SMART MODE:

NORMAL GENOMIC

• Wilson CR, Sauer JM, Carlson GP, Wallin R, Ward MP, Hooser SB
• Species comparison of vitamin K1 2,3-epoxide reductase activity in vitro: kinetics and warfarin inhibition.
• Toxicology. 2003; 189: 191-8
• Display abstract

• A comparative study of vitamin K(1) 2,3-epoxide reductase (VKOR) activity in vitro was conducted across species. The apparent kinetic constants K(m app), V(max), and Cl(int app) were determined in bovine, canine, equine, human, murine, ovine, porcine, and rat hepatic microsomes. In addition to these enzyme kinetic constants, the IC(50) of warfarin for VKOR was determined in human, murine, porcine, and rat hepatic microsomes. Interspecies differences were observed when comparing the K(m app) (range, 2.41-6.46 microM), V(max) (range, 19.5-85.7 nmol/mg/min), and Cl(int app) (range, 8.2-18.4 ml/mg/min) values. Comparison of the IC(50) values of warfarin, across the four species tested, revealed a significant species difference between murine microsomes (0.17 microM) and rat microsomes (0.07 microM). Overall, this study indicates that there are interspecies differences regarding the in vitro reduction of vitamin K(1) 2,3-epoxide by the warfarin-sensitive enzyme vitamin K(1) 2,3-epoxide reductase. Significant differences between the IC(50) values of murine and rat microsomes suggest differences in the susceptibility of these species to warfarin.

• Markussen MD, Heiberg AC, Nielsen R, Leirs H
• Vitamin K requirement in Danish anticoagulant-resistant Norway rats (Rattus norvegicus).
• Pest Manag Sci. 2003; 59: 913-20
• Display abstract

• Resistance to warfarin has been connected to an increase in dietary requirement for vitamin K in British strains of the Norway rat, Rattus norvegicus (Berk). This study examines vitamin K requirement of Danish anticoagulant-resistant Norway rats using a vitamin K deficient feeding test. Wild bromadiolone-resistant rats sampled from different localities in Denmark and rats from bromadiolone-resistant and susceptible laboratory strains were fed on a vitamin K deficient diet over a maximum period of 15 days. Development of vitamin K deficiency, measured as reduced blood-clotting capacity, took place in 43% of the Danish resistant rats and was independent of sex, treatment with supplementary vitamin K3 and sampling locality. Development of deficiency was slower for resistant rats that were supplemented with vitamin K3 prior to the feeding test, suggesting storage of the vitamin K in a vitamin body pool. Intraperitoneal administration of vitamin K1 revealed that 80 microg vitamin K1 kg(-1) bodyweight was sufficient to restore normal blood clotting activity in deficient rats, while 60 microg vitamin K1 kg(-1) bodyweight was insufficient. We conclude that vitamin K requirement is moderately increased in Danish homozygous resistant rats whereas heterozygous resistant rats only have a minor increase in vitamin K requirement compared with susceptible rats. We found no indication of different resistance types being present in our test material since vitamin K requirement was not different between rats from separate sampling localities.

• Kohn MH, Pelz HJ, Wayne RK
• Locus-specific genetic differentiation at Rw among warfarin-resistant rat (Rattus norvegicus) populations.
• Genetics. 2003; 164: 1055-70
• Display abstract

• Populations may diverge at fitness-related genes as a result of adaptation to local conditions. The ability to detect this divergence by marker-based genomic scans depends on the relative magnitudes of selection, recombination, and migration. We survey rat (Rattus norvegicus) populations to assess the effect that local selection with anticoagulant rodenticides has had on microsatellite marker variation and differentiation at the warfarin resistance gene (Rw) relative to the effect on the genomic background. Initially, using a small sample of 16 rats, we demonstrate tight linkage of microsatellite D1Rat219 to Rw by association mapping of genotypes expressing an anticoagulant-rodenticide-insensitive vitamin K 2,3-epoxide reductase (VKOR). Then, using allele frequencies at D1Rat219, we show that predicted and observed resistance levels in 27 populations correspond, suggesting intense and recent selection for resistance. A contrast of F(ST) values between D1Rat219 and the genomic background revealed that rodenticide selection has overwhelmed drift-mediated population structure only at Rw. A case-controlled design distinguished these locus-specific effects of selection at Rw from background levels of differentiation more effectively than a population-controlled approach. Our results support the notion that an analysis of locus-specific population genetic structure may assist the discovery and mapping of novel candidate loci that are the object of selection or may provide supporting evidence for previously identified loci.

• Fregin A, Rost S, Wolz W, Krebsova A, Muller CR, Oldenburg J
• Homozygosity mapping of a second gene locus for hereditary combined deficiency of vitamin K-dependent clotting factors to the centromeric region of chromosome 16.
• Blood. 2002; 100: 3229-32
• Display abstract

• Familial multiple coagulation factor deficiency (FMFD) of factors II, VII, IX, X, protein C, and protein S is a very rare bleeding disorder with autosomal recessive inheritance. The phenotypic presentation is variable with respect to the residual activities of the affected proteins, its response to oral administration of vitamin K, and to the involvement of skeletal abnormalities. The disease may result either from a defective resorption/transport of vitamin K to the liver, or from a mutation in one of the genes encoding gamma-carboxylase or other proteins of the vitamin K cycle. We have recently presented clinical details of a Lebanese family and a German family with 10 and 4 individuals, respectively, where we proposed autosomal recessive inheritance of the FMFD phenotype. Biochemical investigations of vitamin K components in patients' serum showed a significantly increased level of vitamin K epoxide, thus suggesting a defect in one of the subunits of the vitamin K 2,3-epoxide reductase (VKOR) complex. We now have performed a genome-wide linkage analysis and found significant linkage of FMFD to chromosome 16. A total maximum 2-point LOD score of 3.4 at theta = 0 was obtained in the interval between markers D16S3131 on 16p12 and D16S419 on 16q21. In both families, patients were autozygous for 26 and 28 markers, respectively, in an interval of 3 centimorgans (cM). Assuming that FMFD and warfarin resistance are allelic, conserved synteny between human and mouse linkage groups would restrict the candidate gene interval to the centromeric region of the short arm of chromosome 16.

• Wallin R, Sane DC, Hutson SM
• Vitamin K 2,3-epoxide reductase and the vitamin K-dependent gamma-carboxylation system.
• Thromb Res. 2002; 108: 221-6
• Display abstract

• Vitamin K is an essential cofactor for post translational gamma-carboxylation of vitamin K-dependent coagulation factors. The modification is carried out by a system of integral proteins of the endoplasmic reticulum (ER) membrane where the warfarin sensitive vitamin K 2,3-epoxide reductase (VKOR) produces the reduced hydroquinone form of vitamin K (vit.KH(2)) needed by the gamma-carboxylase as the active cofactor. Currently, we have only limited knowledge about how the system functions at the molecular level. VKOR harbors a thiol red/ox center that is essential for electron transfer leading to vitamin K reduction. Reduction of this center with hydrophilic and hydrophobic trialkylphosphines shows that it is located in a hydrophobic environment which must be accessible by an as yet unidentified in vivo reductant of the center. Furthermore, we have addressed the question of whether VKOR or the gamma-carboxylase is the rate-limiting step in the vitamin K-dependent gamma-caboxylation system. A detailed kinetic analysis of an in vitro preparation of the system was undertaken in which gamma-carboxylation of the carboxylase peptide substrate FLEEL was measured as the gamma-carboxylation capacity of the system. Adding VKOR to the test system increased the gamma-carboxylation capacity of the system suggesting that VKOR is the rate-limiting step in the system. This finding puts VKOR in a central position to regulate biosynthesis of biologically active vitamin K-dependent proteins.

