Characterization of Vitamin K epoxide reductase in warfarin resistant roof rats (Rattus rattus)

Warfarin is a commonly used rodenticide worldwide. It inhibits coagulation of blood by inhibiting vitamin K epoxide reductase (VKOR) activity. An inadequate supply of vitamin K blocks the production of prothrombin and cause hemorrhaging. Recently, warfarin-resistant roof rats have been found around Tokyo areas in Japan. However, the mechanism which causes warfarin resistance in roof rat was still unclear. In this study, I focused on the VKOR in roof rats to figure out the mechanisms of warfarin-resistance. The liver was obtained from warfarinsensitive and -resistant roof rats. I cloned VKORC1 gene from roof rats and sequenced. I found novel substitution of leucine to proline at 76 position of VKORC1 amino acid sequence. Then, I determined the kinetic differences between VKOR mutant and wild type. I prepared liver microsomes from-sensitive and -resistant rats. The VKOR-dependent activity was measured over a range of vitamin K epoxide concentration from 12.5 to 100 μM by HPLC in absence and presence of warfarin. Lineweaver-Burk and Hanes plots were used to determine Vmax and Km values, and a constant of inhibition by warfarin (Ki) was calculated according to Dixon plot and Secondary plot. The values of Vmax was significantly lower in resistant rats (9.58±4.35 pmol/min/mg) than those of -sensitive rats (22.06±7.56 pmol/min/mg). I could not find any significant differences in Km values between-resistant (67.59±64.87 μM) and -sensitive (30.36±18.94 μM) rats. Low Vmax values lead to lower enzymatic efficiency (Vmax/Km) inresistant rats (0.31±0.33) than those of -sensitive rats (0.95±0.53). However, since I found large individual differences in Km values in-resistant rats, further study may be need to clarify the VKOR catalytic efficiency at physiological concentration of vitamin K epoxide. In this study, result from warfarin inhibition assay revealed a good exploration for rodents, which acquired the warfarin resistance. The value of Ki was 50- to 100-fold higher in warfarin-resistant than those of -sensitive rats. Finally, I concluded that one of mechanisms which cause warfarin resistance in Japanese roof rat might be low inhibiting effect of warfarin on VKOR mutant.