In the liver, four of the blood clotting factors in the coagulation system (factor II, VII, IX and X) are dependent on vitamin K for their biosynthesis. The vitamin is a necessary cofactor for an enzyme complex, the vitamin K-dependent carboxylase, that converts inactive precursors of the clotting to active clotting factors. This is achieved by Gamma-carboxylation of specific glutamic acid residues in the precursor proteins. In order to function as a cofactor for the carboxylase, vitamin K must be in its reduced form, vitamin K hydroquinone. It appears that in the liver there are two independent pathways for vitamin K reduction. These are: reduction pathway I; a pathway consisting of the coumarin-sensitive enzyme vitamin K-epoxide reductase and reduction pathway II; a pathway consisting of a class of pyridine nucleotide-dependent dehydrogenases that are capable of reducing vitamin K. The pathway II enzymes are less susceptible to inhibition by coumarins and constitute an important pathway for enhanced clotting factor synthesis in cases of coumarin anticoagulant poisoning. The work that is proposed will focus on this clinically important pathway. The individual enzymes in the pathway will be purified and identified and their susceptibility to inhibition by different anticoagulant drugs studied. In addition, experiments are proposed that will measure in vitro recovery of clotting factor synthesis by high concentrations of vitamin K when pathway I is irreversibly blocked by anticoagulant drugs. The results should provide a detailed in vitro model system for the functioning of pathway II as a salvage system for clotting factor synthesis in cases of anticoagulant poisoning. The model will be tested in vivo by administering vitamin K to anticoagulated rats. A second line of work concentrates on an endogenous protein substrate for the carboxylate that follows the enzyme throughout an extensive purification procedure. Methods are proposed which will compare by peptide mapping the purified substrate protein and clotting factors II and X. It will be possible to determine if the endogenous substrate is a precursor form of factor II or X or an unrelated protein.
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