• Begent LA, Hill AP, Steventon GB, Hutt AJ, Pallister CJ, Cowell DC
• Characterization and purification of the vitamin K1 2,3 epoxide reductases system from rat liver.
• J Pharm Pharmacol. 2001; 53: 481-6
• Display abstract

• The enzyme vitamin K1 2,3 epoxide reductase is responsible for converting vitamin K1 2,3 epoxide to vitamin K1 quinone thus completing the vitamin K cycle. The enzyme is also the target of inhibition by the oral anticoagulant, R,S-warfarin. Purification of this protein would enable the interaction of the inhibitor with its target to be elucidated. To date a single protein possessing vitamin K1 2,3 epoxide reductase activity and binding R,S-warfarin has yet to be purified to homogeneity, but recent studies have indicated that the enzyme is in fact at least two interacting proteins. We report on the attempted purification of the vitamin K1 2,3 epoxide reductase complex from rat liver microsomes by ion exchange and size exclusion chromatography techniques. The intact system consisted of a warfarin-binding factor, which possessed no vitamin K1 2,3 epoxide reductase activity and a catalytic protein. This catalytic protein was purified 327-fold and was insensitive to R,S-warfarin inhibition at concentrations up to 5 mM. The addition of the S-200 size exclusion chromatography fraction containing the inhibitor-binding factor resulted in the return of R,S-warfarin inhibition. Thus, to function normally, the rat liver endoplasmic reticulum vitamin K1 2,3 epoxide reductase system requires the association of two components, one with catalytic activity for the conversion of the epoxide to the quinone and the second, the inhibitor binding factor. This latter enzyme forms the thiol-disulphide redox centre that in the oxidized form binds R,S-warfarin.

• Miyake N, Hoshi K, Sano Y, Kikuchi K, Tadano K, Koshihara Y
• 1,25-Dihydroxyvitamin D3 promotes vitamin K2 metabolism in human osteoblasts.
• Osteoporos Int. 2001; 12: 680-7
• Display abstract

• It has been reported that vitamin K2 (menaquinone-4) promoted 1,25-dihydroxyvitamin D3 (1,25(OH)2D3)-induced mineralization and enhanced gamma-carboxyglutamic acid (Gla)-containing osteocalcin accumulation in cultured human osteoblasts. In the present study, we investigated whether menaquinone-4 (MK-4) was metabolized in human osteoblasts to act as a cofactor of gamma-glutamyl carboxylase. Both conversions of MK-4 to MK-4 2,3-epoxide (epoxide) and epoxide to MK-4 were observed in cell extracts of cultured human osteoblasts. The effect of 1,25(OH)2D3 and warfarin on the vitamin K cycle to cultured osteoblasts were examined. With the addition of 1 nM 1,25(OH)2D3 or 25 microM warfarin in cultured osteoblasts, the yield of epoxide from MK-4 increased. However, the conversion of epoxide to MK-4 was strongly inhibited by the addition of warfarin (2.5-25 microM), whereas it was almost not inhibited by 1,25(OH)2D3 (0.1-10 nM). To clarify the mechanism for this phenomenon, a cell-free assay system was studied. Osteoblast microsomes were incubated with 10 microM epoxide in the presence or absence of warfarin and 1,25(OH)2D3. Epoxide reductase, one of the enzymes in the vitamin K cycle was strongly inhibited by warfarin (2.5-25 microM), whereas it was not affected by 1,25(OH)2D3 (0.1-1 nM). Moreover, there was no effect of pretreatment of osteoblasts with 1 nM 1,25(OH)2D3 on the activity of epoxide reductase. However, the activity of epoxidase, that is the gamma-glutamyl carboxylase was induced by the pretreatment of osteoblasts with 1 nM 1,25(OH)2D3. In the present study, it was demonstrated that the vitamin K metabolic cycle functions in human osteoblasts as well as in the liver, the post-translational mechanism, by which 1,25(OH)2D3 caused mineralization in cooperation with vitamin K2 was clarified.

• Wallin R, Hutson SM, Cain D, Sweatt A, Sane DC
• A molecular mechanism for genetic warfarin resistance in the rat.
• FASEB J. 2001; 15: 2542-4
• Display abstract

• Warfarin targets vitamin K 2,3-epoxide reductase (VKOR), the enzyme that produces reduced vitamin K, a required cofactor for g-carboxylation of vitamin K-dependent proteins. To identify VKOR, we used 4'-azido-warfarin-3H-alcohol as an affinity label. When added to a partially purified preparation of VKOR, two proteins were identified by mass spectrometry as calumenin and cytochrome B5. Rat calumenin was cloned and sequenced and the recombinant protein was produced. When added to an in vitro test system, the 47 kDa recombinant protein was found to inhibit VKOR activity and to protect the enzyme from warfarin inhibition. Calumenin was also shown to inhibit the overall activity of the complete vitamin K-dependent g-carboxylation system. The results were repeated in COS-1 cells overexpressing recombinant calumenin. By comparing calumenin mRNA levels in various tissues from normal rats and warfarin-resistant rats, only the livers from resistant rats were different from normal rats by showing increased levels. Partially purified VKOR from resistant and normal rat livers showed no differences in Km-values, specific activity, and sensitivity to warfarin. A novel model for genetic warfarin resistance in the rat is proposed, whereby the concentration of calumenin in liver determines resistance.

• Hanumanthaiah R, Thankavel B, Day K, Gregory M, Jagadeeswaran P
• Developmental expression of vitamin K-dependent gamma-carboxylase activity in zebrafish embryos: effect of warfarin.
• Blood Cells Mol Dis. 2001; 27: 992-9
• Display abstract

• Vitamin K-dependent gamma-carboxylation is an essential posttranslational modification required for the functional activity of coagulation proteins such as factors VII, IX, X, and prothrombin. Warfarin, an inhibitor of vitamin K-dependent gamma-carboxylation, was used in earlier work on adult zebrafish to provide evidence for the presence of vitamin K-dependent carboxylase in zebrafish. Here we demonstrate the presence of vitamin K-dependent carboxylase activity in zebrafish by directly assaying the microsomal fraction prepared from adult, unfertilized eggs, and embryos from different developmental stages. Gamma-carboxylase activity was detected both before and after fertilization of embryos and the activity levels remained relatively constant from 6 h postfertilization (hpf) through other advanced stages of development. The expression of activity in the early embryos (0-6 hpf) may be due to the presence of maternal protein since the activity was detected even in the unfertilized eggs. Gamma-carboxylase activity in the eggs as well as early embryos suggested that vitamin K-dependent carboxylase is important throughout development. The detection of vitamin K-dependent carboxylase mRNA by RT-PCR and inhibitor studies using warfarin confirmed these activity results. Further, these studies provide a basis for selecting warfarin-resistant zebrafish mutants in order to find genes regulating gamma-carboxylase activity including the yet unidentified vitamin K-epoxide reductase.

• Kohn MH, Pelz HJ
• A gene-anchored map position of the rat warfarin-resistance locus, Rw, and its orthologs in mice and humans.
• Blood. 2000; 96: 1996-8
• Display abstract

• The locus underlying hereditary resistance to the anticoagulant warfarin (symbol in the rat, Rw) was placed in relation to 8 positionally mapped gene-anchored microsatellite loci whose positions were known in the genome maps of the rat, mouse, and human. Rw segregated with the markers Myl2 (zero recombinants) and Itgam, Il4r, and Fgf2r (one recombinant each) during linkage analysis in a congenic warfarin- and bromadiolone-resistant laboratory strain of rats. Comparative ortholog mapping between rat, mouse, and human placed Rw onto mouse chromosome 7 at about 60 to 63 cM and onto one of the human chromosomes 10q25.3-26, 12q23-q24.3, and 16p13.1-p11. (Blood. 2000;96:1996-1998)

• Oldenburg J et al.
• Congenital deficiency of vitamin K dependent coagulation factors in two families presents as a genetic defect of the vitamin K-epoxide-reductase-complex.
• Thromb Haemost. 2000; 84: 937-41
• Display abstract

• Hereditary combined deficiency of the vitamin K dependent coagulation factors is a rare bleeding disorder. To date, only eleven families have been reported in the literature. The phenotype varies considerably with respect to bleeding tendency, response to vitamin K substitution and the presence of skeletal abnormalities, suggesting genetic heterogeneity. In only two of the reported families the cause of the disease has been elucidated as either a defect in the gamma-carboxylase enzyme (1) or in a protein of the vitamin K 2,3-epoxide reductase (VKOR) complex (2). Here we present a detailed phenotypic description of two new families with an autosomal recessive deficiency of all vitamin K dependent coagulation factors. In both families offspring had experienced severe or even fatal perinatal intracerebral haemorrhage. The affected children exhibit a mild deficiency of the vitamin K dependent coagulation factors that could be completely corrected by oral substitution of vitamin K. Sequencing and haplotype analysis excluded a defect within the gamma-carboxylase gene. The finding of highly increased amounts of vitamin K epoxide in all affected members of both families indicated a defect in a protein of the VKOR-multienzyme-complex. Further genetic analysis of such families will provide the basis for a more detailed understanding of the structure-function relation of the enzymes involved in vitamin K metabolism.

