Vitamin K epoxide reductase (VKOR) is the target of warfarin, the most widely prescribed anticoagulant for thromboembolic disorders. Although estimated to prevent twenty strokes per induced bleeding episode, warfarin is under-used because of the difficulty of controlling dosage and the fear of inducing bleeding. Although identified in 1974 (ref. 2), the enzyme has yet to be purified or its gene identified. A positional cloning approach has become possible after the mapping of warfarin resistance to rat chromosome 1 (ref. 3) and of vitamin K-dependent protein deficiencies to the syntenic region of human chromosome 16 (ref. 4). Localization of VKOR to 190 genes within human chromosome 16p12-q21 narrowed the search to 13 genes encoding candidate transmembrane proteins, and we used short interfering RNA (siRNA) pools against individual genes to test their ability to inhibit VKOR activity in human cells. Here, we report the identification of the gene for VKOR based on specific inhibition of VKOR activity by a single siRNA pool. We confirmed that MGC11276 messenger RNA encodes VKOR through its expression in insect cells and sensitivity to warfarin. The expressed enzyme is 163 amino acids long, with at least one transmembrane domain. Identification of the VKOR gene extends our understanding of blood clotting, and should facilitate development of new anticoagulant drugs.

We describe a cell-based assay for studying vitamin K-cycle enzymes. A reporter protein consisting of the gla domain of factor IX (amino acids 1-46) and residues 47-420 of protein C was stably expressed in HEK293 and AV12 cells. Both cell lines secrete carboxylated reporter when fed vitamin K or vitamin K epoxide (KO). However, neither cell line carboxylated the reporter when fed KO in the presence of warfarin. In the presence of warfarin, vitamin K rescued carboxylation in HEK293 cells but not in AV12 cells. Dicoumarol, an NAD(P)H-dependent quinone oxidoreductase 1 (NQO1) inhibitor, behaved similarly to warfarin in both cell lines. Warfarin-resistant vitamin K epoxide reductase (VKOR-Y139F) supported carboxylation in HEK293 cells when fed KO in the presence of warfarin, but it did not in AV12 cells. These results suggest the following: (1) our cell system is a good model for studying the vitamin K cycle, (2) the warfarin-resistant enzyme reducing vitamin K to hydroquinone (KH 2 ) is probably not NQO1, (3) there appears to be a warfarin-sensitive enzyme other than VKOR that reduces vitamin K to KH 2 , and (4) the primary function of VKOR is the reduction of KO to vitamin K. (Blood. 2011;117(10):2967-2974) IntroductionVitamin K hydroquinone (KH 2 ) is a cofactor for ␥-glutamyl carboxylase (GGCX), which catalyzes the posttranslational carboxylation of specific glutamic acid residues to ␥-carboxyglutamic acid (gla) in a variety of vitamin K-dependent proteins. 1 ␥-Glutamyl carboxylation is essential for the biologic functions of vitamin K-dependent proteins involved in blood coagulation, bone metabolism, signal transduction, and cell proliferation. Concomitant with ␥-glutamyl carboxylation, KH 2 is oxidized to vitamin K 2,3-epoxide (KO). KO must then be converted back to vitamin K (the quinone form) and then to KH 2 by separate 2 electron reductions to support the carboxylation reaction. The cyclic production of KO and the conversion back to KH 2 constitutes the vitamin K cycle (Figure 1). The only enzymes unequivocally identified as part of the cycle are GGCX and vitamin K epoxide reductase (VKOR). 2 Sherman et al first proposed that the reduction of KO to vitamin K is carried out by a sulfhydryl-dependent epoxide reductase that is sensitive to warfarin inhibition. 3 This enzyme is probably VKOR, the only enzyme thus far shown to reduce KO to vitamin K. On the other hand, some reports suggest that the reduction of vitamin K to KH 2 can be accomplished by at least 2 microsomal enzymes called vitamin K reductases. 4 However, other studies 5,6 suggest that one enzyme serves as both the epoxide reductase and the vitamin K reductase, catalyzing both the reduction of KO to vitamin K and that of vitamin K to KH 2 .Wallin proposed that there are 2 enzymes that reduce vitamin K in support of vitamin K-dependent carboxylation. 7 One enzyme is inhibited by anticoagulant drugs such as warfarin, 8 while the other is an NADH-dependent reductase that is resistant to inhibition by warfarin. 4,[9][10][11] Consistent with the latte...

