Mechanism underlying cofactor-dependent proteolysis of von Willebrand Factor

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PROJECT SUMMARY von Willebrand factor (VWF), a large multimeric plasma protein, plays a critical role in hemostasis. VWF is synthesized and secreted as ultra-large (UL) multimers that contain 25-50 protomers. If not processed by a plasma metalloprotease ADAMTS13, ULVWF can initiate the formation of life- threatening thrombosis as in thrombotic thrombocytopenic purpura (TTP). How the proteolytic cleavage of ULVWF by ADAMTS13 is regulated under physiological conditions is not fully understood. The cleavage site is buried under the central β-sheet within the A2 domain of VWF, and tensile force is required to expose the cleavage site for enzymatic cleavage to occur. Our preliminary studies have demonstrated that coagulation factor VIII (FVIII) may function as a cofactor that facilitates the cleavage of VWF by ADAMTS13 under mechanic shear. Taking advantage of our unique combination of molecular, biochemical and single-molecule biophysical tools available in both laboratories, we will test the hypothesis that the binding of FVIII to VWF-D’D3 and other adjacent domains such as the A2 domain may result in conformational changes in the central A2, thus exposing the cleavage site (Y1605-M1606) more readily to ADAMTS13 under mechanical force. In Aim 1, we will determine the mechanical unfolding profile of A2 with or without other adjacent domains in the absence and presence of FVIII; in Aim 2, we will elucidate the molecular mechanism of A2 and FVIII interactions by investigating their variants and mutants; and in Aim 3, we will determine the physiological relevance of the FVIII-dependent proteolytic cleavage of VWF under force and in animal models and human with heareditary TTP. The completion of the proposed project will help understand the molecular interactions among substrate, enzyme, and protein cofactor under physiological conditions, which provides rationales for the development of novel therapeutics for the prevention and treatment of TTP and other thrombotic and inflammatory disorders.