• Guenthner TM, Cai D, Wallin R
• Co-purification of microsomal epoxide hydrolase with the warfarin-sensitive vitamin K1 oxide reductase of the vitamin K cycle.
• Biochem Pharmacol. 1998; 55: 169-75
• Display abstract

• Vitamin K1 oxide reductase activity has been partially purified from rat liver microsomes. A three-step procedure produced a preparation in which warfarin-sensitive vitamin K1 oxide reductase activity was 118-fold enriched over the activity in intact rat liver microsomes. A major component of the multi-protein mixture was identified as a 50 kDa protein that strongly cross-reacts with antiserum prepared against homogeneous rat liver microsomal epoxide hydrolase. The reductase preparation also had a high level or epoxide hydrolase activity against two xenobiotic epoxide substrates. The K(m) values for hydrolysis by the reductase preparation were similar to those for homogeneous microsomal epoxide hydrolase itself, and the specific hydrolase activities of the reductase preparation were 25-35% of the specific activities measured for the homogeneous hydrolase preparation. Antibodies prepared against homogeneous microsomal epoxide hydrolase inhibited up to 80% of reductase activity of the reductase preparation. Homogeneous microsomal epoxide hydrolase had no vitamin K1 oxide reductase activity. This evidence suggests that microsomal epoxide hydrolase, or a protein that is very similar to it, is a major functional component of a multi-protein complex that is responsible for vitamin K1 oxide reduction in rat liver microsomes.

• Brenner B, Sanchez-Vega B, Wu SM, Lanir N, Stafford DW, Solera J
• A missense mutation in gamma-glutamyl carboxylase gene causes combined deficiency of all vitamin K-dependent blood coagulation factors.
• Blood. 1998; 92: 4554-9
• Display abstract

• To identify potential mutations in the gamma-glutamyl carboxylase gene, the sequence of all exons and intron/exon borders was determined in 4 patients from a consanguineous kindred with combined deficiency of all vitamin K-dependent procoagulants and anticoagulants and results were compared with normal genomic sequence. All 4 patients were homozygous for a point mutation in exon 9 that resulted in the conversion of an arginine codon (CTG) to leucine codon (CGG) at residue 394. Screening of this mutation based on introduction of Alu I site in amplified fragment from normal allele but not from the mutated allele showed that 13 asymptomatic members of the kindred were heterozygous for the mutation. The mutation was not found in 340 unrelated normal chromosomes. The segregation pattern of the mutation which is the first reported in the gamma-glutamyl carboxylase gene fits perfectly with phenotype of the disorder and confirms the suggested autosomal recessive pattern of inheritance of combined deficiency of all vitamin K-dependent procoagulants and anticoagulants in this kindred. The mutated carboxylase protein expressed in Drosophila cells was stable but demonstrated threefold reduced activity compared with WT carboxylase, confirming that the L394R mutation results in a defective carboxylase.

• Cain D, Hutson SM, Wallin R
• Warfarin resistance is associated with a protein component of the vitamin K 2,3-epoxide reductase enzyme complex in rat liver.
• Thromb Haemost. 1998; 80: 128-33
• Display abstract

• Warfarin, the most used drug in the world in long-term anticoagulation prophylaxis, targets the vitamin K 2,3-epoxide reductase (VKOR) of the vitamin K cycle in liver. Recently, the enzyme has been identified as a multicomponent lipid-protein enzyme system in the endoplasmic reticulum (ER) membrane (17). As the first step towards understanding genetic resistance to warfarin, we present in this paper data on VKOR from normal and a strain of warfarin resistant laboratory rats maintained in the United States. Metal induced in vitro assembly of the enzyme complex demonstrates that the glutathione-S-transferase (GST) enzyme component of the complex loses its GST activity upon formation of VKOR. Less VKOR activity is measured upon assembly of the complex from warfarin resistant rats. The GST activity measured in warfarin resistant rats, before assembly of the complex, is 10-fold less sensitive to warfarin inhibition than the GST activity measured in normal rats. Microsomal epoxide hydrolase (mEH) is the second component of VKOR. When incubated with the components of VKOR before assembly of the complex, antibodies raised against mEH prevented formation of the enzyme complex. Sequencing of mEH cDNAs from normal and warfarin resistant rats revealed identical sequences. The data suggest that the mutation responsible for genetic warfarin resistance is associated with the GST component of VKOR.

• Cain D, Hutson SM, Wallin R
• Assembly of the warfarin-sensitive vitamin K 2,3-epoxide reductase enzyme complex in the endoplasmic reticulum membrane.
• J Biol Chem. 1997; 272: 29068-75
• Display abstract

• gamma-Carboxylation of vitamin K-dependent proteins requires a functional vitamin K cycle to produce the active vitamin K cofactor for the gamma-carboxylase which posttranslationally modifies precursors of these proteins to contain gamma-carboxyglutamic acid residues. The warfarin-sensitive enzyme vitamin K epoxide reductase (VKOR) of the cycle reduces vitamin K 2,3-epoxide to the active vitamin K hydroquinone cofactor. Because of the importance of warfarin as an anticoagulant in prophylactic medicine and as a poison in rodent pest control, numerous attempts have been made to understand the molecular mechanism underlying warfarin-sensitive vitamin K 2,3-epoxide reduction. In search for protein components that could be involved in this reaction we designed an in vitro gamma-carboxylation test system where the warfarin-sensitive VKOR produces the cofactor for the gamma-carboxylase. Dissection of this system by chromatographic techniques has identified a member(s) of the glutathione S-transferase gene family as one component of the VKOR enzyme complex in the endoplasmic reticulum membrane. The affinity-purified glutathione S-transferase(s) was sensitive to warfarin but lost its warfarin sensitivity and glutathione S-transferase activity upon association with lipids in the presence of Mn2+ or Ca2+. In the gamma-carboxylation test system, loss of warfarin-sensitive glutathione S-transferase activity coincided with formation of the VKOR enzyme complex. It is proposed that formation of VKOR in the endoplasmic reticulum membrane resembles formation of the lipoxygenase enzyme complex where the glutathione S-transferase-related FLAP protein binds cytosolic lipoxygenase to form a membrane enzyme complex.

• Wallin R, Guenthner TM
• Purification of warfarin-sensitive vitamin K epoxide reductase.
• Methods Enzymol. 1997; 282: 395-408

• Kerr JS et al.
• The characterization of potent novel warfarin analogs.
• Thromb Res. 1997; 88: 127-36
• Display abstract

• Racemic sodium warfarin, Coumadin, is widely used in the prevention of thromboembolic disease. The present study was undertaken to characterize three novel classes of warfarin analogs, and to compare them with the warfarin enantiomers. All three classes of compounds inhibit vitamin K epoxide reductase, the enzyme inhibited by racemic warfarin. The alcohol and the ester analogs have reduced protein binding compared with R-(+)-warfarin. The ester and the fluoro-derivatives have similar in vivo anticoagulant activity in the rat to that of S-(-)-warfarin. Thus, it is possible to synthesize novel warfarin analogs that differ from racemic warfarin or its enantiomers in certain selected properties.

• Liu Y, Nelson AN, Lipsky JJ
• Vitamin K-dependent carboxylase: mRNA distribution and effects of vitamin K-deficiency and warfarin treatment.
• Biochem Biophys Res Commun. 1996; 224: 549-54
• Display abstract

• A cDNA encoding vitamin K-dependent gamma-glutamyl carboxylase was cloned from a human Hep G2 cDNA library. The RNA transcript of the enzyme was found to be widely distributed in various human and rat tissues with liver showing the highest level. The carboxylase transcription in liver was not affected in rats treated with a single dose of warfarin (10 mg/kg) when measured up to 48 hours after the dose, though, at 12 hours, carboxylase activity measured in liver microsomes was elevated 5.4 fold over controls (p < 0.001). In rats fasted for 72 hours there was no affect on transcription in the liver while hepatic carboxylase activity increased 4.1 fold (p < 0.001). These data suggest that the increase in activity of the liver carboxylase in warfarin treated or fasted rats was not regulated by transcription but more likely was due to a posttranscriptional mechanism.

