The long term goal of the proposed studies is to define the molecular mechanisms of the procoagulant regulation of human prothrombin activation by factor Va, and the mechanism of the anticoagulant inactivation of factor Va by the protein C system. The studies address significant gaps in the understanding of three mechanisms: factor V activation, factor Va cofactor activity, and factor Va inactivation. In factor V activation, interactions of factor V and its activation products with exosites I and II of thrombin are hypothesized to mediate recognition of factor V as a substrate and to direct the activation pathway. In the hypothesis for the mechanism of factor Va cofactor activity, the rate and pathway of prothrombin activation are regulated by binding of prothrombin, the activation intermediates, and thrombin to the same site on factor Va in the factor Xa-factor Va complex, in interactions mediated primarily by exosite I present in a low affinity, precursor state on prothrombin (proexosite I). The interactions are modulated by differential expression of the high affinity state of exosite I on the prothrombin activation intermediates, and by interactions of fragment 2 and membranes. Regulation of factor Va inactivation by activated protein C (aPC) and the anticoagulant activity of protein S are hypothesized to result from the interdependence of the assembly of membrane-bound aPC-factor Va- protein S complexes and factor Xa-factor Va complexes. The hypotheses will be evaluated by a combination of equilibrium binding studies of the interactions employing fluorescence techniques, kinetic studies of the proteolytic reactions, and protein structural studies.
Specific aims are: (1) To define the roles of regulatory exosites I and II of thrombin in the mechanism of factor V activation; (2) To determine the mechanism of expression of exosites I and II in the conversion of prothrombin to thrombin and the expression of linkage between the exosites; (3) To define the mechanism of involvement of (pro)exosite I, fragment 2 and membranes in factor Va interactions with prothrombin and its activation products, and the roles of these interactions in factor Va cofactor activity; and (4) To elucidate the mechanism of regulation of factor Va inactivation by aPC involving assembly of membrane-bound factor Xa-factor Va complexes and aPC-factor Va-protein S interactions. The results of the studies are expected to have significance in advancing the understanding of normal hemostatic mechanisms and the molecular pathology of thrombotic vascular disease. Information derived from these studies may provide new molecular targets for anticoagulant therapy.
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