The vitamin K-dependent ␥-glutamyl carboxylase catalyzes the modification of specific glutamates in a number of proteins required for blood coagulation and associated with bone and calcium homeostasis. All known vitamin K-dependent proteins possess a conserved eighteen-amino acid propeptide sequence that is the primary binding site for the carboxylase. We compared the relative affinities of synthetic propeptides of nine human vitamin K-dependent proteins by determining the inhibition constants (K i ) toward a factor IX propeptide/ ␥-carboxyglutamic acid domain substrate. The K i values for six of the propeptides (factor X, matrix Gla protein, factor VII, factor IX, PRGP1, and protein S) were between 2-35 nM, with the factor X propeptide having the tightest affinity. In contrast, the inhibition constants for the propeptides of prothrombin and protein C are ϳ100-fold weaker than the factor X propeptide. The propeptide of bone Gla protein demonstrates severely impaired carboxylase binding with an inhibition constant of at least 200,000-fold weaker than the factor X propeptide. This study demonstrates that the affinities of the propeptides of the vitamin K-dependent proteins vary over a considerable range; this may have important physiological consequences in the levels of vitamin Kdependent proteins and the biochemical mechanism by which these substrates are modified by the carboxylase.The vitamin K-dependent carboxylase catalyzes the posttranslational modification of specific glutamates to ␥-carboxyglutamate (Gla) 1 in a number of proteins. Most vitamin K-dependent proteins are involved in the hemostatic process (prothrombin, factors VII, IX, and X, and proteins C, S, and Z), whereas two others (bone Gla protein and matrix Gla protein) are associated with bone (1-4). Two new putative vitamin K-dependent proteins of unassigned function, proline-rich Gla proteins (PRGP1 and PRGP2), were identified by sequence homology searches and are believed to be membrane proteins (5).A conserved eighteen-amino acid sequence essential for substrate recognition is found in all vitamin K-dependent proteins and was first identified by Pan and Price (6) based on sequence comparisons of the blood and bone vitamin K-dependent proteins. The conserved region is present as a propeptide sequence amino-terminal to the highly conserved Gla domains of the vitamin K-dependent blood proteins and is proteolytically removed to form the mature protein. With bone Gla protein, this sequence is also present as a propeptide amino-terminal to the mature form of the protein, whereas with matrix Gla protein the vitamin K-dependent propeptide-like sequence is part of the mature form of the protein (7). Confirmation of the importance of the propeptide sequence in carboxylation is demonstrated by experiments where deletion of the propeptide abrogates carboxylation of factor IX or protein C expressed in cell culture (8, 9). In addition, mutagenesis studies have identified a number of highly conserved amino acids (e.g. Phe Ϫ16, Ala Ϫ10, Leu Ϫ6) as well as les...

Vitamin K epoxide (or oxido) reductase (VKOR) is the target of warfarin and provides vitamin K hydroquinone for the carboxylation of select glutamic acid residues of the vitamin K-dependent proteins which are important for coagulation, signaling, and bone metabolism. It has been known for at least 20 years that cysteines are required for VKOR function. To investigate their importance, we mutated each of the seven cysteines in VKOR. In addition, we made VKOR with both C43 and C51 mutated to alanine (C43A/C51A), as well as a VKOR with residues C43-C51 deleted. Each mutated enzyme was purified and characterized. We report here that C132 and C135 of the CXXC motif are essential for both the conversion of vitamin K epoxide to vitamin K and the conversion of vitamin K to vitamin K hydroquinone. Surprisingly, conserved cysteines, 43 and 51, appear not to be important for either reaction. For the in vitro reaction driven by dithiothreitol, the 43-51 deletion mutation retained 85% and C43A/C51A 112% of the wild-type activity. The facile purification of the nine different mutations reported here illustrates the ease and reproducibility of VKOR purification by the method reported in our recent publication [Chu, P.-H., Huang, T.-Y., Williams, J., and Stafford, D. W. (2006) Proc. Natl. Acad. Sci. U S A. 103, 19308-19313].

