DESIGN OF MODIFIED PROTEIN C BY MOLECULAR MODELING

THE COAGULATION CASCADE CONSISTS OF A NUMBER OF MULTIDOMAIN SERINE PROTEASES AND PROTEIN COFACTORS. AMONG THESE ARE FACTORS IX, X, AND PROTHROMBIN. PROTEIN C DOWNREGULATES THE INTRINSIC COAGULATION PATHWAY BY INACTIVATING FACTORS VAAND VIIIA. FACTORS IX, X, AND PROTEIN C ARE HIGHLY HOMOLOGOUS PROTEINS. EACH CONTAINS TWO EGF-HOMOLOGOUS DOMAINS AND A N-TERMINAL DOMAIN BEARING MULTIPLE -CARBOXYGLUTAMIC ACID RESIDUES, THE SO-CALLED GLA DOMAIN. COMPETITIVE BINDING EXPERIMENTS HAVE ESTABLISHED THAT FACTORS IX AND X BIND TO RECEPTORS ON ACTIVATED ENDOTHELIAL CELLS THROUGH INTERACTIONS OF THE EGF AND GLA DOMAINS, A PHYSIOLOGICAL MECHANISM FOR DIRECTING COAGULATION TO REGIONS OF DAMAGED ENDOTHELIUM. THE OBJECTIVE IS TO IMPROVE RECOMBINANT PROTEIN C FOR USE ASA SAFE ANTICOAGULANT/ANTITHROMBOTIC AGENT FOR SUPPRESSION OF CLOTTING IN A NUMBER OF VENOUS THROMBOTIC CONDITIONS. SPECIFICALLY, A PROTEIN C VARIANT WITH A HIGH BINDING AFFINITY FOR DAMAGED ENDOTHELIUM WILL BE DESIGNED. THE EGF-HOMOLOGOUS DOMAINS OF PROTEIN C HAVE BEEN IDENTIFIED AS REGIONS THAT CAN BE ENGINEERED TO CONFER BINDING AFFINITYFOR THE FACTOR IX ENDOTHELIAL CELL RECEPTOR. IN PHASE I, MOLECULAR MODELING WILL BE USED TO IDENTIFY RESIDUES IN THE EGF-HOMOLOGOUS DOMAINS OF PROTEIN C THAT CAN BE ALTERED TO MORE CLOSELY MIMIC THE COMPARABLE DOMAIN OF FACTOR IX. IN PHASE II, VARIANTS OF PROTEIN C WITH THESE MUTATIONS WILL BEGENERATED AND THEIR SPECIFIC ACTIONS WITH FACTORS IX, VA AND VIIIA WILL BE DETERMINED.