The ultimate goal of the proposed studies is to define the molecular mechanisms of factor V function in normal hemostasis and in the pathology of thrombosis. The studies address major gaps in understanding the function of proteinase exosites in substrate recognition mechanisms of the procoagulant activation of human factor V, factor Va cofactor activity in prothrombin (Pro) activation, and the anticoagulant inactivation of factor Va by activated protein C (APC). The factor V activation pathway is hypothesized to involve thrombin exosites I and II in substrate recognition of factor V activation species, which control the pathway direction and the expression of specific binding sites for factor Xa and Pro. Factor Va-directed substrate recognition of Pro activation species is hypothesized to occur through exosites expressed on factor Xa within the factor Xa-Va catalytic complex, and between proexosite I on Pro and the factor Va heavy subunit. Binding is coupled to engagement of the factor Xa catalytic site in a conformational change required for proteolytic cleavage. The Pro activation pathway is controlled in part by selective expression of increased (pro) exosite I binding affinity for factor Va on the activation intermediates, modulated by fragments 2 and 1.2, and phospholipid membranes. The mechanism of factor Va inactivation by APC and the cofactor activity of protein S are hypothesized to result from exosite-mediated assembly on membranes of APC-factor Va-protein S and competing factor Xa-Va complexes. The mechanisms will be evaluated in quantitative fluorescence equilibrium binding studies, steady-state and rapid-reaction kinetic studies. Fluorescent Pro derivatives specifically labeled in the zymogen catalytic site have been developed as new probes of the mechanisms.
Specific aims are: (1) To delineate the kinetic mechanism of factor V activation, the expression of binding sites for factor Xa and Pro species, and the roles of thrombin exosites in factor V substrate recognition and pathway direction; (2) To determine the specificity of the binding site on factor Va for Pro activation species, and its role in the individual molecular events in substrate recognition by the factor Xa-Va complex; (3) To define the contribution of proexosite I-mediated Pro-factor Va interactions to the mechanism of regulation of the Pro activation pathway, including substrate channeling; (4) To elucidate assembly of membrane-bound factor Xa-Va and APC-factor Va-protein S complexes in regulation of factor Va inactivation. The results of the studies will contribute to the mechanistic information needed for design of new anticoagulants that act at the level of the exosite-directed regulation of procoagulant and anticoagulant systems. ? ?
Showing the most recent 10 out of 19 publications
