Factor X plays a pivotal role in the coagulation scheme with its activation being at the point of convergence of the intrinsic and extrinsic activation pathways leading to the final stages of hemostasis. Activation of factor X by the intrinsic pathway requires the interaction of factor IXa (enzyme) and factor VIIIa (cofactor) on a phospholipid surface in the presence of calcium. The extrinsic factor X activation complex is composed of factor VII/VIIa (enzyme) assembled with membrane bound tissue factor (cofactor) in the presence of calcium. Once activated, factor Xa (enzyme) associates with factor Va (cofactor) in another membrane surface complex responsible for the activation of prothrombin. The participation of factor X/Xa in these three macromolecular complexes implies that several regions on the surface of this protein control its associations, and provides an unique opportunity to study the structure-function relationships of factor X/Xa with its activators, cofactors, surfaces and substrate. An understanding of the molecular basis of factor X function as it relates to protein-protein interactions in solution and in association with phospholipid (cell) surfaces 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 molecule. Further, antibody biding to antigen requires the antigenic determinant be exposed to the hydrated surface. We will undertake experiments to identify the structural regions on factor X and factor Xa responsible for mediating their interactions with the components of these three complexes by using synthetic peptides corresponding to specific segments of the primary structure within factor X. Physical and biochemical methods will be used to measure peptide binding and inhibition of complex formation and function. Monoclonal antibodies raised to predetermined specificities will be used as an independent avenue to assign an antigenic site with specific function. A third method will be to define the structurally abnormality associated with naturally occurring variant molecules of factor X using immunochemical an biochemical analyses. From the three independent approaches we should be able to assign specific loci on the topology of factor X and factor Xa which mediate their measurable functional activities. Identification of sites of interactions on factor X/Xa may lead to the production of therapeutic agents (peptides and/or monoclonal antibodies) which can impede coagulation and regulate hemostasis.
