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

Abstract

ADAMTS13 is a multidomain protease that limits platelet thrombogenesis through the cleavage of von Willebrand factor (VWF). We previously identified 2 types of mouse Adamts13 gene: the 129/Sv-strain Adamts13 gene encodes the long-form ADAMTS13 having the same domains as human ADAMTS13, whereas the C57BL/6-strain Adamts13 gene encodes the short-form ADAMTS13 lacking the distal C-terminal domains. To assess the physiologic significance of the distal C-terminal domains of ADAMTS13, we generated and analyzed 129/Sv-genetic background congenic mice (Adamts13 S/S ) that carry the short-form ADAMTS13. Similar to wild-type 129/Sv mice (Adamts13 L/L ), Adamts13 S/S did not have ultralarge VWF multimers in plasma, in contrast to 129/Svgenetic background ADAMTS13-deficient mice (Adamts13 ؊/؊ ). However, in vitro thrombogenesis under flow at a shear rate of 5000 s ؊1 was accelerated in Adamts13 S/S compared with Adamts13 L/L . Both in vivo thrombus formation in ferric chlorideinjured arterioles and thrombocytopenia induced by collagen plus epinephrine challenge were more dramatic in Adamts13 S/S than in Adamts13 L/L but less than in IntroductionADAMTS13 is a plasma protease that specifically cleaves von Willebrand factor (VWF). 1 VWF is a multimeric plasma glycoprotein that plays a critical role in platelet adhesion and aggregation on vascular lesions. 2 Endothelial cells and megakaryocytes produce mainly VWF as large multimers that can exceed 20 000 kDa in mass and secrete the multimers into the circulating blood. The adhesive activity of VWF multimers depends on their molecular sizes and in particular the largest multimers, called ultralarge VWF (UL-VWF) multimers, can induce excessive platelet aggregation under shear stress. UL-VWF multimers are normally cleaved by ADAMTS13 to smaller forms, thus restraining platelet thrombus formation. The lack of ADAMTS13 activity allows UL-VWF multimers to persist in the circulation and leads to the development of thrombotic thrombocytopenic purpura (TTP). [3][4][5] ADAMTS13 consists of multiple domains including a metalloprotease domain, a disintegrin-like domain, a thrombospondin type 1 motif (Tsp1) domain, a cysteine-rich domain, a spacer domain, 7 additional Tsp1 domains and 2 complement components C1r/ C1s, urchin epidermal growth factor, and bone morphogenic protein-1 (CUB) domains in order from the N-terminus. So far, the functional roles of ADAMTS13 domains have been studied using in vitro assay systems. [6][7][8][9][10][11][12][13] These studies have shown an essential role of the N-terminal region of ADAMTS13 from the metalloprotease domain to the spacer domain, on the VWF cleavage. However, the results from in vitro studies have lacked consistency on the relative importance of the C-terminal Tsp1 and CUB domains in the substrate recognition and the activity of ADAMTS13. The recombinant human ADAMTS13 mutant lacking the C-terminal Tsp1 and CUB domains maintain almost absolute VWF-cleaving activity under static conditions, indicating that the C-terminal domains are dispensab...

Residues Arg568 and Phe592 contribute to an antigenic surface for anti-ADAMTS13 antibodies in the spacer domain

“…Several studies have shown that the carboxy-terminal TSP2-8 and CUB1-2 domains are important for the processing of VWF by ADAMTS13. 22,30,31 Zheng and coworkers showed that platelet counts on admission were lower in patients with anti-TSP2-8 and/or anti-CUB1-2 IgG. 19 This observation suggests that antibodies directed towards the carboxy-terminal domains inhibit ADAMTS13 activity or alternatively enhance its clearance from the circulation.…”

Amino acid residues Arg659, Arg660, and Tyr661 in the spacer domain of ADAMTS13 are critical for cleavage of von Willebrand factor

“…For example, Banno et al 42 have reported that a naturally occurring mutation in certain strains of mice that truncates murine ADATMS13 after the sixth TSP1 repeat results in significantly reduced efficacy for systemic antithrombosis. 42 A deletion mutation in the TSP1 sixth repeat of ADAMTS13 was identified in a family with hereditary TTP.…”

Pathologic mechanisms of type 1 VWD mutations R1205H and Y1584C through in vitro and in vivo mouse models

“…35 Although similar activity is observed in static assays, this truncated ADAMTS13 demonstrates increased thrombotic occlusions under shear stress conditions, because it is not fully active in vivo. 36 To mitigate any artifacts caused by these differences, we digested both human and mouse substrates with their respective species-specific ADAMTS13. In addition, we performed coinjections of full-length mouse Adamts13 cDNA with the Vwf cDNAs to observe any influences on the VWF multimer profile or clearance rate in vivo.…”