The applicant proposes to study the regulation of prothrombin activation utilizing a combination of rapid reaction kinetics, employing quenched-flow and stopped-flow fluorescence techniques to make thermodynamic and kinetic measurements of the interaction of factor Xa and factor Va in solution and on membranes and to investigate the action of activated protein C (APC) on prothrombinase. Specifically, membrane-dependent events leading to the assembly of factor Xa and factor Va on phospholipids and in solution will be studied using stopped-flow measurements to examine the effect of membrane composition and viscosity on reaction kinetics. Attempts will be made to distinguish between concerted and consecutive cleavages of the two bonds in prothrombin during activation by prothrombinase. The combined reaction catalyzed by the extrinsic factor-X activating complex and by prothrombinase will be probed by assembling these complexes on separate phospholipid vesicles or on a contiguous membrane surface to assess the phenomenon of """"""""channeling"""""""" of reactants between successive enzyme complexes. The kinetic mechanisms of prothrombinase assembly will be studied using stopped-flow fluorescence measurements of prothrombinase assembly and varying membrane composition and viscosity to kinetically isolate the various reactions leading to enzyme complex formation and to test the hypothesis that the enhanced affinity of the factor Xa/factor Va interaction on membranes is a result of the kinetic consequences of oriented binding and approximation of these proteins on the membrane surface. Next the applicant proposes to determine the consequences of the action of activated protein C on the fate of the prothrombinase complex. Experiments will be carried out to determine the contributions of displacement of factor Xa from factor Va by competition with APC versus dissociation of factor Xa from the prothrombinase complex resulting from cleavage of factor Va. Finally, structure-function relationships in factor Va will be studied and evidence sought to delineate the contributions of various individual chains of factor Va on the factor Xa/factor Va interaction to explore the potential contributions of activation peptide excision and conformational alterations accompanying zymogen-protease transition.
