Effect of recombinant ADAMTS13 on microthrombosis and brain

Journal of Thrombosis and Haemostasis, 12: 943–947
DOI: 10.1111/jth.12574
BRIEF REPORT
Effect of recombinant ADAMTS-13 on microthrombosis and
brain injury after experimental subarachnoid hemorrhage
M . D . I . V E R G O U W E N , * † V . L . K N A U P , † J . J . T . H . R O E L O F S , ‡ O . J . D E B O E R ‡ and
J. C. M. MEIJERS†§
*Department of Neurology and Neurosurgery, Brain Center Rudolf Magnus, University Medical Center Utrecht, Utrecht; †Department of
Experimental Vascular Medicine, Academic Medical Center, University of Amsterdam; ‡Department of Pathology, Academic Medical Center,
University of Amsterdam; and §Department of Plasma Proteins, Sanquin-AMC Landsteiner Laboratory, Amsterdam, the Netherlands
To cite this article: Vergouwen MDI, Knaup VL, Roelofs JJTH, de Boer OJ, Meijers JCM. Effect of recombinant ADAMTS-13 on microthrombosis and brain injury after experimental subarachnoid hemorrhage. J Thromb Haemost 2014; 12: 943–7.
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Keywords: ADAMTS13 protein, human; hemostasis;
subarachnoid hemorrhage; thrombosis; von Willebrand
factor.
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Summary. Background: A common complication after
aneurysmal subarachnoid hemorrhage (SAH) is delayed
cerebral ischemia (DCI), which is associated with vasospasm and other mechanisms such as microthrombosis.
ADAMTS-13 activity plays a role in the prevention of
thrombus formation in the cerebral microvasculature.
Previously, we observed that patients with DCI have
lower levels of ADAMTS-13. Objectives: To examine
whether recombinant human ADAMTS-13 (rADAMTS13) reduces cerebral microthrombus formation and brain
injury in an experimental mouse model of SAH including
wild-type and ADAMTS-13/ mice. Methods: Experimental SAH was induced with the prechiasmatic blood
injection model. The following experimental groups were
investigated: (i) C57BL/6J mice (n = 10); (ii) C57BL/6J
mice (n = 10) treated with rADAMTS-13 20 min after
SAH; (iii) ADAMTS-13/ mice (n = 10); and (iv) ADAMTS-13/ mice (n = 10) treated with rADAMTS-13
20 min after SAH. Mice were killed at 48 h. Results are
presented as means with standard errors of the mean.
Results: Infusion with rADAMTS-13 reduced the extent
of microthrombosis by ~ 50% in both wild-type mice
(mean fibrinogen area: 0.28% 0.09% vs. 0.15% 0.04%; P = 0.20) and ADAMTS-13/ mice (mean fibrinogen area: 0.32% 0.05% vs. 0.16% 0.03%; P =
0.016). In addition, rADAMTS-13 reduced brain injury
by > 60% in both wild-type mice (mean microglia area:
0.65% 0.18% vs. 0.18% 0.04%; P = 0.013) and
ADAMTS-13/ mice (mean microglia area: 1.24% 0.36% vs. 0.42% 0.13%; P = 0.077). Conclusions: Our
results support the further study of rADAMTS-13 as a
treatment option for the prevention of microthrombosis
and brain injury after SAH.
Correspondence: Mervyn D.I. Vergouwen, Department of Neurology
and Neurosurgery, Room G03-228, Brain Center Rudolf Magnus,
University Medical Center Utrecht, Heidelberglaan 100, 3584 CX
Utrecht, the Netherlands.
Tel.: +31 88 7550455; fax: +31 30 2542100.
E-mail: [email protected]
Received 3 February 2014
Manuscript handled by: P. de Moerloose
Final decision: P. H. Reitsma, 23 March 2014
Introduction
In 30% of patients with aneurysmal subarachnoid hemorrhage (SAH), the acute hemorrhage is complicated by
delayed cerebral ischemia (DCI) 4–14 days after the
hemorrhage [1]. DCI may progress to cerebral
infarction and increase brain injury after SAH, as
reflected by an increased risk of death or severe disability [1–3].
For decades, it was assumed that DCI is caused by
vasospasm, as vasospasm is strongly associated with DCI
[3]. However, vasospasm is neither a sufficient nor a necessary factor in the development of DCI [4]. More recent
studies have focused on other mechanisms that may contribute to DCI, such as microthrombosis [5–7]. In a previous study, we found that patients with DCI have lower
levels of ADAMTS-13 [8]. ADAMTS-13 rapidly cleaves
ultralarge von Willebrand factor multimers [9], reduces
platelet adhesion and aggregation [10,11], and downregulates inflammation and thrombus formation [12]. ADAMTS-13 activity plays a role among other factors in the
prevention of thrombus formation in the cerebral microvasculature, as illustrated by the occurrence of cerebral
infarcts in patients with thrombotic thrombocytopenic
purpura [13]. With the development of recombinant
human ADAMTS-13 (rADAMTS-13), it has become feasible to prevent thrombus growth in a murine model lacking ADAMTS-13 [10].