• Misenheimer TM, Lund M, Baker EM, Suttie JW
• Biochemical basis of warfarin and bromadiolone resistance in the house mouse, Mus musculus domesticus.
• Biochem Pharmacol. 1994; 47: 673-8
• Display abstract

• Danish mice (Mus musculus domesticus) genetically resistant to the anticoagulant action of two 4-hydroxycoumarins, warfarin and bromadiolone, were examined to determine their mechanism of resistance. The hepatic vitamin K epoxide reductase in the bromadiolone-resistant mice and in one phenotype of warfarin-resistant mice was highly insensitive to in vitro inhibition by warfarin and bromadiolone. The kinetic constants for the epoxide reductase from bromadiolone-resistant mice were also altered. The Vmax for this enzyme was decreased by 40%, and the Km for the reaction reductant, dithiothreitol, was 70% lower than that of normal mice. This phenotype of Danish resistant mice appears to have a resistance mechanism that is similar to that reported for a Welsh strain of warfarin-resistant rats. The other phenotype of Danish resistant mice had a hepatic epoxide reductase that was only slightly less sensitive to warfarin inhibition than normal. The mechanism of warfarin resistance in these mice is not apparent from the available data.

• Thijssen HH, Janssen YP, Vervoort LT
• Microsomal lipoamide reductase provides vitamin K epoxide reductase with reducing equivalents.
• Biochem J. 1994; 297: 277-80
• Display abstract

• This study was undertaken to search for the endogenous dithiol cofactor of the reductases of the vitamin K cycle. As a starting point, the redox-active lipophilic endogenous compounds lipoic acid and lipoamide were looked at. The study shows that microsomes contain NADH-dependent lipoamide reductase activity. Reduced lipoamide stimulates microsomal vitamin K epoxide reduction with kinetics comparable with those for the synthetic dithiol dithiothreitol (DTT). Reduced lipoic acid shows higher (4-fold) Km values. No reductase activity with lipoic acid was found to be present in microsomes or cytosol. The reduced-lipoamide-stimulated vitamin K epoxide reductase is as sensitive to warfarin and salicylate inhibition as is the DTT-stimulated one. Both vitamin K epoxide reductase and lipoamide reductase activity are recovered in the rough microsomes. NADH/lipoamide-stimulated vitamin K epoxide reduction is uncoupled by traces of Triton X-100, suggesting that microsomal lipoamide reductase and vitamin K epoxide reductase are associated. The results suggest that the vitamin K cycle obtains reducing equivalents from NADH through microsomal lipoamide reductase.

• Nakamura K et al.
• Anticoagulant effects of warfarin and kinetics of K vitamins in blood and feces.
• Artery. 1994; 21: 148-60
• Display abstract

• Patients (40 cases) were treated with daily dosage of warfarin of 2-7 mg after being undergone artificial valve replacements. Twenty one days after administration of warfarin, we examined the patients for kinetics of K vitamins and vitamin K-dependent coagulation factors in blood, and intestinal flora in feces, as well as the relationship between K vitamins and coagulation activity. The following results were obtained. (1) In warfarin-administered patients (Group B), blood levels of vitamin K1 and menaquinone-7, a vitamin K2 homologue, were similar to those in non-warfarin-administered patients. Therefore, administration of warfarin did not significantly decreased the levels. (2) In patients selected randomly from Group B (Group C), the vitamin K1 level in feces was higher than that in non-warfarin-administered patients. The menaquinone-7 level in feces was similar to that in non-warfarin-administered patients. For the total counts of bacteria and the detection rate of vitamin K2-producing bacteria, there was no significant difference between Group C and non-warfarin-administered patients. (3) The above mentioned results of (1) and (2) suggest that it is important for development of anticoagulant effects by warfarin to inhibit conversion from vitamin K1 to reduced vitamin K1, as well as to inhibit the reducing process from vitamin K1-epoxide to vitamin K1. (4) Vitamin K1-epoxide, a metabolite of vitamin K1, appeared in blood after administration of warfarin; there was a lower correlation between the blood level of vitamin K1-epoxide and the warfarin dosage. Further, PIVKA-II appeared in blood after administration of warfarin; there was a inverse lower correlation between the level of PIVKA-II and HPT, and between PIVIKA-II and TT. In conclusion, it has been clarified that vitamin K1-epoxide and PIVKA-II are useful parameters to evaluate anticoagulant effect of warfarin.

• Thijssen HH, Janssen YP
• Target-based warfarin pharmacokinetics in the rat: the link with the anticoagulant effect.
• J Pharmacol Exp Ther. 1994; 270: 554-8
• Display abstract

• Warfarin and congeners bind tightly to the target enzyme vitamin K epoxide reductase. In this study the impact of the target binding on the warfarin pharmacokinetics in plasma and liver of the rat was estimated. Furthermore, the effect of warfarin pharmacokinetics on the time course of inhibition of vitamin K epoxide reductase was followed to find a link with the effect in time on the vitamin K-dependent clotting factor synthesis. Biochemical parameters, drug tissue levels and plasma coagulation activity prothrombin time were followed in normal and phenobarbitone (PB)-treated rats for 12 days after a single dose of S-warfarin (0.5 mg/kg). Warfarin accumulated to saturation (40-50 pmol/mg of protein) in liver microsomes to remain prolongedly bound and the half-life of elimination exceeded 7 days. PB-treated rats were not found to differ in this respect. In parallel with the steady increase of microsomal-free warfarin binding sites the ex vivo vitamin K epoxide reductase activity recovered, from 10% control activity at t = 3 hr to 70% at t = 12 days. PB-treated rats showed a 1.8-fold higher recovery rate in free enzyme. Blood coagulation was affected during the time in which the ex vivo vitamin K epoxide reductase activity was less than 20% of normal activity. The ex vivo reductase inhibition showed a sigmoidal effect relationship for plasma S-warfarin. Emax appeared to be significantly less than 100% (95% confidence intervals; 83-91%).(ABSTRACT TRUNCATED AT 250 WORDS)

• Thijssen HH, Drittij-Reijnders MJ
• Vitamin K metabolism and vitamin K1 status in human liver samples: a search for inter-individual differences in warfarin sensitivity.
• Br J Haematol. 1993; 84: 681-5
• Display abstract

• Vitamin K-dependent parameters in human liver samples were investigated to find a clue to the inter-individual differences in sensitivity for oral anticoagulants. Vitamin K epoxide reductase and vitamin K-dependent carboxylase activity differed 2-3-fold between the samples. Microsomal warfarin binding correlated significantly with the reductase activity. Microsomal vitamin K epoxide reductase of the different samples showed equal sensitivity for warfarin inhibition, I50 about 0.1 microM. Vitamin K epoxide reductase activity stimulated by NADH/lipoamide and microsomal lipoamide dehydrogenase activity showed higher inter-subject variability than the reductase activity by itself. Liver vitamin K1 levels varied 4-5-fold. Total and liver microsomal vitamin K1 levels were correlated. One of the liver samples was obtained from a donor anticoagulated with phenprocoumon and additionally treated with vitamin K1. High levels of the vitamin and its epoxide were present. Phenprocoumon was essentially irreversibly bound to the microsomes. In general the results confirm inter-individual differences in the hepatic vitamin K-dependent systems; the differences as such were found to be small. However, as the various parameters can work synergistically in the same direction, they may well account for the wide dose range observed in oral anticoagulant therapy.

• Morris DP, Soute BA, Vermeer C, Stafford DW
• Characterization of the purified vitamin K-dependent gamma-glutamyl carboxylase.
• J Biol Chem. 1993; 268: 8735-42
• Display abstract

• Vitamin K-dependent carboxylase, purified from bovine liver, has properties similar to those reported for the carboxylase activity present in crude, solubilized microsomes. The purified carboxylase was found to possess the vitamin K epoxidase activity, believed to be essential for vitamin K-dependent carboxylation, but did not contain vitamin K epoxide reductase activity. Kinetic studies of the carboxylase done under defined conditions were complicated by the non-Michaelis-Menten kinetic behavior observed for reactions with two of the enzymes substrates, FLEEL and vitamin K1 hydroquinone. Initial rate experiments with the substrate FLEEL demonstrated behavior consistent with substrate inhibition and gave half-maximal activity at 1 mM FLEEL. Experiments with the substrate vitamin K1 hydroquinone also displayed non-Michaelis-Menten kinetics, as maximal activity was reached prematurely in relation to behavior at lower concentrations. Half-maximal activity was observed at 35 microM vitamin K1 hydroquinone. Initial rate experiments with varying NaH14CO3 concentration displayed Michaelis-Menten kinetics and gave a Km(app) of 0.29 mM. At cosubstrate concentrations chosen to obtain near-maximal activity, initial rate studies with varying NaH14CO3 concentration indicated a kcat near 1.0 s-1. Removal of the fourth substrate, oxygen, resulted in the loss of more than 99% of carboxylase activity. The sulfhydryl reagent N-ethylmaleimide inhibited carboxylase irreversibly, as did the anticoagulant warfarin.

