SUBENDOTHELIAL BINDING OF VON WILLEBRAND FACTOR

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Injury to vascular endothelium results in the rapid adhesion of platelets to the exposed underlying subendothelium (SE), an early step in the initiation of hemostasis and of atherogenesis. This platelet adhesion requires von Willebrand factor (vWf), which we previously demonstrated to be present within SE. We identified three vWf binding proteins in SE extracts, with apparent molecular weights of 150,000, 200,000 and 80,000, and succeeded in isolating the 150,000 protein. We found that its characteristics match those of a newly identified form of collagen, type VI collagen, the building block of microfibrils. We have initiated studies with purified type VI collagen and have evidence that vWf binds to type VI collagen, and also, preliminarily that: 1) the binding domain on vWf is different from those reported for fibrillar collagens, and, 2) binding is mediated by a globular domain on the type VI collagen molecule. We will now pursue two major avenues: 1) Having identified the 150kDa protein, we plan to use similar methods, including SDS-PAGE, preparative electro-elution, radio-ligand and dot-blotting, iso- electric focusing amino acid analysis, Western blotting to isolate and characterize the 200 and 80kDa proteins, and 2) we will investigate the molecular basis of the binding between vWf and VI collagen with an eye toward contributing information which will be useful in developing ways of modulating this binding. The mechanism of binding will be dissected with monoclonal antibodies to specific domains on vWf, and enzymatic and CNBr fragments of vWf, along with candidate peptides and other adhesive glycoproteins. The binding requirements on type VI collagen will be similarly studied. Fluorescence immuno-localization studies will be used to determine whether vWf, type VI collagen and other relevant extracellular matrix proteins and adhesive glycoproteins co-localize in blood vessels and in tissue culture. We will begin to examine the functional significance of vWf binding to type VI collagen in static and flow systems, using de-endotheliazed blood vessels. Elucidation of the binding of vWf to SE would be an important advance in understanding the mechanism of platelet adhesion and the pathogenesis of atherosclerosis. This knowledge may contribute to new means of regulating the attachment of platelets to injured blood vessels.