The coagulation cascade consists of a number of multi-domain serine proteases and protein cofactors. Among these are factors IX, X, and prothrombin. Protein C down-regulates the intrinsic coagulation pathway by inactivating factors Va and VIIIa. Factors IX, X and protein C are highly homologous proteins. Each contains two EGF-homologous domains and an N-terminal domain bearing multiple gamma-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. This is one physiological mechanism for directing coagulation to regions of damaged endothelium. The objective of this proposal is to improve recombinant protein C for its intended use as a safe anticoagulant/antithrombotic agent for suppression of clotting in a number of venous thrombotic conditions. Specifically, the investigators would like to design a protein C variant with a high binding affinity for damaged endothelium. They have identified the EGF-homologous domains of protein C as regions that can be engineered to confer binding affinity for the factor IX endothelial cell receptor. In Phase I of this project, they will use molecular modelling 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, they will generate variants of protein C with these mutations and determine their affinity for the factor IX receptor as well as their efficiency in the catalytic inactivation of factors Va and VIIIa.
