A new method for isolation of high molecular weight DNA from eukaryotes is presented. This procedure allows preparation of DNA from a variety of tissues such as calf thymus or human placenta and from cells which were more difficult to lyse until now (e.g. Crypthecodinium cuhnii, a dinoflagellate). The DNA obtained in such a way has an average molecular weight of about 200 X 10(6) d and contains very few, if any, single strand breaks.

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.

Hemophilia B, or factor IX deficiency, is an X-linked recessive disorder occurring in about 1 in 25,000 males. Affected individuals are at risk for spontaneous bleeding into many organs; treatment mainly consists of the transfusion of clotting factor concentrates prepared from human blood or recombinant sources after bleeding has started. Small- and large-animal models have been developed and/or characterized that closely mimic the human disease state. As a preclinical model for gene therapy, recombinant adeno-associated viral vectors containing the human or canine factor IX cDNAs were infused into the livers of murine and canine models of hemophilia B, respectively. There was no associated toxicity with infusion in either animal model. Constitutive expression of factor IX was observed, which resulted in the correction of the bleeding disorder over a period of over 17 months in mice. Mice with a steady-state concentration of 25% of the normal human level of factor IX had normal coagulation. In hemophilic dogs, a dose of rAAV that was approximately 1/10 per body weight that given to mice resulted in 1% of normal canine factor IX levels, the absence of inhibitors, and a sustained partial correction of the coagulation defect for at least 8 months.

Summary.  Post‐translational modification of glutamate to gamma carboxyl glutamate is required for the activity of vitamin K‐dependent proteins. Carboxylation is accomplished by the enzyme gamma glutamyl carboxylase (GGCX) which requires the propeptide‐containing substrate and three co‐substrates: reduced vitamin K, CO2, and O2. Most propeptides bind tightly to GGCX and all of the Glu residues that will be modified are modified during one binding event. Complete carboxylation is thus dependent upon the rate of carboxylation and the dissociation rate constant of the substrate from the GGCX enzyme. If the propeptide is released before carboxylation is complete, partially carboxylated vitamin K‐dependent proteins are produced. The rate of carboxylation is mainly controlled by the level of reduced vitamin K available for the reactions while the dissociation rate constant is dependent upon both the propeptide and the Gla domain of the substrate. In addition, there are allosteric effects that increase the rate of dissociation of the fully carboxylated substrates. Carboxylation requires the abstraction of a proton from the 4‐carbon of glutamate by reduced vitamin K and results in the conversion of vitamin K to vitamin K epoxide. The vitamin K epoxide must be recycled to vitamin K before it can be reused, a reaction catalyzed by the enzyme vitamin K epoxide reductase (VKOR). The gene for VKOR has recently been identified but the enzyme itself has not been purified to homogeneity. It appears, however, that most of the variability observed in patients response to warfarin may be attributed to variability in the VKOR gene.

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...