Coagulation proteases are a series of structurally similar trypsin-like enzymes, which normally circulate in plasma as inactive precursors and are only activated when the integrity of the blood vessel is compromised by either injury or inflammation. The cascade is initiated and propagated by the successive activations of the vitamin K-dependent zymogens, factors VII, IX, X and prothrombin;and the non- enzymatic cofactors Va and VIlla that culminate in generation of the fibrinogen clotting enzyme thrombin. Although these proteases all have similar three-dimensional structures, they exhibit a very high degree of specificity in interaction with their related cofactors and substrates. The molecular basis of coagulation protease/cofactor/substrate specificity is not well understood. We hypothesize that a limited number of divergent residues on the conserved surface loops of these proteins provide specific recognition sites for the assembly of these molecules on membrane surfaces. It is established that cofactors also dictate specificity by altering the topography of the membrane-bound coagulation enzymes in the activation complexes. We further hypothesize that cofactors can also alter the topography of the membrane-bound substrates to maintain scissile bonds at distances and orientations optimal for interaction with the active- sites of the proteases in the activation complexes. To test these hypotheses, we propose to investigate the molecular determinants of the specificity of factor X/Xa (fX/fXa) assemblies into the respective activation complexes using wild-type and mutant proteins. Specifically, we propose i) to investigate the functional significance of the first epidermal growth factor (EGF)-like domain of fXa and its contribution to the specificity of factor Va (fVa) recognition in the prothrombinase complex, ii) to determine whether the cofactor function of fVa in the prothrombinase complex alters the topography of the membrane-bound substrate, iii) to determine whether the first EGF domain of fX possesses an interactive site for tissue factor in the extrinsic Xase complex, and iv) to investigate the contribution of the post-translationally attached oligosaccharide chains of the fX activation peptide to the recognition specificity of the zymogen in the intrinsic Xase complex. These studies will utilize fluorescence spectroscopy, enzyme kinetics, and competitive binding methods to provide critical information about the kinetic and thermodynamic constants that define the specific interaction of fX/fXa with the components of the respective physiological activation complexes. Understanding the mechanism by which clotting factors interact with each other in the activation complexes is essential for design and development of therapeutic drugs and inhibitors for use in treatment of abnormalities (thrombosis and hemorrhage) in blood.
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