Functional imaging of shear-dependent activity of ADAMTS13 in regulating mural thrombus growth under whole blood flow conditions

Abstract

The metalloprotease ADAMTS13 is assumed to regulate the functional levels of von Willebrand factor (VWF) appropriate for normal hemostasis in vivo by reducing VWF multimer size, which directly represents the thrombogenic activity of this factor. Using an in vitro perfusion chamber system, we studied the mechanisms of ADAMTS13 action during platelet thrombus formation on a collagen surface under whole blood flow conditions. Inhibition studies with a functionblocking anti-ADAMTS13 antibody, combined with immunostaining of thrombi with an anti-VWF monoclonal antibody that specifically reflects the VWFcleaving activity of ADAMTS13, provided visual evidence for a shear ratedependent action of ADAMTS13 that limits thrombus growth directly at the site of the ongoing thrombus generation process. Our results identify an exquisitely specific regulatory mechanism that prevents arterial occlusion under high shear rate conditions during mural thrombogenesis. IntroductionThe adhesive protein von Willebrand factor (VWF) plays a major role in platelet thrombogenesis, a process crucial for hemostasis. However, the excessive function of VWF is thought to increase the risk of fatal arterial thrombosis. 1,2 The thrombogenic activity of VWF is strictly dependent upon its multimeric structure, which is thought to be regulated in vivo by the metalloprotease ADAMTS13 through its cleavage of the A2 domain of the VWF subunit. 3,4 Indeed, patients with congenital deficiency of ADAMTS13 suffer repeated thrombotic complications attributed to excessive function of the ultra-large VWF (ULVWF) multimer, which is not found in normal blood circulation. [3][4][5][6] This concept was recently confirmed by knock-out mouse studies, in which ADAMTS13 Ϫ/Ϫ mice exhibited enhanced thrombogenicity in the ex vivo or in vitro experimental blood flow conditions tested. 7,8 The mechanisms by which ADAMTS13 regulates VWF remain poorly understood. However, recent studies showing that ADAMTS13 under flow conditions can rapidly cleave ULVWF secreted from and anchored to cultured endothelial cell layers 9,10 have raised the possibility that blood flow is critical in activating ADAMTS13. 11 Indeed, the VWF-cleaving activity of ADAMTS13 cannot be reproduced in vitro under static conditions unless the substrate VWF molecule is somewhat modified (eg, denatured by guanidine-HCl or urea). 3,4 Further, the question arises of whether ADAMTS13, in addition to its known action on ULVWF freshly released from endothelial cells, might also act directly at the local sites of thrombus generation to regulate thrombus growth.To address these issues, we analyzed the role and mechanisms of ADAMTS13 action in mural platelet thrombogenesis on a collagen-coated glass surface in an in vitro perfusion chamber system. Our visual evidence demonstrates that ADAMTS13 cleaves VWF and down-regulates mural thrombus growth at the site of ongoing thrombus generation in a shear rate-dependent manner under whole blood flow conditions. Methods Blood collectionThe present work was approved by the i...

The distal carboxyl-terminal domains of ADAMTS13 are required for regulation of in vivo thrombus formation

“…It has also been shown that ADAMTS13 can cleave platelet-bound VWF multimers 37 and limit thrombus formation through the cleavage of VWF at the surface of forming thrombi 28 in in vitro flow chamber systems. Therefore, in our experimental settings, ADAMTS13 attenuates thrombus growth, possibly through the cleavage of VWF multimers bound on the surface of platelet-rich thrombi under high shear rate.…”

“…27 In addition, ADAMTS13 cleaves VWF and down-regulates thrombus formation in shear rate-dependent manner. 28 Based on these observations, we further examined thrombus formation at a higher shear rate of 5000 s Ϫ1 . As expected, thrombus formation in Adamts13 Ϫ/Ϫ mice was significantly elevated compared with Adamts13 L/L mice at 5000 s Ϫ1 ( Figure 3B).…”

Acquired von Willebrand Syndrome in Patients With an Axial Flow Left Ventricular Assist Device

“…5 Specifically, structural changes can be induced by high shear stress in the von Willebrand molecule exposing the bond between amino acids 842 and 843 6 to the specific von Willebrand protease (ADAMTS13). 7 This results in acquired type 2 vWS, which is characterized by the loss of the largest von Willebrand multimers, which are most effective in platelet-mediated hemostasis. 8,9 The incidence of bleeding events in patients with aortic stenosis within 6 months before valve replacement was 21%.…”

Pathology and Pathophysiology of Atherothrombosis: Virchow's Triad Revisited

“…The study also showed cleavage of large sized VWF multimer during platelet thrombus formation under a high shear rate. The VWFcleaving site by ADAMTS-13 localized on the surface of platelet thrombus, and the ADAMTS-13 activity was shear dependent manner (Shida et al 2008). Thus, ADAMTS-13 may work at the site of ongoing thrombus generation and limit thrombus growth.…”