Summary. Background: The VKORC1 gene codes for the VKORC1 enzyme, which is responsible for the reduction of vitamin K epoxide into vitamin K. VKORC1 enzyme is the target of vitamin K antagonists (VKA). Twenty‐eight rare single mutations in the VKORC1 coding sequence have been reported from resistant patients receiving unusually high doses of VKA to achieve therapeutic anticoagulation.
Objectives: It has been suggested that these mutations are responsible for the resistant phenotype, while biochemical consequences of these mutations on the VKORC1 enzyme have not yet been evaluated. Therefore, the aim of this study was to investigate the causality of the VKORC1 mutations in the resistance phenotype.
Methods: Wild‐type VKORC1 and its spontaneous mutants were expressed in Pichia pastoris and susceptibility to VKA was assessed by the in vitro determination of kinetic and inhibition constants.
Results and Conclusions: The in vitro analysis revealed that six mutations only (A26P, A41S, V54L, H68Y, I123N and Y139H) were associated with increase in Ki, suggesting their involvement in the resistance phenotype observed in patients. A41S and H68Y led to selective resistance, respectively, to indane‐1,3‐dione and 4‐hydroxycoumarine derivatives. The other mutations did not increase the Ki. Furthermore, 10 mutations (S52L, S52W, W59L, W59R, V66M, V66G, G71A, N77S, N77T and L128R) led to an almost complete loss of activity. These results suggest the existence of other resistance mechanisms.
Background: Effective involvement of VKORC1L1 in vitamin K epoxide reductase activity, target of vitamin K antagonists (VKAs), is still unclear. Results: VKORC1L1 is not inhibited by VKAs and catalyzes VKOR activity in extrahepatic tissues. Conclusion: During long term anticoagulation the limited unwanted side effects of VKAs are due to VKORC1L1. Significance: Potential pharmaco-toxicologic effects of specific VKORC1L1 inhibitors should be assessed.
This biomolecular approach to studying and detecting resistance is easier to carry out than the phenotypic approach measuring blood coagulation time because it can be conducted on biological samples from dead animals, and it is less dangerous for the operator.
A warfarin-resistant strain and a warfarin-susceptible strain of wild rats (Rattus norvegicus) maintained in enclosures of the National Veterinary School of Lyon (France) were studied to determine the mechanism of the resistance to anticoagulant rodenticides. A low vitamin K epoxide reductase (VKOR) activity has been reported for many resistant rat strains. As recently suggested, mutations in the vitamin K epoxide reductase subunit 1 (VKORC1) gene are the genetic basis of anticoagulant resistance in wild populations of rats from various locations in Europe. Here we report, for our strain, one of the seven described mutations (Tyr139Phe) for VKORC1 in rats. In addition, a low expression of mRNA encoding VKORC1 gene is observed in resistant rats, which could explain their low VKOR activity. We calculated kinetic parameters of VKOR in the warfarin-resistant and warfarin-susceptible rats. The V(max) and the K(m) of the VKOR obtained in resistant rats were lowered by 57 and 77%, respectively, compared to those obtained in susceptible rats. As a consequence, the enzymatic efficiency (V(m)/K(m)) of the VKOR was similar between resistant and susceptible rats. This result could be a good explanation to the observation that no clinical signs of vitamin K deficiency was observed in the warfarin-resistant strain, while a low VKOR activity was found. VKOR activity in warfarin-resistant rats was poorly inhibited by warfarin (K(i) for warfarin is 29 microM and 0.72 microM for resistant and susceptible rats, respectively).
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