Understanding vitamin K-dependent proteins will require insight into their biosynthesis and regulation. Unique for these proteins is their vitamin K-dependent post-translational modification. The modification is carried out by the _-carboxylase, an integral protein of the ER membrane which uses the reduced form of vitamin K (vitamin KH2) as cofactor. The reduced cofactor is produced by the enzyme Vitamin K 3,4-Epoxide reductase (VKOR) which is the target for the anticoagulant drug warfarin. Despite numerous attempts to purify VKOR, the molecular components that constitute the enzyme have not been identified. The applicants have been able to show that VKOR is an enzyme complex in the ER membrane and they propose that assembly of the complex is similar to assembly of the lipoxygenase complex on the nuclear envelope membrane. Both enzymes incorporates a member of the glutathione-S-transferase gene family as part of the lipid-protein enzyme complex. Experiments are proposed to complete the model of the VKOR enzyme complex and unveil the basis for genetic resistance to warfarin. Recombinant VKOR will be used to increase the capacity of cell lines to produce functional clotting factors VII and IX. It is our hypothesis that vitamin KH2 cofactor production by VKOR is a limiting factor in cellular production of these recombinant proteins. Although 8-10 different vitamin K-dependent proteins have been shown to be made extrahepatically only some have been identified. They include the coagulation factors prothrombin and protein S, the growth arrest specific gene 6 product Gas6 and the bone and cartilage resident proteins, osteocalsin and matrix GLA protein (MGP). In the last specific aim of this application experiments are proposed to identify new vitamin K-dependent proteins by vitamin K-dependent radioactive labeling. The proposed experiments will provide a better understanding of vitamin K metabolism and vitamin K function and have an impact on prophylactic medicine concerned with recombinant vitamin K-dependent coagulation factors and warfarin anticoagulation. The experiments should also contribute to the exciting, rapidly expanding area of research on extra-hepatic vitamin K-dependent proteins.
