18 The oxidized status of VKORC1 is maintained in the ER, which contains a redox buffer comprising a high concentration (∼15 mM) of reduced glutathione (GSH) and oxidized glutathione (GSSG). 18,19 Indeed, this membrane oxidoreductase is primarily oxidized in cells, with ∼40% of it in the fully oxidized state and 50% in partially oxidized state. 13-17 This large discrepancy is probably because DTT reduces VKORC1 almost completely, whereas warfarin preferably inhibits oxidized VKORC1. In the presence of DTT, however, the 50% inhibitory concentration (IC 50) of warfarin is at millimolar concentration, 11,12 which is much higher than the nanomolar therapeutic range of warfarin. VKORC1 activity was traditionally analyzed in vitro, using dithiothreitol (DTT) to mimic the reducing equivalent in cells. Thus, warfarin dosage control should use VKORC1 level as a major indicator, and improved antidotes may be designed based on their competition with warfarin. Taken together, warfarin is a competitive inhibitor that binds VKORC1 tightly and inhibits at a stoichiometric (1:1) concentration, whereas exceeding the VKORC1 level results in warfarin overdose. The competition occurs also in cells, resulting in rescued VKORC1 activity that augments the antidotal effects of vitamin K. However, warfarin is released from purified VKORC1-warfarin complex with increasing amount of vitamin K, indicating competitive inhibition. The kinetics data can be fitted with the Morrison equation, giving a nanomolar inhibition constant and demonstrating that warfarin is a tight-binding inhibitor. Characterization of the inhibition kinetics required the use of microsomal VKORC1 with a native reductant, glutathione, that enables effective warfarin inhibition in vitro. Here we find that effects of the major predictor of warfarin dosage, SNP −1639 G>A, follow a general correlation that warfarin 50% inhibitory concentration decreases with cellular level of vitamin K epoxide reductase (VKORC1), suggesting stoichiometric inhibition. Improving the dosage management and antidotal efficacy requires mechanistic understanding. Dose control of warfarin is a major complication in anticoagulation therapy and overdose is reversed by the vitamin K antidote.
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