleeding time remained prolonged. In contrast, the
vWF:CBA activity remained decreased. This findingmay
indicate a correlation between the collagen-binding activity
and the bleeding time in this patient.
There was a strikingly rapid clearance of the vWF-related
activities vWF:Ag, vWF:RCoF, and vWFCBA and the high
molecular weight multimers of v W F after infusion. As increased proteolysis of vWF and binding of v W F to
lymphoma cells were ruled out, the decrease of high molecular weight multimers of v W F is probably explained bya
preferential complex formation between the inhibitor and the
high molecular weight multimers of vWF, as observed by
others.3@’These multimers are known to possess the ristocetin cofactor activity and have numerous collagen-binding
domains. Subsequently, these vWF-inhibitor complexes are
rapidly cleared from the circulation by the mononuclear
phagocytic system.
vWF has two domains involved in the interaction with
collagen type I and
They reside on part of the GPIbbinding domain (aa 422-826) known as the A1 domain and
the A3 domain (aa 909-1112). With the use of several separate rvWF fragments in the immunoprecipitation experiments, we could show that the inhibitor was reactive with
the GPIb and A3 domains of vWF. Despite a considerable
heterogeneity between the two collagen-binding domains of
vWF, the inhibitor recognized an epitope occurring on both
domains. Several mouse monoclonal antibodies (MoAbs)
that also inhibit the binding of vWF to collagen have been
des~ribed.’~-~’
Most of them were reactive either with the
AlZ5or the A3
Our autoantibody resembles the
characteristics of the mouse MoAb described by Roth et alZ4
that was also reactive with both collagen-binding domains.
To our knowledge, this is the first antibody of human
origin inhibiting the binding of vWF to collagen, resulting
in a virtual absence of functional vWF and a concomitant
bleeding tendency. In contrast to severe congenital vWD, in
which there is a comparable absence of functional vWF,
our patient did not suffer from muscle or joint bleedings.
Apparently the amount ofFV1II:C present was sufficient
From www.bloodjournal.org by guest on January 20, 2015. For personal use only.
VAN GENDERENET AL
3382
- 180
- 116
- 84
- 58
- 48.5
- 36.5
- 26
1
2
3
4
1
2
3
4
Fig 4. Immunoprecipitation of metabolically labeled culture supernatant of recombinant vWF fragments with patient serum. Precipitates
were separated using 4% t o 15% SDS-PAGE and autoradiographed for 20 days. (A) Lane 1, immunoprecipitation of patient's serum with
labeled GPlb domain supernatant (the arrow indicates the position of the precipitated
GPlb domain); lane 2, labeled A2 domain supernatant;
lane 3, labeled A3 domain supernatant (the arrow indicates the position of the precipitated
A3 domain); lane 4, labeled D4 domain supernatant.
(B) Competition immunoprecipitation experiment
with excess unlabeled GPlb, A3 domains, or excess BHK culture medium. Lane l,
immunoprecipitation of labeled AAl-rvWF, a mutant lacking theA1 domain of vWF, in the presence of an excess BHK culture medium; lane 2, same as
lane 1, but labeled AAl-rvWF was precipitated in the presence of an excess mixture of unlabeled GPlb and A3 domain fragments; lane 3,
labeled rvWF precipitated in the presence of an excess BHK culture medium; lane 4, same as lane 3, but labeled rvWF was precipitated in the
presence of an excess of unlabeled GPlb and A3 domain fragments. The t w o visible bands in lanes 1 and 3 represent the propolypeptide and
the mature subunit of vWF, which are due t o inefficient processing in the BHK cells. Note the size difference between the deletion mutant
AAl-rvWF, missing 239 amino acids, in lane 1 and the rvWF in lane 3.
enough to prevent these hemophilia-like bleeding complications.
In conclusion, in this study a case of acute vWD is described caused by an IgM autoantibody that inhibits the bind-
ing of vWF to collagen andthat reacts with both the GPlb
and A3 domains of vWF. Although several investigators
using cultured endothelial cells have recently shown that it
is unlikely that subendothelial collagen is the binding site
From www.bloodjournal.org by guest on January 20, 2015. For personal use only.
