The mechanical force-induced activation of the adhesive protein von Willebrand Factor (VWF), which experiences high hydrodynamic forces, is essential in initiating platelet adhesion. The importance of the mechanical force-induced functional change is manifested in the multimeric VWF’s crucial role in blood coagulation, when high fluid shear stress activates plasma VWF (pVWF) multimers to bind platelets. Here we showed that a pathological level of high shear stress exposure of pVWF multimers results in domain conformational changes, and the subsequent shifts in the unfolding force allow us to use force as a marker to track the dynamic states of multimeric VWF. We found that shear-activated pVWF multimers (spVWF) are more resistant to mechanical unfolding than non-sheared pVWF multimers, as indicated in the higher peak unfolding force. These results provide insight into the mechanism of shear-induced activation of pVWF multimers.
Summary Background and Objective Upon stimulation, endothelial cells release von Willebrand factor (VWF) enriched in ultra-large (UL) forms that are rapidly cleaved by ADAMTS-13. The zinc metalloprotease fits in the consensus for members of the ADAMTS family, but also contains two unique C-terminal CUB domains. There are 5 and 2 cysteine residues in the CUB-1 and CUB-2 domains, respectively, instead of four as deducted from the consensus. In this study, we investigated the role of cysteine residues in the CUB-1 domain in ADAMTS-13 synthesis and activity. Methods and Results CUB-1 and cysteine mutations were expressed in mammalian cell lines and examined for synthesis, secretion, stability, and VWF-cleaving activity. When expressed as isolated domain, CUB-1, but not CUB-2, covalently aggregated. Converting any of the four cysteines that fit in the CUB consensus (C1192, C1213, C1236 and C1254) reduced the secretion of the mutants to the conditioned medium, but not to extracellular matrix. The mutations also resulted in a moderate increase in proteolytic degradation and decrease in cleaving plasma VWF under static, but not flowing conditions. In contrast, replacing C1275, which was found to be in the thiol form, with a serine residue prevented covalent aggregation of CUB-1, but had no effect on secretion and VWF-cleaving activity. C1275S is also markedly resistant to proteolytic degradation. Conclusion The data illustrate the importance of consensus cysteines in the secretion and proteolytic activity of ADAMTS-13. They also identify an ADAMTS-13 mutant that is resistant to proteolytic degradation, while maintaining a normal VWF-cleaving activity.
Von Willebrand factor (VWF) multimers are large adhesive proteins that are essential to the initiation of hemostatic plugs at sites of vascular injury. The binding of VWF multimers to platelets, as well as VWF proteolysis, is regulated by shear stresses that alter VWF multimeric conformation. We used single molecule manipulation with atomic force microscopy (AFM) to investigate the effect of high fluid shear stress on soluble dimeric and multimeric forms of VWF. VWF dimers are the smallest unit that polymerizes to construct large VWF multimers. The resistance to mechanical unfolding with or without exposure to shear stress was used to evaluate VWF conformational forms. Our data indicate that, unlike recombinant VWF multimers (RVWF), recombinant dimeric VWF (RDVWF) unfolding force is not altered by high shear stress (100 dynes/cm 2 for 3 min at 37 • C). We conclude that under the shear conditions used (100 dynes/cm 2 for 3 min at 37 • C), VWF dimers do not self-associate into a conformation analogous to that attained by sheared large VWF multimers.
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