Mechanism of coumarin action: sensitivity of vitamin K metabolizing enzymes of normal and warfarin-resistant rat liver.

The in vitro effects of two coumarin anticoagulants, warfarin and difenacoum, on rat liver microsomal vitamin K dependent carboxylase, vitamin K epoxidase, vitamin K epoxide reductase, and cytosolic vitamin K reductase (DT-diaphorase) from the livers of normal and a warfarin-resistant strain of rats have been determined. Millimolar concentrations of both coumarins are required to inhibit the carboxylase and epoxidase activities in both strains of rats. Sensitivity of DT-diaphorase to coumarin inhibition differs when a soluble or liposomal-associated substrate is used, but the diaphorases isolated from both strains of rats have comparable sensitivity. The anticoagulant difenacoum is an effective rodenticide in the warfarin-resistant strain of rats, and the only enzyme studied from warfarin-resistant rat liver that demonstrated a significant differential inhibition by the two coumarins used was the vitamin K epoxide reductase. This enzyme also showed the greatest sensitivity to coumarin inhibition among the enzymes studied. These results support the hypothesis that the physiologically important site of action of coumarin anticoagulants is the vitamin K epoxide reductase.