© 2014 International Society on Thrombosis and Haemostasis
12/06/2014
944 M. D. I. Vergouwen et al
The purpose of this study was to examine whether rADAMTS-13 reduces cerebral microthrombus formation
and brain injury after experimental SAH.
48 h in 4% paraformaldehyde. Coronal cuts were made
with a mouse brain matrix (Zivic Instruments, Pittsburgh,
PA, USA), embedded in paraffin, and cut into 5-lm sections with a microtome.
Materials and methods
Outcome measures
Mice
This study was approved by the Institutional Animal
Care and Use Committee. The ADAMTS-13/ mice
used in this study were on a C57BL/6J background [12].
The control wild-type mice on a C57BL/6J background
were purchased from Charles River (France). All animals
were male, weighed 22–30 g, and were housed in a facility
with controlled light/dark cycles.
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The following experimental groups were investigated: (i)
C57BL/6J mice (n = 10) with intracisternal injection of
60 lL of blood from a donor C57BL/6J mouse (n = 10);
(ii) C57BL/6J mice (n = 10) with intracisternal injection
of 60 lL of blood from a donor C57BL/6J mouse
(n = 10) and with subsequent intravenous injection of rADAMTS-13 (in the tail vein 20 min after creation of
SAH, at a dose of 3460 U kg1); (iii) ADAMTS-13/
mice (n = 10) with intracisternal injection of 60 lL of
blood from a donor ADAMTS-13/ mouse (n = 10); and
(iv) ADAMTS-13/ mice (n = 10) with intracisternal
injection of 60 lL of blood from a donor ADAMTS-13/
mouse (n = 10) and with subsequent intravenous injection
of rADAMTS-13 (in the tail vein 20 min after creation of
SAH, at a dose of 3460 U kg1). The different types of
experiment were performed in random order. The number
of mice (n = 10) was based on an assumed mortality rate
of 20% (leaving eight mice available for analysis), a minimum difference in extent of microthrombosis of 30%
between groups with and without treatment with rADAMTS-13, a standard deviation of 20% in both groups,
5% error, and 80% power. rADAMTS-13 was prepared
as described previously [14].
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Experimental groups
Outcome measures were survival rate, the amount of
microthrombosis as reflected by the degree of fibrin(ogen)
staining (rabbit anti-mouse fibrinogen IgG, 1 : 100 000;
Gentaur, Kampenhout, Belgium), and the amount of brain
injury as reflected by the degree of microglia activation
(rabbit-a-Iba-1, 1 : 1000 in phosphate-buffered saline
[PBS]; Wako Chemicals, Richmond, VA, USA). The secondary antibody was Bright Vision Poly-horseradish peroxidase–anti-rabbit IgG (1 : 1 in PBS; Immunologic BV,
Duiven, The Netherlands). From coronal 5-lm sections
that were taken 3 mm anterior to the cerebellum, we
selected two predefined areas of cerebral cortex and one
area of hippocampus per cerebral hemisphere (Leica
DM 5000B; Leica, Wetzlar, Germany [9 10 magnification]). To compensate for longitudinal sectioning of thrombosed arterioles, for each mouse we analyzed three coronal
sections that were 30 lm apart. Images were analyzed with
0
FIJI software (IMAGE-J-WIN32). In short, 3,3 -diaminobenzidine-positive cells were segmented by use of the color deconvolution plugin. The total immunopositive area was
measured in all 18 areas, expressed as the percentage of the
total area, and subsequently pooled to calculate a mean
immunopositive area per mouse.
SAH model
For SAH creation, we used the prechiasmatic blood injection model as described previously, with injection of
60 lL of blood [15–17]. Body temperature was maintained at 37 °C. Cerebral blood flow (CBF) was measured
between 7.5 min prior to SAH creation up to 15 min
after blood injection, with a laser Doppler flow meter
(BLF22; Transonics Systems, New York, NY, USA). The
success of SAH creation was confirmed by a sharp reduction in CBF during blood injection. Mice were killed at
48 h, as the extent of microthrombosis after SAH in mice
is most pronounced after 48 h [18]. After intracardiac
perfusion–fixation, brains were removed and fixed for
Statistical analysis
CBF after blood injection was expressed as a percentage
of baseline flow and presented as the median with interquartile range (IQR). Median CBF during blood injection
was compared between all four groups by use of a Kruskal–Wallis ANOVA, and, 15 min after injection, between
wild-type and ADAMTS-13/ mice by use of a Mann–
Whitney U-test. The results of fibrinogen and microglia
staining are presented as means with standard errors of
the mean. Differences between two groups were calculated
with an independent samples t-test. Probability values of
< 0.05 were considered to be of statistical significance.
