Cellular disruption resulting from trauma and cellular stimulation by a variety of biological agonists can initiate the expression of tissue factor, an obligatory integral membrane protein cofactor for factor VII activity, leading to hemostasis and thrombosis. Factor VII is the first zymogen or, in its activated form, factor VIIa is the first serine protease which in association with tissue factor and calcium form the extrinsic pathway macromolecular complex responsible for initiating blood coagulation. Substrates for this enzyme complex are factors IX and X, and factor VII can be activated by factors IXa, Xa or XIIa. Regulation of this initiation complex can be by the exposure and interaction of tissue factor to blood, the dynamics of factor VII and factor VIIa to compete with near equal affinity for binding to the cofactor and the inhibition of the formed complex by factor X(Xa) and a serum lipoprotein. Factor VIIa may also be inhibited by antithrombin III in the presence of heparin. Thus, the multiple interactions of factor VII implies that several regions on the surface of this molecule control its association, and provides the basis to study the structure function relationships of this protein with its activators, cofactor, substrates and inhibitors. Identification of these molecular loci on factor VII is the overall goal of this project. Protein-protein interactions require that the regions mediating the associations leading to specific function must be at the solvent accessible surface of the molecules. Further, antibody binding to antigen requires the antigenic determinant be exposed to the hydrated surface. We will undertake experiments to identify the structural regions on factor VII/VIIa with synthetic peptides corresponding to specific segments of the primary structure of factor VII. Physical and biochemical studies will used to measure peptide binding and inhibition of complex formation and function. Monoclonal antibodies raised to predetermined specificities will be used as an independent approach to assign an antigenic site with specific function. Definition of a structural abnormality on naturally occurring variants of factor VII will provide a third avenue of investigation. From these three independent approaches we should be able to assign specific loci on the topology of factor VII/VIIa which mediate its functional activities. Identification of sites of interaction may lead to the production of therapeutic agents (peptides and/or monoclonal antibodies) which can impede coagulation and regulate hemostasis.
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