Warfarin is a widely used oral anticoagulant medicine and rodenticide which antagonizes vitamin K dependent-Gamma-carboxylation of specific N-terminal glutamate residues in at least six essential coagulation factors including factor II (prothrombin), VII, IX, and X. Glutamate Gamma-carboxylation in proteins of a number of other tissues such as developing bone, kidney, lung and placenta is also dependent upon vitamin K. Recently warfarin was shown to exhibit anticancer activity in animal models and in man which can be related to its antivitamin K activity. The ultimate goal of this project is to determine the molecular bases for the antivitamin K activity of warfarin. Determination of the metabolic pathways utilized by vitamin K, which are essential to its Gamma-carboxylation activity, is a further, associated goal of the project.
The specific aims of this proposal are: 1) to purify the 14 to 17,000 dalton protein involved in vitamin K and vitamin K 2,3-epoxide (KO) metabolism and identify whether the catalytically active disulfide is in a prosthetic group or is formed by sulfhydryl containing amino acids; 2) to determine if warfarin binding to the 14 to 17,000 dalton protein is its physiologically relevant site of action; 3) to identify and purify other enzymes and proteins that participate in vitamin K and vitamin KO metabolism and study them in reconstituted systems; and 4) to ascertain the physiologic reductant for the vitamin K and vitamin KO metabolizing enzymes. Protein and enzyme isolation will be accomplished by combinations of electrophoretic, high performance liquid chromatography (HPLC), and conventional and affinity chromatographic techniques. Physiologically relevant sites of warfarin action will be determined by comparison of its affinity for proteins isolated from warfarin-sensitive and resistant rats. Effects of various cofactors and recombination techniques on the rates of vitamin K and vitamin KO metabolism will be assessed by HPLC. Results obtained from these investigations will resolve the outstanding questions concerning the number of enzymes involved in vitamin K and vitamin KO metabolism, the nature of their catalyticaly active groups, and the factors regulating their inhibition by warfarin. Ultimately they will aid in the rational design of better, more specific antivitamin K drugs.
