Anticoagulant protein C--structural and functional studies

Abstract: Anticoagulant protein C is a vitamin K-dependent factor circulating in plasma as a zymogen of a serine protease. It is converted to its active form (APC) by the thrombin-thrombomodulin complex on the surface of endothelial cells. APC inhibits the generation of thrombin by selective proteolytic inactivation procoagulant factors Va and VIIIa. Protein S, another vitamin K-dependent protein, serves as a cofactor to APC in the down-regulation of the coagulation cascade. In the studies upon which this thesis is based, we have tried to elucidate the structure and function of the protein C molecule. We found intact factor V to be yet another cofactor to APC. Human plasma factor V was demonstrated to stimulate the inactivation of the FVIIIa-FIXa complex in the presence of APC and protein S in a purified system. Thus, factor V also manifests anticoagulant properties. The species-specific interaction of APC, protein S and factor V was studied in systems containing proteins from human and bovine origins. The results suggest the species specificity of APC to be due to the interaction between APC and protein S, but not factor V. Vitamin K-dependent proteins contain 9-13 g-carboxyglutamic acids in the Gla domain, which are crucial for membrane and calcium binding. Despite very high levels of amino acid sequence identity, naturally occurring vitamin K-dependent proteins appear to be characterized by a wide (about 1000-fold) range of membrane affinity. The recombinant bovine protein C mutant H10, and human protein C mutants, G11E32D33, Q10G11E32D33 and G11N12E32D33 were demonstrated to manifest greatly enhanced membrane affinity and anticoagulant activity, as compared to their respective wild type forms. Loop148 in the serine protease domain of bovine protein C lacks four amino acids, as compared with the corresponding loop in human protein C. Study of recombinant human protein C containing bovine loop-148 and bovine protein C with human loop-148 indicated human protein C with bovine loop-148 to manifest increased catalytic activity against small synthetic and natural substrates. This suggests loop148 to be crucially involved in the catalytic activity of the enzyme. Using a recombinant human protein C mutant (K62N/K63D), we studied the interaction of APC with protein C inhibitor (PCI) in the absence and presence of heparin. The results suggest that residues K62 and K63 not only interact directly with PCI but are also involved in heparin binding.