Vitamin K epoxide reductase (VKOR) is essential for the production of reduced vitamin K that is required for modification of vitamin K-dependent proteins. Three- and four-transmembrane domain (TMD) topology models have been proposed for VKOR. They are based on in vitro glycosylation mapping of the human enzyme and the crystal structure of a bacterial (Synechococcus) homologue, respectively. These two models place the functionally disputed conserved loop cysteines, Cys-43 and Cys-51, on different sides of the endoplasmic reticulum (ER) membrane. In this study, we fused green fluorescent protein to the N or C terminus of human VKOR, expressed these fusions in HEK293 cells, and examined their topologies by fluorescence protease protection assays. Our results show that the N terminus of VKOR resides in the ER lumen, whereas its C terminus is in the cytoplasm. Selective modification of cysteines by polyethylene glycol maleimide confirms the cytoplasmic location of the conserved loop cysteines. Both results support a three-TMD model of VKOR. Interestingly, human VKOR can be changed to a four-TMD molecule by mutating the charged residues flanking the first TMD. Cell-based activity assays show that this four-TMD molecule is fully active. Furthermore, the conserved loop cysteines, which are essential for intramolecular electron transfer in the bacterial VKOR homologue, are not required for human VKOR whether they are located in the cytoplasm (three-TMD molecule) or the ER lumen (four-TMD molecule). Our results confirm that human VKOR is a three-TMD protein. Moreover, the conserved loop cysteines apparently play different roles in human VKOR and in its bacterial homologues.

The majority of cases of human hemophilia B are the result of missense mutations in the coagulation factor IX gene and defective circulating factor IX is detectable in most patients. The available mouse factor IX knockout models of hemophilia B (FIXKO mouse) reproduce the bleeding phenotype of human hemophilia B, but because the models produce no factor IX they fail to reproduce the dominant human phenotype. We have created a human factor IX mouse model of hemophilia B (R333Q-hFIX mouse) by homologous recombination in embryonic stem cells. The mouse expresses no mouse factor IX, but instead expresses a missense mutant human factor IX from the mouse FIX promoter. Mutant human factor IX mRNA transcript and circulating human factor IX are detectable throughout development, but factor IX activity is less than 1% and the mouse exhibits the hemophilic phenotype. When R333Q-hFIX mice were challenged by intramuscular injection of adeno-associated virus expressing human factor IX, factor IX expression without the development of antibodies was observed. In contrast, given the same treatment, FIXKO mice consistently develop antibodies. Our R333Q-hFIX mice strain will complement the FIXKO mice for studying factor IX circulating kinetics and gene therapy. (Blood. 2004;104:1733-1739)

Residue K5 in factor IX ␥-carboxyglutamic acid (Gla) domain participates in binding endothelial cells/collagen IV. We injected recombinant factor IX containing mutations at residue 5 (K5A, K5R) into factor IX-deficient mice and compared their behavior with that of wild-type factor IX. The plasma concentration of factor IX that binds to endothelial cells/collagen IV (recombinant wild type and K5R) was consistently lower than that of the one that does not bind (K5A). Mice treated with wild type or K5R had 79% of the injected factor IX in the liver after 2 minutes, whereas 17% remained in circulation. In mice injected with K5A, 59% of the injected factor IX was found in liver and 31% was found in plasma. When we blocked the liver circulation before factor IX injection, 74% of K5A and 64% of K5R remained in the blood. When we treated the mouse with EDTA after injecting exogenous factor IX, the blood levels of factor IX that bind to endothelial cells/collagen IV increased, presumably because of release from endothelial cell/collagen IV binding sites. In contrast, the levels of the mutants that do not bind were unaffected by EDTA. In immunohistochemical studies, factor IX appears on the endothelial surfaces of mouse arteries after factor IX injection and of human arteries from surgical specimens. Thus, we have demonstrated that factor IX binds in vivo to endothelial cell-collagen IV surfaces. Our results suggest that factor IX Gla-domain mediated binding to endothelial cells/ collagen IV plays a role in controlling factor IX concentration in the blood. IntroductionFactor IX is the zymogen of a serine protease involved in blood coagulation. Activated factor IX (factor IXa) contains 4 identifiable structural domains. Starting from the N-terminus, these are the ␥-carboxyglutamic acid (Gla) domain, the 2 epidermal growth factor-like domains, and the proteolytic domain. Factors IX and IXa bind to a specific site on the surface of cultured vascular endothelial cells. The binding is calcium-dependent, saturable, and reversible in vitro. [1][2][3] Our previous studies on cultured endothelial cells demonstrate that the binding regions in factor IX are in the Gla domain. 4 Furthermore, mutations within the Gla domain, including lysine 5 to alanine (K5A) and valine 10 to lysine (V10K), produced factor IX that did not have measurable affinity for endothelial cells but retained normal clotting activity. In contrast, changing lysine 5 to arginine (K5R) increased factor IX affinity 3-fold for cultured bovine vascular endothelial cells. 5 Cheung et al 6 demonstrated that collagen IV was a strong candidate for the factor IX endothelial cell-associated binding site. Further, results from atomic force scanning microscopy indicate that factor IX molecules bind specifically to 2 sites on collagen IV, 98 and 50 nm from its C-terminus. 7 Although the binding of factor IX to endothelial cells/collagen IV is well documented, little is known about the physiologic significance of binding. The hemophilia B mouse produced using gene-targeting te...

