Blood coagulation Factor X occupies a central role in the clotting cascade. It can be activated by either Factor VIIa-tissue factor (extrinsic tenase) or by Factor IXa-VIIIa-phospholipid (intrinsic tenase). Once activated, it participates in formation of the prothrombinase complex, composed of Factor Xa (F.Xa) and the cofactor Factor Va (F.Va) on a membrane surface in the presence of calcium ions. The prothrombinase complex catalyzes the conversion of prothrombin to the active serine protease thrombin. Recent work has also established that, in addition to its role in the coagulation cascade, F.Xa serves as a ligand for the cellular receptor Effector Cell Protease Receptor-1 (EPR-1), a membrane- bound protein found on monocytes, lymphocytes, endothelial cells and vascular smooth muscle cells. This observation suggests that F.Xa, like thrombin and protein S, can serve as a link between the coagulation system and other host defense systems such as inflammation and wound repair. The goal of the experiments in this proposal is to determine the molecular sites of interaction between F.Xa and its cofactor F.Va, and between F.Xa and its cellular receptor EPR-1. To carry this out, we will prepare recombinant variants of F.Xa using site-directed mutagenesis and a mammalian expression system. We have recently worked out a multi-step procedure for isolating fully carboxylated rF.Xa from the expression system. Both chimeric proteins (domain exchanges) and point mutations will be synthesized and tested. Choice of mutations for analysis will be based on the defined 3-dimensional structure of F.Xa and on previous experimental findings including work with proteolytic fragments, with synthetic peptides, and with chimeric proteins. Binding to F.Va will be assessed using optical biosensor technology and competitive inhibition assays. To determine sites on F.Xa required for interaction with EPR-1, recombinant variants will be radiolabelled and tested in binding assays to EPR+ HUVEC's and to CHO cells transfected with EPR-1. This work will contribute to our understanding of the molecular interactions by which F.Xa exerts its biological effects in enzyme complexes and through cellular receptors.
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