Results and discussion
Median CBF during blood injection dropped to ≤ 15% of
baseline in all four groups (P = 0.64), which is a reflection
of an acute increase in intracranial pressure and an indication that blood was injected correctly. Median CBF at
15 min was lower in wild-type mice than in ADAMTS13/ mice (wild-type mice, 40.0 [IQR 31.5–45.2]; ADAMTS-13/ mice, 55.6 [IQR 42.5–80.2]; P = 0.003). The
survival rate was similar in wild-type mice (10/10 without
rADAMTS-13 vs. 10/10 with rADAMTS-13; P > 0.99)
© 2014 International Society on Thrombosis and Haemostasis
12/06/2014
Recombinant ADAMTS-13 in subarachnoid hemorrhage 945
and ADAMTS-13/ mice (9/10 without rADAMTS-13
vs. 8/10 with rADAMTS-13; P > 0.99). In wild-type mice,
infusion with rADAMTS-13 20 min after SAH induction
resulted in a trend towards less microthrombosis (mean
fibrinogen area: 0.28% 0.09% vs. 0.15% 0.04%;
P = 0.20), whereas infusion or rADAMTS-13 in ADAMTS-13/ mice resulted in a statistically significant reduction in the extent of microthrombosis (mean fibrinogen
area: 0.32% 0.05% vs. 0.16% 0.03%; P = 0.016)
(Fig. 1). In addition, rADAMTS-13 resulted in a statistically significant reduction in brain injury (> 60%) in wildtype mice (mean microglia area: 0.65% 0.18% vs.
0.18% 0.04%; P = 0.013) and a trend towards less brain
injury in ADAMTS-13/ mice (mean microglia area:
1.24% 0.36% vs. 0.42% 0.13%; P = 0.077) (Fig. 2).
Our results indicate that rADAMTS-13 provides a protective effect after experimental SAH. Several recent studies have shown that rADAMTS-13 is also a promising
treatment in other types of stroke [19,20]. In experimental
ischemic stroke, treatment with rADAMTS-13 in a wildtype mouse immediately before reperfusion reduced
infarct volume and improved functional outcome without
producing hemorrhagic transformation [19–22]. The proposed mechanism is that ADAMTS-13 protects the brain
from acute cerebral inflammation, decreases neuroinflammation in brain ischemia–reperfusion injury, reduces the
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P = 0.077
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Mean microglia area (%)
P = 0.013
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Mean fibrinogen area (%)
4
100 µm
100 µm
C57/BI6J treated
with rADAMTS13
ADAMTS13 –/– treated
with rADAMTS13
100 µm
100 µm
Fig. 1. Effect of recombinant ADAMTS-13 (rADAMTS-13) on microthrombosis after experimental subarachnoid hemorrhage. (A) Bar
graphs showing mean fibrinogen area ( standard error of the mean)
per group. (B) Representative fibrinogen staining in the cortex per
group.
C57/BI6J
ADAMTS13 –/–
100 µm
100 µm
C57/BI6J treated
with rADAMTS13
ADAMTS13 –/– treated
with rADAMTS13
100 µm
100 µm
Fig. 2. Effect of recombinant ADAMTS-13 (rADAMTS-13) on
brain injury after experimental subarachnoid hemorrhage. (A) Bar
graphs showing mean microglia area ( standard error of the mean)
per group. (B) Representative microglia staining in the hippocampus
per group.
© 2014 International Society on Thrombosis and Haemostasis
12/06/2014
946 M. D. I. Vergouwen et al
Rottensteiner and F. Scheiflinger (Baxter Innovations
GmbH, Vienna, Austria) for kindly providing rADAMTS-13, and D. Ginsburg (University of Michigan, Ann
Arbor, MI, USA) for providing the ADAMTS-13/
mice. M. D. I. Vergouwen is financially supported by a
personal grant from the Dutch Heart Foundation
(2011T18).
Disclosure of Conflict of Interests
The authors state that they have no conflict of interest.
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However, in our study, we did not find evidence that
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Our predefined probability level was not reached in
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This probably resulted from the fact that there was
more variation within the groups than assumed in our
initial sample size calculation. We found that some mice
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despite a sharp decrease in cerebral perfusion during
blood injection, which is an indicator that blood was
injected correctly. We decided not to exclude these mice
from our results, because in clinical practice there are
patients with aneurysmal SAH who do not develop
delayed cerebral ischemia and have excellent clinical outcomes, despite the presence of large amounts of subarachnoid blood.
In conclusion, our results support the further study of
rADAMTS-13 as a treatment option for the prevention
of microthrombosis and brain injury after SAH. Before
our results can be extrapolated to a clinical trial in
humans, it should first be determined whether rADAMTS-13 reduces delayed cerebral infarctions in an animal
model of SAH.
Addendum
M. D. I. Vergouwen and J. C. M. Meijers designed the
study. V. L. Knaup performed the experiments. M. D. I.
Vergouwen, V. L. Knaup, J. J. T. H. Roelofs, and O. J.
de Boer analyzed the results. M. D. I. Vergouwen wrote
the first draft of the manuscript. V. L. Knaup, J. J. T. H.
Roelofs, O. J. de Boer, and J. C. M. Meijers performed
critical revisions of the manuscript for important intellectual content.
Acknowledgements
This study was funded by a grant from the Netherlands
Thrombosis Foundation (2010-4). The authors thank H.
© 2014 International Society on Thrombosis and Haemostasis
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