• Ouafik LH, Mattei MG, Giraud P, Oliver C, Eipper BA, Mains RE
• Localization of the gene encoding peptidylglycine alpha-amidating monooxygenase (PAM) to human chromosome 5q14-5q21.
• Genomics. 1993; 18: 319-21
• Display abstract

• Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) is a multifunctional protein containing two enzymes that act sequentially to catalyze the alpha-amidation of neuroendocrine peptides. Southern blot analysis of human placental DNA demonstrated that PAM is encoded by a single gene. The chromosomal localization of the PAM gene was established using in situ hybridization. A 2.2-kb human PAM cDNA hybridized to human metaphase chromosomes revealed a significant clustering of silver grains over chromosome 5 bands q14-q21. The gene encoding another enzyme important in the post-translational processing of neuroendocrine precursors, prohormone convertase 1 (PC1), is localized in the same region (5q15-q21).

• Mosterd JJ, Thijssen HH
• The relationship between the vitamin K cycle inhibition and the plasma anticoagulant response at steady-state S-warfarin conditions in the rat.
• J Pharmacol Exp Ther. 1992; 260: 1081-5
• Display abstract

• The relationship between the inhibition of the vitamin K cycle and the inhibition of the vitamin K-dependent clotting factor synthesis was studied in the rat under a controlled rate of S-warfarin administration. Steady state of drug disposition was achieved from beyond day 2 and stable anticoagulation was achieved from beyond days 3 to 4. Doses up to 0.5 micrograms/kg/h were without effect, whereas 3 micrograms/kg/h reduced prothrombin complex activity to about 10%. Factor II and factor VII activity were equally suppressed as prothrombin complex activity. The concentration-effect relationship for steady-state S-warfarin plasma concentration and the inhibition of clotting factor synthesis revealed a steep sigmoidal response relationship (IC50 = 0.21 +/- 0.01 micrograms/ml, Hill slope = 2.07 +/- 0.3). Contrary to this, the target enzyme vitamin K1 2,3-epoxide reductase showed a dose-dependent response for the entire dose range. The sigmoidal effect relationship for plasma S-warfarin and enzyme inhibition showed a slope of 0.81 +/- 0.07 with IC50 = 16 +/- 1 ng/ml. The results demonstrate a reserve capacity for the coumarin-sensitive reductase; at least 70% of the hepatic vitamin K1 2,3-epoxide reductase activity has to be eliminated before the vitamin K-dependent carboxylation of the clotting factors objectively becomes compromised. In the study, the microsomal warfarin binding sites were compared with the vitamin K1 2,3-epoxide reductase activity, and a strict 1 to 1 relationship was found supporting the relationship between both.(ABSTRACT TRUNCATED AT 250 WORDS)

• Mosterd JJ, Thijssen HH
• The long-term effects of the rodenticide, brodifacoum, on blood coagulation and vitamin K metabolism in rats.
• Br J Pharmacol. 1991; 104: 531-5
• Display abstract

• 1. The long-term (30 days) effects of a single dose of brodifacoum (0.2 mg kg-1, orally) on blood clotting activity and on liver parameters of the vitamin K cycle were investigated in rats. Maximal effect on blood clotting activity was seen on day one. On day seven blood clotting activity had returned to normal. 2. Liver microsomal vitamin KO reductase activity was maximally suppressed (10% of control activity) on day one, steadily recovered to about 40% on day 15 to remain at that level. The same time course was seen for the number of microsomal warfarin binding sites. 3. The persistent inhibition of the vitamin K cycle was also verified in vivo; following vitamin K administration (10 mg kg-1, i.v.) on day 30, the brodifacoum-treated rats accumulated vitamin KO in the liver. 4. Although clotting factor synthesis was normal, brodifacoum-treated rats were highly sensitive to warfarin. 5. Brodifacoum rapidly accumulated in the liver until the saturation of the microsomal binding site. Brodifacoum binding to the target prevented its elimination from the liver; liver content on day 30 was not different from day 7. 6. The results show (1) an over capacity for the hepatocellular vitamin K cycle, (2) a dissociation of the vitamin K epoxidation and the vitamin K-dependent carboxylation, (3) the 'superwarfarin' rodenticides to be extremely persistent due to their binding to the target.

• Hallak HO, Wedlund PJ
• Vitamin K epoxide reductase activity and its inhibition by warfarin in young and old rats.
• Drug Metab Dispos. 1991; 19: 278-9

• Ross PJ, Shearer MJ, Diplock AT, Schey SA
• A fibroblast cell culture model to study vitamin K metabolism and the inhibition of vitamin K epoxide reductase by known and suspected antagonists.
• Br J Haematol. 1991; 77: 195-200
• Display abstract

• The metabolism and antagonism of vitamin K has been studied in cultured fibroblasts. Monolayers of 3T3 mouse fibroblasts (grown in the absence or presence of warfarin or other putative antagonists) were incubated for 24 h with [1',2'-3H2]phylloquinone (K1) or [1',2'-3H2]phylloquinone epoxide (K1O), the cells harvested and lipid extracts fractionated by high performance liquid chromatography. [3H]K1 was converted to [3H]K1O (about 20% of [3H] lipids) and to unidentified polar metabolites (30%). [3H]K1O was converted to [3H]K1 (3%) and to polar metabolites (50%). Cells grown with warfarin showed a marked increase in the [3H]K1O:K1 ratio and in the proportion of polar metabolites. The metabolic interconversion of K1 and K1O and inhibitory response to warfarin provide evidence for a fibroblast pathway analogous to the vitamin K-epoxide cycle in the liver. From the K1O:K1 ratios it was possible to grade the antagonism of vitamin K epoxide reductase activity by known and suspected inhibitors. Inhibitory ratios were seen for racemic warfarin down to 10(-8) M. S-warfarin was a more potent antagonist than the R-enantiomer. Consistently low K1O:K1 ratios were observed for N-methyl-thiotetrazole and antibiotics with (moxalactam) or without (cefotaxime) this side chain suggesting that none of these compounds are direct inhibitors of vitamin K epoxide reductase. Fibroblasts grown in cell culture provide a useful model to study the extrahepatic role of vitamin K and the mode of action of vitamin K antagonists.

• Uotila L
• The metabolic functions and mechanism of action of vitamin K.
• Scand J Clin Lab Invest Suppl. 1990; 201: 109-17
• Display abstract

• Vitamin K functions in animal cells as the cofactor of the enzyme vitamin K-dependent carboxylase which catalyzes the post-translational formation of gamma-carboxyglutamyl (Gla) residues in specific vitamin K-dependent proteins. These proteins include four blood coagulation factors (prothrombin and Factors VII, IX and X), other plasma proteins (protein C, protein S and protein Z), two proteins from bone (osteocalcin or bone Gla-protein and matrix Gla-protein), and other proteins from lung, kidney, spleen, testis, placenta and other tissues. In the proteins involved in blood coagulation the Gla residues are mandatory for the activation of the inactive proenzymes; this process occurs on phospholipid surfaces to which the proenzymes are bound via Gla residues and calcium ions. The energy needed in the carboxylation reaction is obtained from the oxidation of vitamin K hydroquinone to 2,3-epoxide of the vitamin. Specific enzymes, vitamin K epoxide reductase and vitamin K quinone reductases, catalyze consecutive reactions in which the vitamin K hydroquinone is regenerated, thus allowing continued use of the vitamin K molecule for the carboxylations. The oral anticoagulants, derivatives of 4-hydroxycoumarin and indan-1,3-dione, used as therapeutic agents in thromboembolic disease, are antagonists to vitamin K preventing the catalytic use of vitamin K in the carboxylations by irreversibly inhibiting vitamin K epoxide reductase.

