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.

The systematic use of antivitamin K anticoagulants (AVK) as rodenticides caused the selection of rats resistant to AVKs. The resistance is mainly associated to genetic polymorphisms in the Vkorc1 gene encoding the VKORC1 enzyme responsible for the reduction of vitamin K 2,3-epoxide to vitamin K. Five major mutations, which are responsible for AVK resistance, have been described. Possible explanations for the biological cost of these mutations have been suggested. This biological cost might be linked to an increase in the vitamin K requirements. To analyze the possible involvement of VKORC1 in this biological cost, rVKORC1 and its major mutants were expressed in Pichia pastoris as membrane-bound proteins and their catalytic properties were determined for vitamin K and 3-OH-vitamin K production. In this report, we showed that mutations at Leu-120 and Tyr-139 dramatically affect the vitamin K epoxide reductase activity. Moreover, this study allowed the detection of an additional production of 3-hydroxyvitamin K for all the mutants in position 139. This result suggests the involvement of Tyr-139 residue in the second half-step of the catalytic mechanism corresponding to the dehydration of vitamin K epoxide. As a consequence, the biological cost observed in Y139C and Y139S resistant rat strains is at least partially explained by the catalytic properties of the mutated VKORC1 involving a loss of vitamin K from the vitamin K cycle through the formation of 3-hydroxyvitamin K and a very low catalytic efficiency of the VKOR activity.

Vitamin K plays a crucial role in the regulation of vascular calcifications by allowing activation of matrix Gla protein. The dietary requirement for vitamin K is low because of an efficient recycling of vitamin K by vitamin K epoxide reductase (VKORC1). However, decreased VKORC1 activity may result in vascular calcification. More than 30 coding mutations of VKORC1 have been described. While these mutations have been suspected of causing anticoagulant resistance, their association with an increase in the risk of vascular calcification has never been considered. We thus investigated functional cardiovascular characteristics in a rat model mutated in VKORC1. This study revealed that limited intake in vitamin K in mutated rat induced massive calcified areas in the media of arteries of lung, aortic arch, kidneys and testis. Development of calcifications could be inhibited by vitamin K supplementation. In calcified areas, inactive Matrix Gla protein expression increased, while corresponding mRNA expression was not modified. Mutation in VKORC1 associated with a limited vitamin K intake is thus a major risk for cardiovascular disease. Our model is the first non-invasive rat model that shows spontaneous medial calcifications and would be useful for studying physiological function of vitamin K.