ADAMTS13 gene deletion aggravates ischemic brain damage: a possible neuroprotective role of ADAMTS13 by ameliorating postischemic hypoperfusion

von Willebrand factor (VWF) is a large multimeric protein that plays a key role in thrombus formation by tethering platelets at sites of vascular injury.¹  Smaller VWF multimers are less active, and the potent thrombogenic activity of ultra-large VWF (ULVWF) secreted from endothelium is regulated in vivo through cleavage by ADAMTS13.^2,3  The importance of this mechanism for normal hemostasis is supported by evidence that patients with deficiency of ADAMTS13 function, diagnosed with thrombotic thrombocytopenic purpura, have ULVWF in circulating blood and VWF-dependent microvascular thrombosis.²  Recently, we demonstrated that ADAMTS13 cleaves VWF on the surface of platelet thrombi in a shear force–dependent manner, which limits thrombus growth in vitro.⁴  These data suggest that ADAMTS13 is a key molecule that maintains a physiologic balance between hemostasis and thrombosis through regulation of VWF function in vivo.

ADAMTS13 function is crucial for preventing thrombosis in the cerebral microvasculature, as indicated by the occurrence of neurologic deficits in thrombotic thrombocytopenic purpura, but the role of ADMTS13 in the pathogenesis of reperfusion injury after arterial thrombosis has not been established. To address this issue, we investigated the role of ADAMTS13 in a transient middle cerebral arterial occlusion (MCAO) model of ischemia-reperfusion injury in the mouse brain⁵  using Adamts^−/− mice.⁶ 

Because brain ischemia-reperfusion injury is dependent on the platelet glycoprotein Ib–VWF axis⁷  and platelet thrombosis adversely affects the postischemic cerebral microcirculation^8-11  leading to secondary brain damage,¹⁰  ADAMTS13 may reduce platelet thrombus growth and thereby ameliorate ischemic brain injury by improving the postischemic no-reflow phenomenon.¹²  Here we demonstrate that Adamts13 gene deletion aggravates postischemic cerebral blood reflow, resulting in larger infarct volume. This result suggests that ADAMTS13 may indeed suppress excessive platelet thrombus growth in vivo.

The effect of Adamts13 gene deletion on brain ischemia was studied using male Adamts13^−/− (−/−) mice and wild-type (+/+) mice generated by our study group.⁶  We used male mice only to obtain consistent results because female mice are known to be more resistant to stroke. The experimenters were blinded to the genotype of each animal until all studies had been finished. This study was approved by the institutional ethics committee of Fukuoka University.

Focal cerebral ischemia (MCAO by intraluminal thread) was induced in Adamts13^−/− and wild-type mice as previously described.^5,13,14  Preliminary experiments using 1-hour MCAO gave excessively high mortality of 4 of 5 for one group and 1 of 4 for another. Therefore, we reduced the time of MCAO to 30 minutes. Body temperature was maintained at 36.5°C to 37.0°C during surgery. The success of the left MCAO was confirmed according to the following criteria: (1) regional cerebral blood flow (rCBF) in the left cerebral cortex at the thread insertion less than 20% of pre-MCAO rCBF; and (2) consistent presence of significant ischemic neurologic symptoms of the left cerebral hemisphere, including right forepaw paralysis and right circling behavior during 30-minute MCAO.

The rCBF was measured by LASER Doppler flowmetry (LDF; ALF21, Advance Co) as previously described.⁵  The LDF probe was placed in the left cerebral cortex stereotaxically. The rCBF was monitored in all animals during the period between 30 minutes before MCAO and immediately after reperfusion.

The brains were sectioned coronally (four 2-mm-thick slices) according to a mouse brain matrix 24 hours after MCAO or sham operation. The infarct area was measured using an image-analysis system (National Institutes of Health Image software, Version 1.63) in each slice stained with 2,3,5 triphenyltetrazolium chloride, and the infarct volume was calculated.^5,13  Paraffin-embedded brains were stained with phosphotungstic acid hematoxylin (PTAH) to demonstrate fibrin in thrombi or incubated with anti-VWF antibody (sc-8068; Santa Cruz Biotechnology), followed by a standard avidin-biotin-peroxidase complex technique to demonstrate VWF in thrombi.

Neurologic deficit was assessed at 24 hours after MCAO using a neurologic score as previously described,¹³  and the survival rates were also measured at 24 hours after MCAO.

Data are mean plus or minus SEM. For multiple pairwise comparisons, 2-way analysis of variance followed by Scheffé test was performed. When only 2 groups were compared, Student t test was used. Probability values less than .05 were considered to be of statistical significance.

