Both the intrinsic and extrinsic coagulation pathways terminate with the activation of the blood protein Factor X. Activated Factor X in turn forms a supramolecular complex, termed prothrombinase, consisting of an enzyme (Factor X alpha), a protein cofactor (Factor V alpha), calcium ion, and a phospholipid surface. This assembled complex catalyzes the proteolytic activation of prothrombin to the protease thrombin. The expedient formation of thrombin at wound sites thus requires the precise recognition and binding of the enzyme to a cofactor, a surface, and a substrate. Each of these interactions is mediated by distinct regions on the surface of Factor X. The approach outlined in this proposal will define those peptide sites on Factor X involved in recognition and binding to prothrombin, Factor V alpha, and phospholipid. The approach is to use isolated peptide fragments of Factor X, synthetic peptides from the known sequence of Factor X, and antibodies to specific topographic sites on Factor X in well- defined functional assays for Factor X. Peptides that will be synthesized include the variable region sequences of Factor X and sequences from the Gla domain. Several available monoclonal antibodies will be examined for their ability to inhibit Factor X activity and the antigenic determinant recognized by the inhibitory antibody alpha Beta F chi-2 beta will be localized. Antibodies produced to the synthetic peptides will also be examined for their ability to bind Factor X and to inhibit prothrombin activation, prothrombinase complex assembly or Factor X binding to phospholipid vesicles. This combined approach on the structure/function of Factor X using peptide fragments, synthetic peptides, and site- specific antibodies will provide more information about the relevant surface features of the Factor X molecule important for its function than would each approach used separately. Such topographic information will be extremely valuable in understanding the assembly, function, and regulation of prothrombinase, and at the same time, assist the design and modification of genetically engineered blood coagulation proteins.
