Von Willebrand factor (vWF) is a large glycoprotein secreted by endothelial cells and platelets into the plasma and vascular connective tissue. In addition to binding to and stabilizing Factor VIII, vWF provides a bridge through which platelets adhere to the subendothelial connective tissue in the event that the endothelium is wounded. This interaction permits platelet aggregation and is one of the earliest events in hemostasis. The normal interaction between platelets and vWF depends in part on the size and structure of vWF, which is a multimeric protein composed of 2 to >20 disulfide-linked protomers. The protomers comprise two identical polypeptide chains of about 200 kDa each. In normal individuals, the majority of circulating vWF is in the form of intermediate to very large multimers, which have been shown to be more effective in mediating platelet adhesion and aggregation than the smaller forms of vWF. A number of mutations of the vWF gene cause a selective loss of the high molecular weight forms of circulating vWF, leading to a bleeding disorder termed Type IIA von Willebrand's disease. There is evidence that these variants of vWF are more susceptible to proteolysis, most likely by a calpain-like enzyme, but the protease has not been characterized. In order to identify the responsible protease, we have modified the standard method of analyzing vWF monomers to increase speed and sensitivity. With this technique, we will use purified, high molecular weight multimers of vWF to assay for protease activity in normal and Type IIA vWD plasma. The protease will then be purified and analyzed structurally (partial sequencing, peptide mapping and immunochemical) and kinetically. Kinetic analyses may be extended to synthetic peptides containing the cleavage site and/or recombinant domains of normal and variant vWF. The effect of shear stress on the cleavage of normal and variant vWF by the protease will be of particular interest.