3383
ACQUIRED VON WILLEBRANDDISEASE
3. MannucciPM, Lombardi R, Bader R, HorellouMH, Finazzi
G, Besana C, Conard J, Samama M: Studies of the pathophysiology
of acquired von Willebrand’s disease in seven patients with lymphoproliferative disorders or monoclonal gammapathies. Blood 64:614,
I984
4. Richard C, Cuadrado MA, Prieto M. Battle J, Ldpez Fernindez
MF. Rodriguez Salazar ML, Bello C, Recio M, Santoro T, Gomez
Casares MT, Zubizarreta A: Acquiredvon Willebrand disease in
multiple myeloma secondary to absorption of von Willebrand factor
by plasma cells. Am J Hematol 35:114, 1990
S. HandinRI.MartinV,
Moloney WC: Antibody-induced von
Willebrand’s disease: A newlydefined inhibitor syndrome. Blood
48:393, 1976
6. Mohri H, Noguchi T, Kodama F, Itoh A, Ohkubo T: Acquired
von Willebrand disease due to inhibitor of human myeloma protein
specific for von Willebrand factor. A m J Clin Pathol 87:663, 1987
7. Zettervall 0, Nilsson IM: Acquired von Willebrand’s disease
caused by a monoclonal antibody. Acta MedicaScand 20452 I , 1978
8. Can TE, Sawers RJ, Koutts J: Pathogenesis of antibody-induced acquired von Willebrand syndrome. Am J Hematol 9:363,
I980
9. Joist JH, Cowan JF. Zimmerman TS: Acquired von Willebrand’s disease. Evidence for a quantitative and qualitative factor
VI11 disorder. N Engl J Med 298:988, 1978
IO. Ivy AC, Nelson D, Bucher G: The standardization of certain
factors in the cataneous ‘venostasis’ bleeding time technique. J Lab
Clin Med 26:1812, 1941
I I . Cejka J: Enzyme immunoassay for WlII-rclotcd antigen. Clin
Chem 28: 1356, 1982
12. Macfarlane DE, Stibbe J, Kirby EP. Zucker MB, Grant RA,
McPherson JA: A method for assaying von Willebrand factor (RistoFig 5. Immunoprecipitation of‘2sI-vWF with the patient’s serum,
cetin cofactor). Thromb Diath Haemorrh 34306, 1975
in competition with the four recombinant fragments of vWF and cold
13. Brown JE, Bosak JO: An ELlSA test for the binding of von
rvWF, showing that both the GPlb and A3 domain of vWF, as well
Willebrand antigen to collagen. Thromb Res 43:303, 1986
as full-length rvWF, were able to compete with the binding of t h e
14. Brosstad F, Kjonniksen 1, Ronning B, Stormorken H: Visualinhibitor to vWF, whereas the A2 and D4 domain of vWF and excess
ization of von Willebrand factor multimers by enzyme-conjugated
BHK culture medium were not. Precipitates were analyzed using 5%
secondary antibodies. Thromb Haemost S5:276, 1986
PAGE, scanned using a Phospholmager and analyzed using Im15. Kasper CK. Aledon LM, Counts RB, Edson JR. Fratantoni J.
agequant software. Lane 1, excess BHK culture medium (CM);lane
Green D, Hampton JW, Hilgartner MW, Lazerson J, Levine PH,
2,20-fold excess of cold vWF (vWFI; lane 3, excess GPlb (GPlb)culture
McMillan CW. Pool JG, Shapiro SS. Shulman NR, van Eys J: A
medium; lane 4, excess A2 (A21 culture medium; lane 5, excess A 3
(A31 culture medium; lane 6, excess D4 (D4) culture medium.
more uniform measurement of factor VI11 inhibitors. Thromb Diath
Haemorrh 342369,1975
16. Palmiter RD. Behringer RR, Quaife CJ, Maxwell F. Maxwell
IH, Brinster RL:Cell lineage ablation in transgenic mice by cellfor vWF”.” this patient shows that disturbing the interaction specific expression of a toxin gene. Cell S0:435, 1987
ofvWFwithcollagentype
I in vivoresults in a severe
17. Sixma JJ, Schiphorst ME, Verweij CL, Pannekoek H: Effect
bleeding tendency. Apparently, collagen located elsewhere
of deletion of the AI domain of von Willebrand factor on its binding
to heparin. collagen and platelets in the presence of ristocetin. Eur
in the vessel wall plays a pivotal role in maintaining proper
J Biochem 196369, 1991
hemostasis by providing a binding site for vWF.
