The specific proteolytic activation steps of blood coagulation are catalysed by serine proteinases that are homologous to each other and to the archetypic serine proteinases of digestion. In many cases, explanations for the molecular basis of the distinctive macromolecular substrate specificity of the coagulation reactions remain speculative and incomplete. Evidence suggests that prothrombin activation by the prothrombinase complex, results from interactions at an extended macromolecular recognition site (Exosites) on the enzyme followed by active site interactions prior to cleavage and product release. We will use prothrombin activation catalysed by the prothrombinase complex as a paradigm for specific macromolecular substrate recognition and cleavage by coagulation complexes to investigate the basis for its substrate specificity and function. Using prethrombin 2 as a substrate analog, binding and stopped flow kinetic measurements will be used to provide a complete kinetic and thermodynamic description of the stepwise interactions that lead to substrate recognition and cleavage. We will test the hypothesis that exosite, rather than active site interactions, determine substrate affinity and binding specificity for the macromolecular substrate. The significance of these findings towards prothrombinase function will be tested in studies of prothrombin and meizothrombin cleavage. By comparing the function of factor Xa with prothrombinase, we will determine if modulation of binding interactions can explain the increased Vmax for the reaction which accompanies prothrombinase assembly. Recombinant activation site mutants of the substrate will be used to determine if binding specificity determined by exosite interactions directs cleavage at the scissile bond. By the use of recombinant mutants of factor Xa, we will test the hypothesis that sites removed from the catalytic site of the protease are responsible for determining substrate binding specificity. We will also extend approaches developed in studies with prothrombinase to other coagulation reactions to determine if exosite-dependent substrate recognition is a prevalent mechanism through which coagulation proteinases achieve their specificity. We believe that the approaches contained in this proposal will provide unanticipated insights into the function of the coagulation enzymes. The delineation of the determinants of macromolecular substrate specificity will likely suggest novel approaches for therapeutic targeting of these reactions in thrombotic and vascular disease states.
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