• Wallin R, Rossi F, Loeser R, Key LL Jr
• The vitamin K-dependent carboxylation system in human osteosarcoma U2-OS cells. Antidotal effect of vitamin K1 and a novel mechanism for the action of warfarin.
• Biochem J. 1990; 269: 459-64
• Display abstract

• An osteoblast-like human osteosarcoma cell line (U2-OS) has been shown to possess a vitamin K-dependent carboxylation system which is similar to the system in human HepG2 cells and in liver and lung from the rat. In an 'in vitro' system prepared from these cells, vitamin K1 was shown to overcome warfarin inhibition of gamma-carboxylation carried out by the vitamin K-dependent carboxylase. The data suggest that osteoblasts, the cells involved in synthesis of vitamin K-dependent proteins in bone, can use vitamin K1 as an antidote to warfarin poisoning if enough vitamin K1 can accumulate in the tissue. Five precursors of vitamin K-dependent proteins were identified in osteosarcoma and HepG2 cells respectively. In microsomes (microsomal fractions) from the osteosarcoma cells these precursors revealed apparent molecular masses of 85, 78, 56, 35 and 31 kDa. When osteosarcoma cells were cultured in the presence of warfarin, vitamin K-dependent 14C-labelling of the 78 kDa precursor was enhanced. Selective 14C-labelling of one precursor was also demonstrated in microsomes from HepG2 cells and from rat lung after warfarin treatment. In HepG2 cells this precursor was identified as the precursor of (clotting) Factor X. This unique 14C-labelling pattern of precursors of vitamin K-dependent proteins in microsomes from different cells and tissues reflects a new mechanism underlying the action of warfarin.

• Misenheimer TM, Suttie JW
• Warfarin resistance in a Chicago strain of rats.
• Biochem Pharmacol. 1990; 40: 2079-84
• Display abstract

• A warfarin-resistant strain of rats trapped in Chicago was studied to determine the mechanism of the warfarin resistance. The Chicago-resistant rats (CR) differ from a Welsh-resistant strain (WR) which has a vitamin K epoxide reductase that is insensitive to warfarin. The epoxide and dithiol-dependent quinone reductases of the CR rats were as sensitive to warfarin as the normal enzyme. Unlike the irreversible warfarin inhibition seen in normal rats, the warfarin inhibition of the epoxide reductase from the CR strain was partially reversible in vitro. In this respect, the CR rats appeared similar to a Scottish warfarin-resistant strain. The same steady-state level of warfarin (40 ng/mg protein) in liver microsomes could be achieved in normal and CR strain rats following a few days ingestion of a diet containing 50 ppm warfarin, but clearance of warfarin (1 mg/kg) from the liver microsomes was more rapid in the CR strain than in normal rats, and the recovery of epoxide reductase activity and prothrombin levels was more rapid. The mechanism of warfarin resistance in the CR strain differed from the warfarin resistance mechanisms of both the Scottish- and Welsh-resistant rat strains. The combination of an increased rate of warfarin clearance and the partially reversible inhibition of the epoxide reductase would be sufficient to allow the rats to survive a limited exposure to warfarin.

• Yamanaka Y, Yamano M, Yasunaga K, Shike T, Uchida K
• Effect of warfarin on plasma and liver vitamin K levels and vitamin K epoxide reductase activity in relation to plasma clotting factor levels in rats.
• Thromb Res. 1990; 57: 205-14
• Display abstract

• Changes in plasma and liver vitamin K1 and vitamin K1 epoxide levels, liver microsomal vitamin K epoxide reductase activity, and plasma clotting factor II and VII levels were determined in rats after a single injection of warfarin (2.5 mg/kg, s.c.). The plasma and liver vitamin K1 levels gradually decreased after warfarin injection, attaining the lowest values at 2-3 hrs and remaining low for 48 hrs. They then returned to the control levels at 72 hrs. The changes in vitamin K1 epoxide levels were opposite, with an increase being seen soon after the warfarin injection, the highest values at 3 hrs and a gradual decrease to the initial levels occurring subsequently. The combined levels of vitamin K1 plus vitamin K1 epoxide, however, remained almost constant in both plasma and liver after the warfarin injection. The liver vitamin K epoxide reductase activity decreased to its lowest level soon after the injection and then gradually increased after 12 hrs, but the activity at 72 hrs was only about 30% of the initial activity. The plasma clotting factor levels gradually decreased after the injection, bottomed at 24 hrs and then began to increase, recovering almost to the initial levels at 72 hrs. A positive correlation was found between plasma and liver levels for both vitamin K1 and vitamin K1 epoxide, and the slope of the vitamin K1 epoxide curve was steeper than that for vitamin K1 in the warfarin-treated rats. A similar positive correlation was found for both vitamin K1 and vitamin K1 epoxide after vitamin K1 injection in normal untreated rats, but the slope of the vitamin K1 epoxide curve was much shallower. These results suggest that warfarin inhibits vitamin K epoxide reductase and decreases blood clotting factor synthesis, thus increasing plasma and liver vitamin K1 epoxide levels. A vitamin K epoxide reductase activity one third of that in normal rats is sufficient to maintain normal reduction of vitamin K1 epoxide and synthesis of blood clotting factors.

• Soulban G, Labrecque G, Belanger PM
• Temporal variation in the effects of warfarin on the vitamin K cycle.
• Chronobiol Int. 1990; 7: 403-11
• Display abstract

• In this in vivo study, the time-dependent effect of oral sodium warfarin was studied in male rats synchronized under a 12-hr light-dark cycle (light 0600-1800). Groups of 5 animals received an oral dose of 500 micrograms/kg of warfarin or saline at 0600 or 1800 and 1 mg/kg of vitamin K 8 hr later and the rats were sacrificed 240 min after vitamin K administration. The activities of the vitamin K reductase and vitamin K epoxide reductase were measured indirectly by determining the content of vitamin K1 and vitamin K epoxide reductase in the plasma and liver. The data obtained in control rats indicated that vitamin K and vitamin K 2,3 epoxide concentrations in plasma and liver were higher (P less than 0.05) at 1800 than at 0600. Warfarin had a greater (P less than 0.05) inhibitory effect on the vitamin K and vitamin K-epoxide reductases at 0600 compared to 1800; plasma levels of S- and R-warfarin did not vary with time of administration. The findings suggest that the activity of both reductases under control conditions, and the warfarin-induced inhibition of these enzymes varied depending on the time of drug administration.

• Bick D, Curry CJ, McGill JR, Schorderet DF, Bux RC, Moore CM
• Male infant with ichthyosis, Kallmann syndrome, chondrodysplasia punctata, and an Xp chromosome deletion.
• Am J Med Genet. 1989; 33: 100-7
• Display abstract

• We report on a male infant with X-linked ichthyosis, X-linked Kallmann syndrome, and X-linked recessive chondrodysplasia punctata (CPXR). Chromosome analysis showed a terminal deletion with a breakpoint at Xp22.31, inherited maternally. This patient confirms the localization of XLI, XLK, and CPXR to this region of the X chromosome and represents an example of a "contiguous gene syndrome." A comparison of the manifestations of patients with CPXR, warfarin embryopathy, and vitamin K epoxide reductase deficiency shows a remarkable similarity. However, vitamin K epoxide reductase deficiency does not appear to be the cause of CPXR. We propose that CPXR may be due to a defect in a vitamin K-dependent bone protein such as vitamin K-dependent bone carboxylase, osteocalcin, or matrix Gla protein.

• Thijssen HH, Baars LG
• Microsomal warfarin binding and vitamin K 2,3-epoxide reductase.
• Biochem Pharmacol. 1989; 38: 1115-20
• Display abstract

• Rat liver microsomal 4-hydroxycoumarin binding was studied by assaying specific [14C]warfarin binding. Microsomes of warfarin-sensitive rats contained about 40 pmole of specific binding sites per mg of microsomal protein. There was no difference for R- or S-[14C]warfarin. Neither was there any difference between the enantiomers of acenocoumarol and phenprocoumon to prevent the in vitro racemic [14C]warfarin binding. Pretreatment of the microsomes with dithiothreitol, the in vitro reductor for microsomal vitamin K epoxide reductase activity, reduced the warfarin binding. Vitamin K epoxide nor vitamin K affected the warfarin binding. Microsomes of the Welsh warfarin resistant genotype showed weak warfarin binding properties. The Scottish resistant variant, on the other hand, did not differ from sensitive microsomes. Warfarin binding was reduced in microsomes of rats to which S-warfarin was administered. The reduction in warfarin binding was linear with the inhibition of microsomal vitamin K epoxide reductase activity and was linear with the amount of S-warfarin present in the microsomes. The results show the microsomal 4-hydroxycoumarin binding to be related to the target enzyme vitamin K epoxide reductase.