18. van Mourik JA, Mochtar IA: Purification of human anti-hemophilic factor (FVITI)by gelchromotography. Biochim Biophys
ACKNOWLEDGMENT
Acta 221:677. 1970
We thank Drs Jet Bakker and Peter P. Viergever (Zuiderzieken19. Fraker PJ. Speck JC: Protein and cell membrane iodinations
huis. Rotterdam, The Netherlands) for referral of the patient and Dr
with a sparingly soluble chloramide, I , 3, 4, 6,-tetrachloro-3, 6Beatrice Fournier for critically reading the manuscript. Klaas Last,
diphenyl glycolurol. Biochem Biophys Res Commun 80349, 1978
Sonja van de Luytgaarde. and Marion Schiphorst are acknowledged
20. Laemmli UK: Cleavage of structural proteins during the asfor technical assistance.
sembly of the head of the bacteriophage T4. Nature 227:680, 1970
21. Sakariassen KS, Bolhuis PA, Sixma JJ: Human blood platelet
REFERENCES
adhesion to artery subendothelium is mediated by factor VIII-von
Willebrand factor boundto the subendothelium. Nature 279:636,
I . Ruggeri ZM, Ware J: The structure and function of von WilleI979
brand factor. Thromb Haemost 67594. 1992
22. Santoro S: Preferential binding ofhigh molecular weight
2. Sixma JJ, Sakariassen KS, Beeser-Visser NH:Adhesionof
forms of von Willebrand factor to fibrillar collagen. Biochim Bioplatelets to human artery subendothelium. Effect of factor VIII-von
phys Acta 756: 123. 1983
Willebrand factor of various multimeric composition. Blood 63:128.
23. De Groot PG, Ottenhof-Rovers M. van Mourik JA, Sixma JJ:
I984
II
1
2
13
3
4
5
6
From www.bloodjournal.org by guest on January 20, 2015. For personal use only.
3384
Evidence that the primary binding site of von Willebrand factor that
mediates platelet adhesion on subendothelium is not collagen. J Clin
Invest 82:65, 1988
24. Roth GJ, Titani K, Hoyer LW, Hickey MJ: Localization of
binding sites within human von Willebrand factor for monomeric
type I11 collagen. Biochemistry 25:8357, 1986
25. Pareti FI, Niija K, McPherson JM, Ruggeri ZM: Isolation and
characterization of two domains ofhumanvon Willebrand factor
that interact with fibrillar collagen type I and 111. J Biol Chem
262:13835, 1987
26. Girma J-P, Kalafatis M, Pietu G, Lavergne J-M, Chopek MW,
VAN GENDEREN ET AL
Edgington TS, Meyer D: Mapping of distinct von Willebrand factor
domains interacting with platelet GPIb and GPIIb/IIIa and withcollagen using monoclonal antibodies. Blood 67:1356, 1986
27. Stel HV, Sakariassen KS, Scholte BJ, Veerman ECI, van der
Kwast TH, De Groot PG, Sixma JJ, van Mourik JA: Characterization
of 25 monoclonal antibodies to factor VIII-von Willebrand factor:
Relationship between ristocetin-induced platelet aggregation and
platelet adherence to subendothelium. Blood 63:1408, 1984
28. Wagner DD, Urban-Pickering M, Marder V: von Willebrand
factor binds to extracellular matrices independently of collagen. Proc
Natl Acad Sci USA 81:471, 1984
From www.bloodjournal.org by guest on January 20, 2015. For personal use only.
1994 84: 3378-3384
Acquired von Willebrand disease caused by an autoantibody
selectively inhibiting the binding of von Willebrand factor to collagen
PJ van Genderen, T Vink, JJ Michiels, MB van 't Veer, JJ Sixma and HH van Vliet
Updated information and services can be found at:
http://www.bloodjournal.org/content/84/10/3378.full.html
Articles on similar topics can be found in the following Blood collections
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American
Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.

Download Report