Vitamin K 2,3-epoxide reductase: the basis for stereoselectivity of 4-hydroxycoumarin anticoagulant activity.

1. The administration of S-warfarin (1 mg kg-1 i.v.) to rats that were pre-loaded 48 h before with tracer doses (6 micrograms) of 14C-labelled R- or S-warfarin caused the plasma levels of these compounds to increase. This is due to the substitution of the microsomal (vitamin K 2,3-epoxide (K0) reductase) bound R- or S-[14C]-warfarin by the unlabelled 4-hydroxycoumarin administered. The rate of reappearance was 3-4 fold higher for R- than for S-warfarin; t1/2 of release: 1.2 +/- 0.04 and 3.7 +/- 0.6 h, respectively. 2. Liver microsomes prepared from rats pretreated with R- or S-[14C]-warfarin, released these compounds only in the presence of dithiothreitol (DTT; 10 mM). The rate of release was higher for R- than for S-warfarin-treated microsomes. 3. Liver microsomes treated in vitro with R- or S-acenocoumarol could be reactivated by DTT (10 mM). Reactivation was higher for the R- than for the S-acenocoumarol-treated microsomes. 4. The microsomal vitamin K0 reductase activity under 'normal' assay conditions ([DTT] = 2 mM) was as sensitive for R- as for S-4-hydroxycoumarins. At elevated DTT concentrations (= 42 mM) the rate of vitamin K0 conversion was about 1.5 fold higher in the presence of the R-isomers than in the presence of the S-isomers. For instance, at 2 mM DDT the reductase activities in the presence of 2.6 microM R- and S-warfarin were about 15% of control. At 42 mM DTT the activities were 90 and 65% of control, respectively. 5. In the in vitro experiments acenocoumarol appeared to be more potent than warfarin and phenprocoumon. 6. The following mechanism is proposed: vitamin K0 reductase becomes oxidized during substrate reduction. The oxidized (i.e. inactive) form binds equally to the R- and S-enantiomers of 4- hydroxycoumarins. The attached (covalently bound?) coumarin is released by the reactivation (i.e. reduction) of the enzyme. However, the rate of reactivation is strongly attenuated by the attached coumarin. This effect is more pronounced for the S-configuration of the 4-hydroxycoumarin anticoagulants.