• Preusch PC, Hazelett SE, Lemasters KK
• Sulfaquinoxaline inhibition of vitamin K epoxide and quinone reductase.
• Arch Biochem Biophys. 1989; 269: 18-24
• Display abstract

• Sulfaquinoxaline (N1-(2-quinoxalinyl)sulfanilamide) has been shown to be a potent (Ki = 1 microM) freely reversible inhibitor of the dithiothreitol-dependent reduction of both vitamin K epoxide and vitamin K quinone by rat liver microsomes in vitro. This observation provides an explanation for the hemorrhagic syndrome occasionally seen in poultry on medicated feed and the efficacy of sulfaquinoxaline in anticoagulant based rodenticides. Sulfaquinoxaline inhibition resembled inhibition by coumarin anticoagulants (e.g., warfarin) and hydroxynaphthoquinones (e.g., lapachol). Inhibition was observed in assays using microsomes from control strain rats, but the enzyme was resistant to sulfaquinoxaline in microsomes from warfarin-resistant rats. Steady-state kinetics inhibition patterns were nearly competitive versus dithiothreitol and nearly uncompetitive versus vitamin K epoxide as is observed for warfarin and lapachol. These results suggest that this inhibitor binds to the oxidized form of vitamin K epoxide reductase in the same way as suggested for the coumarins and hydroxyquinones. Of 10 other sulfa drugs tested, none were inhibitors, and of fragments and related compounds tested, only 2-aminoquinoxaline benzenesulfonamide was active. These results provide a probably orientation in the binding site in relation to that for warfarin and lapachol.

• Hazelett SE, Preusch PC
• Tissue distribution and warfarin sensitivity of vitamin K epoxide reductase.
• Biochem Pharmacol. 1988; 37: 929-34
• Display abstract

• The distribution of vitamin K epoxide reductase activity and its sensitivity to warfarin have been examined in whole microsomes from tissues of both control and warfarin-resistant strain rats. The distribution of activity roughly paralleled that previously shown for the vitamin K-dependent carboxylase. Activity on a per gram tissue basis was highest in kidney, adrenal, spleen, lung, testes, and epididymis at a level about 1/20th of that present in liver microsomes. Vitamin K quinone formation by microsomes from warfarin-resistant rats was approximately half that of control strain samples. In addition, hydroxy vitamin K was formed by warfarin-resistant strain microsomes to about the same extent as vitamin K quinone in all tissues. The Km values for dithiothreitol (DTT) and vitamin K epoxide were similar in all tissues (range = 0.1-0.2 mM DTT at 40 microM vitamin K epoxide, and 10-30 microM vitamin K epoxide at 2 mM DTT). The sensitivities to warfarin were similar for all control strain rat tissues (I50 = 10-20 microM at 2 mM DTT and 40 microM vitamin K epoxide) and similarly elevated for all warfarin-resistant rat tissues (I50 = 30 to greater than 80 microM). These results suggest that the identical enzyme is expressed in all tissues and that tissue specific isozymes do not occur.

• Choonara IA et al.
• The relationship between inhibition of vitamin K1 2,3-epoxide reductase and reduction of clotting factor activity with warfarin.
• Br J Clin Pharmacol. 1988; 25: 1-7
• Display abstract

• 1 The effect of low dose steady state warfarin (0.2 mg and 1 mg daily) on clotting factor activity and vitamin K1 metabolism was studied in seven healthy volunteers. 2 Steady state plasma warfarin concentrations were 41-99 ng ml-1 for the 0.2 mg dose and 157-292 ng ml-1 for the 1 mg dose. 3 There was a significant prolongation of the mean prothrombin time (0.9 s) after 1 mg warfarin daily, but no significant change in prothrombin time after 0.2 mg warfarin daily. There was no significant change in individual clotting factor activity (II, VII, IX or X) with either dose of warfarin. 4 Following the administration of a pharmacological dose of vitamin K1 (10 mg), all seven volunteers had detectable levels of vitamin K1 2,3-epoxide with both doses of warfarin (Cpmax 31-409 ng ml-1). 5 Both the Cpmax and the AUC for vitamin K1 2,3-epoxide were significantly greater on 1 mg of warfarin daily than 0.2 mg daily (P less than 0.01). 6 The apparent dissociation between inhibition of vitamin K1 2,3-epoxide reductase and reduction of clotting factor activity, produced by warfarin, may reflect the insensitivity of functional clotting factor assays to a small reduction in clotting factor concentration.

• Thijssen HH, Baars LG, Vervoort-Peters HT
• Vitamin K 2,3-epoxide reductase: the basis for stereoselectivity of 4-hydroxycoumarin anticoagulant activity.
• Br J Pharmacol. 1988; 95: 675-82
• Display abstract

• 1. The administration of S-warfarin (1 mg kg-1 i.v.) to rats that were pre-loaded 48 h before with tracer doses (6 micrograms) of 14C-labelled R- or S-warfarin caused the plasma levels of these compounds to increase. This is due to the substitution of the microsomal (vitamin K 2,3-epoxide (K0) reductase) bound R- or S-[14C]-warfarin by the unlabelled 4-hydroxycoumarin administered. The rate of reappearance was 3-4 fold higher for R- than for S-warfarin; t1/2 of release: 1.2 +/- 0.04 and 3.7 +/- 0.6 h, respectively. 2. Liver microsomes prepared from rats pretreated with R- or S-[14C]-warfarin, released these compounds only in the presence of dithiothreitol (DTT; 10 mM). The rate of release was higher for R- than for S-warfarin-treated microsomes. 3. Liver microsomes treated in vitro with R- or S-acenocoumarol could be reactivated by DTT (10 mM). Reactivation was higher for the R- than for the S-acenocoumarol-treated microsomes. 4. The microsomal vitamin K0 reductase activity under 'normal' assay conditions ([DTT] = 2 mM) was as sensitive for R- as for S-4-hydroxycoumarins. At elevated DTT concentrations (= 42 mM) the rate of vitamin K0 conversion was about 1.5 fold higher in the presence of the R-isomers than in the presence of the S-isomers. For instance, at 2 mM DDT the reductase activities in the presence of 2.6 microM R- and S-warfarin were about 15% of control. At 42 mM DTT the activities were 90 and 65% of control, respectively. 5. In the in vitro experiments acenocoumarol appeared to be more potent than warfarin and phenprocoumon. 6. The following mechanism is proposed: vitamin K0 reductase becomes oxidized during substrate reduction.(ABSTRACT TRUNCATED AT 250 WORDS)

• Trivedi LS, Rhee M, Galivan JH, Fasco MJ
• Normal and warfarin-resistant rat hepatocyte metabolism of vitamin K 2,3-epoxide: evidence for multiple pathways of hydroxyvitamin K formation.
• Arch Biochem Biophys. 1988; 264: 67-73
• Display abstract

• Vitamin K and 3- (and/or 2)-hydroxy-2,3-dihydro-2-methyl-3-phytyl-1,4-naphthoquinone (hydroxyvitamin K) have been identified as metabolites of vitamin K 2,3-epoxide incubated with hepatocytes isolated from normal and warfarin-resistant rats. Dithiothreitol added to the extracellular medium differentially enhanced the formation of both metabolites: hydroxyvitamin K formation, almost undetectable in the absence of dithiothreitol, was particularly affected. Addition of the vitamin K 2,3-epoxide reductase inhibitors warfarin (5 to 100 microM) and brodifacoum (1 to 5 microM) to normal rat hepatocyte cultures produced a slight increase in hydroxyvitamin K formation and a marked inhibition of vitamin K formation. Brodifacoum was a weak inhibitor of hydroxyvitamin K formation at higher concentrations. Hepatocytes from warfarin-resistant rats catalyzed hydroxyvitamin K formation 1.5 to 2 times faster and vitamin K formation 1.5 to 2 times slower than did normal rat hepatocytes. The addition of warfarin to these cultures had no effect on epoxide metabolism to hydroxyvitamin K and only partially diminished metabolism to vitamin K. In contrast, brodifacoum (1 microM) addition produced 50% inhibition of hydroxyvitamin K formation and almost complete inhibition of vitamin K formation. These data suggest that in resistant, but not in normal rat hepatocytes, the vitamin K 2,3-epoxide reductase makes a significant contribution to hydroxyvitamin K formation. A second sulfhydryl-dependent pathway, present in both strains, is also involved in the formation of this metabolite. They also suggest that in resistant rats, warfarin inhibition of the vitamin K 2,3-epoxide reductase, and presumably the sulfhydryl-dependent vitamin K reductase, is incomplete and independent of concentration.

