This proposal outlines a series of experiments designed to better delineate sites of specific intermolecular interactions between human coagulation factor X (FX) and its functional cofactors, phospholipid membranes, human factor Va (FVa), and a specific inhibitor, tissue factor pathway inhibitor (TFPI). The experiments proposed are designed to better delineate amino acid residues of human coagulation factor X which are involved in molecular interactions with its cofactor in the prothrombinase complex (FVa); phospholipid membrane (which is required for the normal activation and activity of FX); and with its specific inhibitor, TFPI. Using site- directed mutagenesis, individual amino acid residues of FX in regions thought to be involved with these intermolecular interactions will be modified and rFX expressed in mammalian cells. Purified recombinant variant proteins will be characterized in clotting assays; a functional prothrombinase assay using purified components (to determine interaction with the cofactor, FVa); and in kinetic and binding assays for the TFPI interaction with FXa and the TFPI-FXa inhibition of FVIIa/TF.
| Camire, R M; Larson, P J; Stafford, D W et al. (2000) Enhanced gamma-carboxylation of recombinant factor X using a chimeric construct containing the prothrombin propeptide. Biochemistry 39:14322-9 |
| Larson, P J; Camire, R M; Wong, D et al. (1998) Structure/function analyses of recombinant variants of human factor Xa: factor Xa incorporation into prothrombinase on the thrombin-activated platelet surface is not mimicked by synthetic phospholipid vesicles. Biochemistry 37:5029-38 |
