Funding is requested studies on the role of von Willebrand factor (vWF) in hemostasis and pathological arterial thrombosis.
Four specific aims are envisioned to develop the overall goal of defining the mechanisms through which vWF participates in the initiation and progression of thrombus formation. 1) The solved three-dimensional structures of the vWF A1 domain will be used to build a model of the contact interface with the platelet receptor, glycoprotein (GP) Ibalpha. The putative participation of individual residues in receptor binding will be tested in a flow system evaluating the consequences of selected mutations on the ability of recombinant A1 domain to tether platelets to a surface. These studies will provide a functional map of vWF with respect to platelet binding. 2) Isolated A1 domain with mutations or conformational alterations resulting in distinct functional attributes will be expressed and characterized. Larger fragments containing vWF domains A1, A2, and A3 will also be expressed for functional characterization. Crystallization trials will be performed as the first step towards solution of the three-dimensional structure of relevant molecules. These studies will provide information necessary to understand how interactions between the matrix, vWF, and platelets are regulated. 3) Hemodynamic forces may influence the immobilization of soluble vWF onto reactive surfaces, thus affecting recruitment and tethering of platelets. Varying flow conditions will be used to test vWF binding to collagen fibrils and subendothelial matrix, and to evaluate how modulation of this process relates to subsequent functions. The interactions of vWF with substrates and of platelets with substrate-bound vWF will be analyzed independently, to determine the effects of flow on each of these initial steps of thrombus formation. 4) Experimental evidence indicates that domains A1 of matrix vWF and A3 of plasma vWF are required for efficient platelet arrest and thrombus formation onto a subendothelial surface, suggesting the hypothesis that both domains influence vWF binding to matrix components and membrane receptors. Monoclonal antibodies and site directed mutagenesis will be used to evaluate the role of domains A1 and A3 in vWF interaction with collagen and subendothelial matrix, as well as on subsequent platelet adhesion and aggregation. The proposed research will have an impact on public health by providing mechanistic information relevant to the identification of novel targets for anti-thrombotic intervention. The results obtained should ultimately benefit patients at risk of cardiovascular and cerebrovascular events.
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