Evidence for Control of von Willebrand Factor Multimer Size by Intramolecular Thiol-Disulfide Exchange.

Plasma von Willebrand factor (VWF) is a multimeric glycoprotein from endothelial cells and platelets that mediates adhesion of platelets to sites of vascular injury. In the shear force of flowing blood, however, only the very large VWF multimers are effective in capturing platelets. The multimeric size of VWF is controlled by proteolysis at the Tyr⁸⁴²-Met⁸⁴³ peptide bond by ADAMTS13. It is apparent, however, that VWF multimers are processed to a significant degree in the absence of ADAMTS13 in mice and humans. This finding implies that another mechanism of controlling VWF multimer size must exist. We noticed similarities between the structural control of α-keratin and VWF. Both are multimeric elongated proteins whose substrates are linked by disulfide-bonds and both proteins undergo shear-dependent conformational change. Keratin shape is controlled by thiol-mediated events and we have shown that the free thiol of thrombospondin-1 can facilitate cleavage of the disulfide-bonds that hold VWF multimers together. The structure of α-keratin is controlled by shear-dependent intramolecular thiol-disulfide exchange. Free thiols in keratin from unpaired cysteines exchange with nearby disulfides when the α-keratin fibre is exposed to shear. The thiol-disulfide exchange process continues until the shear across the fibre is resolved. We hypothesized that a similar mechanism may be operating in VWF. Intramolecular thiol-disulfide exchange is dependent on the presence of at least one unpaired cysteine thiol in the protein. Using a biotin-linked maleimde that specifically alkylates free thiols, we have shown that plasma VWF does contain unpaired cysteine residues. VWF was coated onto the interior of glass capillary tubes and exposed to shear using a precision syringe pump. VWF eluted from the tube when shear was stepped, but not when the shear was constant, and the eluted VWF was of smaller multimer size than that bound to the tube. These observations imply that VWF multimer size can be controlled by intramolecular thiol-disulfide exchange. We suggest that this mechanism may be responsible for the processing of VWF multimer size observed in the absence of ADAMTS13.