Coumarins target blood coagulation via inhibition of the vitamin K epoxide reductase multiprotein complex (VKOR). This complex recycles vitamin K 2,3-epoxide to vitamin K hydroquinone, an essential cofactor for the post-translational gamma-carboxylation of several blood coagulation factors. Recently, two groups including ours identified a key component of the VKOR which we named Vitamin K Epoxid Reductase Subunit 1 (VKORC1). The corresponding gene comprises a 5 kb genomic region and consist of three exons encoding a small 163 aa transmembrane protein. Since VKOR was hypothesized to be involved in two heritable diseases, combined deficiency of vitamin K dependent clotting factors type 2 (VKCFD2) and resistance to coumarin-type drugs (warfarin/phenprocoumon resistance, WR) we sequenced the VKORC1 gene in patients with these phenotypes. The same homozygous missense mutation (R98W) was found in two families from Germany and Lebanon with a mild VKCFD2 phenotype. In 7 patients from 5 families with various degrees of WR (weekly warfarin dose ranged from 115 mg to 280 mg, two patients exhibited complete WR) different heterozygous missense mutations distributed throughout the gene (V29L, V45A, R58G, V66M, L128R) were identified. Furthermore, we proved a heterozygous nonsense mutation (W5Stop) in a young female patient with highly increased phenprocoumon sensitivity. Over-expression of the wild-type protein in HEK 293 cells leads to a striking increase in VKOR activity which is sensitive to warfarin inhibition. In contrast to the wild-type protein, the VKCFD2 variant exhibited a dramatically decrease of VKOR activity. The WR variants showed a varying reduction of the VKOR activity, but unexpectedly, were sensitive to warfarin in the HEK cell expression system The reason for the differing WR phenotypes in human and in the HEK cell expression system are still to be elucidated. In conclusion, VKORC1 protein most likely represents the molecular target of coumarins, which have prescribed for oral anticoagulant therapy since more than 60 years. Mutations in the VKORC1 gene are causative for the hereditary conditions of VKCFD2, warfarin resistance and also warfarin sensitivity. Our findings may provide a basis for a rational design of novel anticoagulants targeting VKOR in the future.

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