Molecular Basis of Platelet Adhesion

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DESCRIPTION (provided by applicant): Platelet adhesion to vascular subendothelium is the initial step in both normal hemostasis and arterial thrombosis. The plasma protein von Willebrand Factor (VWF) plays an important role in platelet thrombus formation, because it binds strongly to the collagen in the subendothelium and to the platelet receptor GPIb. This bridge function is critical to stabilize the interaction between platelets and subendothelium under high shear and high flow conditions present in the arterial and microcirculation. The A1, A2, and A3 domains mediate key interactions for VWF. The A1 domain binds to GPIb, heparin, sulphatides and collagen Type VI. Conformational changes are thought to expose the VWF-A1 domain after the binding of other regions in VWF to collagen. The VWF-A2 domain contains a physiological cleavage site that is associated with the metalloprotease ADAMTS13 in plasma. The VWF-A3 domain contains the major collagen-binding site for fibrillar collagens. The importance of the A domains in VWF is underscored by the inherited bleeding disorder Type 2 von Willebrand disease (VWD), which corresponds to functional abnormalities in VWF. The mutated residues that cause the variants of Type 2A, 2B, and 2M VWD have been found in the VWF-A1 and VWF-A2 domains. The goal of this grant is to understand the molecular mechanism by which the VWF-collagen interaction induces the VWF-A1 to assume the active conformation that results in binding to platelet GPIb. In the first aim, we plan to describe the residues that make contact with collagen within the VWF-A3 domain. We will also test the hypothesis that the VWF-A1 and VWF-A3 domains have a direct interaction. In the second aim we will make recombinant VWF-A2, VWF-AIA2, and VWF-A2A3 proteins to analyze the role of the VWF-A2 domain in the VWF-binding to both GPIb and collagen. We will also study the molecular mechanism by which Type 2A mutations increase the sensitivity of the VWF-A2 domain for proteolysis. The third aim will characterize the binding of the recombinant VWF-AIA2A3 protein to GPIb and collagen and the interaction with plasma metalloprotease. We will study the effects of interesting mutants identified in the two previous aims together with some Type 2B mutations on the affinity and regulation of the VWF-A1A2A3. For the last two specific aims, we plan to analyze the role of glycosylation on the GPIb and collagen binding and in plasma metalloprotease activity. The new information obtained from this work will establish the structural basis by which VWF multimers is activated and regulated. It may also contribute to the development of an effective assay to detect the activity of the metalloprotease in plasma. It will provide molecular guidance for the generation of reagents to treat arterial thrombosis.