The rCBF decreased to less than 20% of the baseline value during 30-minute MCAO and returned to baseline immediately after reperfusion in both Adamts13^−/− and wild-type mice. However, during the subsequent 30 minutes, the rCBF for both groups decreased, suggesting that ischemia-reperfusion had induced thrombosis. The rCBF for Adamts13^−/− mice progressively decreased compared with wild-type mice (significantly decreased at 20 and 30 minutes after reperfusion, P < .05, Scheffé test, Figure 1).

The survival rates of the Adamts13^−/− and wild-type mice did not differ (17 of 20 vs 16 of 20, respectively). However, Adamts13^−/− mice had larger brain infarctions compared with wild-type mice 24 hours after MCAO (P < .01; Figure 2A), which is reflected by a difference in neurologic score assessing left hemisphere function (Figure 2B). Histologic and immunohistochemical examinations revealed that more thrombi containing fibrin and VWF were observed in Adamts13^−/− mice (Figure 2C), which may contribute to the lowered rCBF and increased infarct volume in Adamts13^−/− mice. These results indicate that Adamts13 gene deletion aggravates ischemic brain damage.

Our results indicate that ADAMTS13 is crucial in vivo to protect the brain from ischemia-reperfusion injury by ameliorating postischemic hypoperfusion. The possible neuroprotective effect of ADAMTS13 may result from the cleavage of ULVWF secreted from endothelium activated by ischemia^3,15  and cleavage of VWF multimers on the surface of thrombi formed on the ischemic endothelial cells.⁴  Without adequate ADAMTS13, progressive platelet thrombus growth may narrow the microvascular lumen, increasing fluid shear stress locally. Without negative feedback regulation by proteolysis of VWF, ischemia-reperfusion injury may cause a vicious cycle in which the VWF-platelet thrombosis and fluid shear stress enhance each other and contribute to the progressive deterioration of cerebral blood reflow, as observed in the Adamts13^−/− mice. ADAMTS13 may, therefore, prevent microvascular occlusion under high shear stress and augment the cerebral blood flow after ischemia-reperfusion in vivo.

In addition to VWF-platelet thrombus formation,^10,11,16,17  microvascular plugging by activated leukocytes^18-20  can lead to no-reflow phenomena in brain ischemia.¹¹  Importantly, platelet-ULVWF strings support leukocyte tethering on the endothelium under high fluid-shear stress.^21,22  A recent study suggested that deficiency of ADAMTS13 can increase leukocyte adhesion on the vessels and extravasation.²³  Thus, ADAMTS13 deficiency may enhance the leukocyte activation in the ischemic cerebral vasculature after reperfusion and thereby aggravate ischemic brain damage. Indeed, Adamts13^−/− mice accumulated more inflammatory cells in the brain tissue than wild-type mice after MCAO (Figure 2C), suggesting that ADAMTS13 may reduce inflammation as well as thrombosis associated with ischemia-reperfusion injury.

In conclusion, ADAMTS13 deficiency causes progressive decline of postischemic rCBF, with a resultant exacerbation of ischemic brain injury, suggesting an important role of ADAMTS13 in neuroprotection. The regulation of VWF-platelet interactions by supplementation with ADAMTS13 may ameliorate ischemia-reperfusion injury of the brain. Because ADAMTS13 tends to dissolve excessive VWF-platelet thrombi with increasing efficiency as the flow path narrows, treatment of acute ischemic stroke with ADAMTS13 might have a relatively low risk of hemorrhagic complications.

After submission of our paper, the complementary paper by Zhao et al²⁴  appeared in Blood, which also demonstrated the important role of ADAMTS13 for brain reperfusion injury.

The authors thank Dr Kouko Tatsumi and Dr Takahiko Kasai at the Laboratory of the Department of Anatomy and Neuroscience, and Diagnostic Pathology, respectively, in Nara Medical University, for histologic assistance.

Contribution: M. Fujioka and K.H. performed most of the animal experiments and prepared the manuscript; A.K., K.I., S.H., T.N., and C.M. helped to perform the animal experiments; H.F. and M.S. worked on the experimental design and data analysis; K.M., M. Fujiwara, K.O., and K.N. provided direction throughout the work, made the overall experimental design, and edited the manuscript; and F.B., K.K., and T.M. produced the ADAMTS13 gene-deleted mice.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Kenji Nishio, Department of Emergency and Critical Care Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8522, Japan; e-mail: knishio@naramed-u.ac.jp.