Macromolecular Substrate Recognition in Blood Coagulation

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The specific proteolytic activation steps of blood coagulation are catalyzed by serine proteinases that are homologous to each other and to the archetypal serine proteinases <of digestion. We will use prothrombin activation catalyzed by the prothrombinase complex as a paradigm for specific macromolecular substrate recognition and cleavage by coagulation complexes to investigate the basis for enzymic function. Our studies are focused on the central role of extended interactions between substrate and enzyme in driving the action of prothrombinase on the two cleavage sites in prothrombin. A series of approaches will assess the role of the transition between zymogen and proteinase that accompanies the initial cleavage of prothrombin in regulating presentation of the tethered substrate to the enzyme and in providing a rate-limiting step in the process of thrombin formation. Novel fluorescent derivatives of the interacting species will be used in resonance energy transfer studies to establish physical correlates of changes in substrate presentation following proteinase formation and the role of substrate-cofactor interactions in enzymic function. Binding studies will establish the thermodynamic basis for the kinetic behavior of prothrombin as a compound substrate for prothrombinase. These approaches will assess the role of geometric constraints arising from exosite tethering that permit the enzyme to discriminate between the two cleavage sites in the substrate. Additional studies will assess the role of the Nterminal domain of the substrate in participating in the exosite-dependent tethering process. Mechanistic insights obtained under defined conditions will be extended to understand the function of prothrombinase on activated platelets and endothelial cells with the hypothesis that the cleavage pathway and intermediate produced is dependent on cell type, determined by the ability of the cells to support prothrombin binding. Overall, our strategies are based on novel concepts derived from an expanded understanding of substrate recognition by prothrombinase. The proposed approaches will shed new light on important yet poorly understood facets of the biochemistry and biology of enzyme function relevant to normal hemostasis, in disease states and for therapeutic targeting of this reaction in thrombotic and vascular disease.