Key Points• CRISPR-Cas9-mediated GGCX knockout cell-based assay clarifies the correlation between GGCX genotypes and their clinical phenotypes.• A GGCX mutation decreases clotting factor carboxylation and abolishes MGP carboxylation, causing 2 distinct clinical phenotypes.Vitamin K-dependent coagulation factors deficiency is a bleeding disorder mainly associated with mutations in g-glutamyl carboxylase (GGCX) that often has fatal outcomes. Some patients with nonbleeding syndromes linked to GGCX mutations, however, show no coagulation abnormalities. The correlation between GGCX genotypes and their clinical phenotypes has been previously unknown. Here we report the identification and characterization of novel GGCX mutations in a patient with both severe cerebral bleeding disorder and comorbid Keutel syndrome, a nonbleeding malady caused by functional defects of matrix g-carboxyglutamate protein (MGP). To characterize GGCX mutants in a cellular milieu, we established a cell-based assay by stably expressing 2 reporter proteins (a chimeric coagulation factor and MGP) in HEK293 cells. The endogenous GGCX gene in these cells was knocked out by CRISPR-Cas9-mediated genome editing. Our results show that, compared with wild-type GGCX, the patient's GGCX D153G mutant significantly decreased coagulation factor carboxylation and abolished MGP carboxylation at the physiological concentration of vitamin K. Higher vitamin K concentrations can restore up to 60% of coagulation factor carboxylation but do not ameliorate MGP carboxylation. These results are consistent with the clinical results obtained from the patient treated with vitamin K, suggesting that the D153G alteration in GGCX is the causative mutation for both the bleeding and nonbleeding disorders in our patient. These findings provide the first evidence of a GGCX mutation resulting in 2 distinct clinical phenotypes; the established cell-based assay provides a powerful tool for studying the clinical consequences of naturally occurring GGCX mutations in vivo. (Blood. 2016;127(15):1847-1855 Introduction Vitamin K-dependent carboxylation is a posttranslational modification that converts specific glutamate (Glu) residues to g-carboxyglutamate (Gla) residues in vitamin K-dependent proteins. It is required for the functioning of numerous vitamin K-dependent proteins involved in a broad range of biological functions, including blood coagulation. Carboxylation is catalyzed by the enzyme g-glutamyl carboxylase (GGCX), which uses a reduced form of vitamin K (KH 2 ) as a cofactor. Concomitant with each glutamate modification, KH 2 is oxidized to vitamin K epoxide (KO). Because humans cannot synthesize vitamin K, KO must be converted back to KH 2 by the enzymes KO reductase and the as-yet unknown vitamin K reductase in a pathway known as the vitamin K cycle. 1Defects of vitamin K-dependent carboxylation have long been known to cause bleeding disorders, referred to as combined vitamin Kdependent coagulation factors deficiency (VKCFD). Patients with VKCFD have decreased levels of mul...