A Novel Mutation in VKORC1 and Its Effect on Enzymatic Activity in Japanese Warfarin-Resistant Rats

Abstract

ABSTRACT. Warfarin is a rodenticide commonly used worldwide. It inhibits coagulation of blood by inhibiting vitamin K 2,3-epoxide reductase (VKOR) activity. An inadequate supply of vitamin K blocks the production of prothrombin and causes hemorrhage. Recently, warfarin-resistant brown rats (Rattus norvegicus) were found around the Aomori area of Japan. There is no significant difference in the metabolic activity of warfarin in sensitive and resistant brown rats. To clarify the mechanism underlying warfarin resistance, we cloned the VKORC1 gene from rats and identified a novel substitution of arginine to proline at position 33 of the VKORC1 amino acid sequence. Then, we determined the differences in kinetics of VKOR activity between warfarin-resistant and sensitive rats. Hepatic microsomal VKOR-dependent activity was measured over a range of vitamin K epoxide concentrations from 6.25 to 150 µM. The V max values of resistant rats (0.0029 ± 0.020 nmol/min/mg) were about one tenth of those of sensitive rats (0.29 ± 0.12 nmol/min/mg). The K m values of resistant rats (47 ± 32 µM) were similar to those of sensitive rats (59 ± 18 µM). Warfarin-sensitive rats exhibited enzyme efficiencies (V max /K m ) which were ten-fold greater than those observed in resistant rats. It may mean that VKOR activity of warfarin-resistant Aomori rats is almost lost, because their enzymatic efficiencies are very low even without warfarin. Further studies are needed to clarify how these rats can survive with a markedly reduced VKOR activity and how they simultaneously exhibit warfarin resistance.

Dicoumarol: A Drug which Hits at Least Two Very Different Targets in Vitamin K Metabolism

“…Based on the structure of the cyanobacterial enzyme, it was suggested that warfarin binds at the same site at the quinone seen in the structure (and that vitamin K epoxide would also interact at this site) [47]. This is consistent with the locations of some of the mutations which alter the sequence of rat VKOR conferring resistance to warfarin and those which are associated with hereditary clotting deficiencies in humans [55,[62][63][64][65][66][67]. Many of these variant forms of VKOR have lower catalytic activity than the wild-type and it is presumed that this loss of fitness is a "price worth paying" for resistance to rat poison [68].…”

“…Using a homology model based on the cyanobacterial structure, docking with AutoDock Vina as implemented in Chimera [69,70], predicted that both dicoumarol and warfarin would bind at the base of the cleft with partially overlapping binding sites ( Figure 3). These models predict that the ligands bind close to some residues implicated in warfarin resistance, notably Ala-26, Trp-59, Glu-67, His-68, Gly-71, Leu-120 and Tyr-139 [55,[62][63][64][65][66][67]71].…”

Low warfarin resistance frequency in Norway rats in two cities in China after 30 years of usage of anticoagulant rodenticides

“…28,41 Studies on the genetic basis of warfarin resistance led to the development and usage of molecular methods for identifying and screening rodents, which are more convenient and economical than the LFP and BCR methods currently used in China. 2,9,14,15,[24][25][26] Studies of the molecular resistance mechanism in rats could facilitate the development of molecular tools for rapid screening of rats for anticoagulant resistance.…”

“…18 In Norway rats, at least 10 Vkorc1 mutations were suggested to be associated with anticoagulant resistance based on in vivo, in vitro and in silico studies. 9,14,15,[19][20][21][22][23][24][25][26] Some Vkorc1 mutations, for example Tyr139Cys/Phe/Ser, have been widely used as genetic markers for screening or monitoring resistant commensal rodent species in Europe. 2,14 However, those genetic markers may not be applicable to rodents in other regions as other resistance Vkorc1 mutations may be present in different local populations.…”

“…2,14 However, those genetic markers may not be applicable to rodents in other regions as other resistance Vkorc1 mutations may be present in different local populations. 4,9,14,15,[24][25][26] www.soci.org X Ma et al…”