The cooperative activity between the carboxyl-terminal TSP1 repeats and the CUB domains of ADAMTS13 is crucial for recognition of von Willebrand factor under flow

Abstract

ADAMTS13 cleaves von Willebrand factor (VWF) between Tyr 1 Deficiency of plasma ADAMTS13 activity, due to either inherited mutations of the ADAMTS13 gene 2-9 or acquired autoantibodies against the ADAMTS13 protein, 10,11 results in thrombotic thrombocytopenic purpura (TTP).ADAMTS13 is primarily synthesized in hepatic stellate cells, 12-14 endothelial cells, 15,16 and megakaryocytes or platelets. 17,18 The plasma ADAMTS13 in healthy individuals ranges from 0.5 to 1 mg/L. 19,20 ADAMTS13 consists of metalloprotease, disintegrin, first thrombospondin type 1 (TSP1) repeat, and Cys-rich and spacer domains. 2,21 The C-terminus of ADAMTS13 has additional TSP1 repeats and 2 CUB (Cis/Cis/Urinary epidermal growth factor, Bone morphogenetic protein) domains. 2,21 Previous studies have shown that the N-terminus of ADAMTS13 is required and sufficient for recognition and cleavage of denatured multimeric VWF [22][23][24] or peptide substrate . 22 More recent studies have demonstrated that the spacer domain of ADAMTS13 binds the exosite (E 1660 APDLVLQR 1668 ) near the C-terminus of the VWF-A2 domain. 25,26 However, the role of the middle and distal C-terminal domains of ADAMTS13 in substrate recognition remains controversial. On the one hand, ADAMTS13 mutant lacking the CUB domains or truncated after the spacer domain cleaved multimeric VWF with similar efficiency as the full-length ADAMTS13 under static and denatured conditions 23,24 ; the mutant truncated after the spacer domain, when mixed with ADAMTS13-depleted plasma, was found to be "hyperactive" in cleaving a "stringlike" structure, which represents platelets attached to the newly released VWF on the endothelial cell surface in a parallelflow chamber-based assay. 27 These data suggest that the distal portion of ADAMTS13 molecule may be dispensable under static and denatured conditions but may play a role in modulating ADAMTS13-VWF interaction under flow. On the other hand, synthetic peptides or recombinant fragments derived from the CUB domains 28 appeared to block the cleavage of the stringlike structure on endothelial cells, suggesting that the CUB domains may directly participate in binding or recognition of VWF under flow. Although the parallel-flow chamber assay may mimic physiological condition, its complexity involving live endothelial cells, histamine stimulation, and platelets makes the quantitation less accurate and kinetic determination of ADAMTS13 and VWF interaction impossible.In the present study, we have developed a simple flow assay based on mechanical-induced shear stress on a mini vortexer or a laminar flow in a BIAcore (Uppsala, Sweden) system to determine the role of the C-terminal ADAMTS13 in recognition and cleavage of multimeric VWF. Our data demonstrate directly and quantitatively that the cooperative activity between the middle C-terminal TSP1 repeats and the distal C-terminal CUB domains of ADAMTS13 may be crucial for productive binding and cleavage of VWF under flow. Materials and methodsThe human study has been approved by the Institutional Re...

Von Willebrand factor-binding protein is a hysteretic conformational activator of prothrombin

“…Localized depletion of Fbg in an endocarditis vegetation may result in shutting off ProT⅐VWbp by the more rapid reversal of the conformational change. Additional results with VWbp show no significant effect of the plasma concentration of VWF (150 nM) on the hysteretic mechanism of ProT activation, for either untreated VWF or the extended conformation induced by vortexing VWF under conditions that enhance VWF cleavage by ADAMTS13 (28).…”

Factor VIII accelerates proteolytic cleavage of von Willebrand factor by ADAMTS13

“…The requirement for denaturants such as urea and guanidine and the use of buffers at nonphysiological pH and ionic strength could all obscure contributions of other components to proteinase function. The development of a simple shear stress-based assay (18) has provided an opportunity to investigate cofactor-dependent regulation of ADAMTS13 using the native macromolecular substrate and buffer conditions that are more consistent with the physiologic milieu. We show that FVIII accelerates the action of ADAMTS13 on VWF at concentrations that are consistent with the expected marginal saturation of VWF monomers with FVIII in blood.…”

“…pVWF was purified from cryoprecipitate as described (27). Recombinant ADAMTS13 containing a V5-His tag at the C terminus was expressed in HEK293 cells and purified according to published procedures (18). Thrombin was prepared from prothrombin and purified as described (28).…”

“…Purified pVWF or rVWF (37.5 g/ml or 150 nM) was incubated at 25°C for 3 min or the indicated times with 50 nM recombinant ADAMTS13 in the absence or presence of FVIII, FVIII-SQ, FVIII-2RKR, or FVIIIa (0 -40 nM) in 20 mM Hepes, 0.15 M NaCl, 5 mM CaCl2, and 0.5 mg/ml BSA (pH 7.5) under constant vortexing at 2,500 rpm. Experiments were performed in 0.2-ml thin-walled PCR tubes (Fisher Scientific, Newark, DE) with a final reaction volume of 20 l as described previously (18). The reaction was quenched at various times by adding an equal volume of 125 mM Tris, 10% (vol/vol) glycerol, 2% (wt/vol) SDS, and 0.01% (wt/vol) bromophenol blue (pH 6.8), followed by heating at 100°C for 5 min.…”

“…Recombinant ADAMTS13 was coupled to a carboxymethylated dextran plasmon resonance chip (Ϸ2,000 response units; 2-10 ng/mm 2 ) by using methods described previously (18). Casein was immobilized in a similar way in the control channel, and both surfaces were blocked by using 1 M ethanolamine (pH 8.5).…”