This ongoing project proposes to extend studies of the role(s) of PS allosteric regulation of blood coagulation factors Xa and Va. The working hypothesis is that PS exposure is critical in regulating prothrombin activation because it apparently serves to assemble the prothrombinase, activate factor Xa, place the appropriate peptide bond of prothrombin near the factor Xa active site, and then channel the activation intermediate back to factor Xa so a second peptide bond can be cleaved. In the next grant period, this hypothesis will be tested by more thoroughly assessing the role of platelet membrane in regulating blood clotting by: 1) defining the rate and pathway of prothrombin proteolysis by factor Xa upon binding a soluble form of PS; 2) employ soluble synthetic PS analogues to search for an effective inhibitor of factor Xa activation; 3) map the PS binding site(s) on factor Xa and prothrombin; and 4) explore the use of multi-dimensional NMR for determining the structure of PS binding domains. A prediction of this hypothesis is that PS binding helps direct prothrombin activation through meizothrombin, a role previously reserved for factor Va. Thus, the role of Va will be readdressed along the lines of its ability to maintain the intermediate in the neighborhood of the factor Xa active site thereby facilitating cleavage of a second peptide bond to liberate the thrombin catalytic domain from the rest of prothrombin. The role of Va will be investigated by: 1) defining the mechanism of factor Va membrane binding; 2) comparison of the kinetics of the whole prothrombinase complex with previous studies of factor Xa alone; and 3) development of a structural model for the juxtaposition of prothrombin and meizothrombin to factors Xa, Va, and the PS membrane during prothrombin activation.
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