• Thijssen HH
• Warfarin resistance. Vitamin K epoxide reductase of Scottish resistance genes is not irreversibly blocked by warfarin.
• Biochem Pharmacol. 1987; 36: 2753-7
• Display abstract

• The dithiothreitol-dependent vitamin K 2,3 epoxide (vitamin KO) reductase activity in liver microsomes of Scottish-derived warfarin-resistant Wistar rats (Tolworth Laboratory) was compared to that of susceptible Wistar rats. Under the test conditions, reductase activities in liver homogenates and in liver microsomes were comparable for both strains. The in vitro i50 of S-warfarin for microsomal reductase activity was 1 to 2 microM in both strains. The effect of in vivo S-warfarin was investigated after single doses, i.e. 0.2 and 1 mg/kg for the susceptible rats, and 1 and 5 mg/kg for the resistant rats. At 20 hr following the warfarin administrations in the susceptible strain, microsomal reductase was suppressed to about 30% of control. Microsomal reductase activity in the resistant strain was not reduced. Tissue and microsomal warfarin concentrations, however, were comparable in both strains. Wash experiments with microsomes which were treated in vitro with S-warfarin showed that vitamin KO reductase of the warfarin-resistant strain was not irreversibly inactivated by warfarin. The reactivation was mediated by DDT. The results suggest the following characteristic of the vitamin KO reductase of the Scottish resistance gene: contrary to the "normal" enzyme, the tight complex between the inhibitor and the resistant enzyme is liable to reactivation by reduction of the disulfide bridge in the active centre of the enzyme. This property explains the resistance for 4-hydroxycoumarin anticlotting activity.

• de Boer-van den Berg MA, Uitendaal MP, Vermeer C
• Direct measurement of vitamin K-dependent enzymes in various isolated and cultured tumor and non-tumor cells.
• Mol Cell Biochem. 1987; 75: 71-6
• Display abstract

• A modification of the assay for vitamin K-dependent carboxylase is described with which the enzyme could be detected in relatively low amounts of cells (n = 10(6)). Using this assay, we could demonstrate vitamin K-dependent carboxylase activity in hepatocytes, renal tubular cells, osteoblasts, endothelial cells and macrophages, but not in lymphocytes or platelets. The cultured tumor cells UMR-106, B16 and 5583 also contained vitamin K-dependent carboxylase activity. Vitamin K epoxide reductase activity was demonstrated only in cells where vitamin K-dependent carboxylase activity was present. The tumor cells possessed remarkably less K epoxide reductase activity than the normal cells. When cells were cultured in medium containing warfarin, the K epoxide reductase activity was found to be decreased and the amount of non-carboxylated precursor proteins had increased, suggesting an analogous vitamin K mechanism as in liver.

• Wallin R, Martin LF
• Warfarin poisoning and vitamin K antagonism in rat and human liver. Design of a system in vitro that mimics the situation in vivo.
• Biochem J. 1987; 241: 389-96
• Display abstract

• The present paper describes a system in vitro that has been developed to mimic vitamin K metabolism and vitamin K function in liver. In this system the two pathways that are known to participate in vitamin K reduction are active and the vitamin K-dependent carboxylase accepts a synthetic pentapeptide as substrate. With this system in vitro the effect of warfarin on both pathways was examined under conditions which simulated a warfarin-poisoned liver. Identical experiments were completed with rat and human liver. All activities currently associated with vitamin K metabolism and vitamin K function were similar in the rat and human systems. Warfarin neutralized the ability of pathway I (the vitamin K epoxide reductase pathway) to produce reduced and active vitamin K cofactor for the carboxylase. In both the rat and the human system, however, when warfarin was present, reduced vitamin K cofactor was produced by pathway II (the dehydrogenase pathway). The data are consistent with observations in vivo on the effect of vitamin K1 when used as an antidote. This suggests that the system in vitro reflects the mechanism in vivo by which vitamin K1 overcomes warfarin poisoning.

• Suttie JW
• The biochemical basis of warfarin therapy.
• Adv Exp Med Biol. 1987; 214: 3-16
• Display abstract

• Vitamin K is required for a liver microsomal enzyme that catalyzes the posttranslational conversion of specific glutamyl residues in precursors of the vitamin K-dependent clotting factors to gamma-carboxyglutamyl residues in the plasma form of these proteins. A second product of this carboxylation reaction is the 2,3-epoxide of the vitamin. The anticoagulant warfarin blocks the enzyme which reduces this epoxide to vitamin K quinone, and also blocks one of the microsomal pathways which converts the quinone to the active coenzyme form of the vitamin, the hydroquinone. Warfarin anticoagulation therefore reduces the activity of the vitamin K-dependent carboxylase and results in the secretion of vitamin K-dependent clotting factors that are under carboxylated. The presence of gamma-carboxyglutamyl residues is essential for the normal Ca++/phospholipid-mediated activation of prothrombin, and the des-gamma-carboxy forms of prothrombin secreted by patients receiving warfarin therapy lack biological activity and have a reduced thrombotic potential.

• Wilson AC, Fasco MJ
• Vitamin-K-dependent proteins in microsomes of primary Lewis lung tumors.
• Int J Cancer. 1986; 38: 877-82
• Display abstract

• Microsomes isolated from Lewis lung (LL) primary tumors raised in C57BL/6 mice have been shown to (i) contain a 4-hydroxycoumarin (warfarin)-sensitive cycle of vitamin K metabolism which is at least qualitatively similar to that of liver, and (ii) catalyze the incorporation of NaH14 CO3 into endogenous protein in a vitamin-K hydroquinone-dependent reaction to produce gamma-carboxyglutamate. As in liver microsomes, LL microsomal reduction of vitamin K 2,3-epoxide to vitamin K was greatly enhanced by exogenous dithiols such as dithiothreitol, but under identical conditions the former was 10-fold faster. The R(+) and S(-) warfarin enantiomers were highly and equally effective inhibitors of both the liver and tumor vitamin K 2,3-epoxide reductases-the average I50 against the tumor enzyme was 0.25 microM. Partially purified reductases isolated by centrifugation of sodium-cholate-treated liver and LL tumor microsomes over a discontinuous sucrose gradient were also inhibited by the sulfhydryl reagent N-ethylmaleimide following their reduction by dithiothreitol. Like the activity of the epoxide reductase, that of the gamma-carboxylase was much lower in tumor than in liver microsomes and was only detectable in microsomes isolated from tumor-bearing mice previously administered S(-) warfarin. In view of the reported inhibition of LL tumor metastasis by warfarin and diet-induced vitamin-K deficiency, vitamin-K-dependent proteins may play a role in the spread and/or subsequent growth of LL cells.

• Creedon KA, Suttie JW
• Effect of N-methyl-thiotetrazole on vitamin K epoxide reductase.
• Thromb Res. 1986; 44: 147-53
• Display abstract

• Clinical use of antibiotics containing a N-methyl-thiotetrazole (NMTT) side chain has been reported to be associated with an increased incidence of a vitamin K-responsive hypoprothrombinemia. Administration of NMTT to rats decreased the activity of the liver microsomal vitamin K epoxide reductase, increased the liver ratio of vitamin K epoxide to vitamin K, and decreased the rate of metabolism of injected vitamin K epoxide. These responses are the same as those observed following the administration of coumarin anticoagulants. In contrast to the effect of coumarin anticoagulants, NMTT did not inhibit the vitamin K epoxide reductase in vitro. These data suggest that the hypoprothrombinemia which has been observed following use of these antibiotics results from the inactivation of the liver vitamin K epoxide reductase by NMTT or a NMTT metabolite.

• Wallin R, Patrick SD, Ballard JO
• Vitamin K antagonism of coumarin intoxication in the rat.
• Thromb Haemost. 1986; 55: 235-9
• Display abstract

• An in vitro system which expresses all enzyme activities related to vitamin K-dependent carboxylation of blood clotting factors was prepared from livers of rats overdosed with warfarin, difenacoum and dicumarol respectively. In this system, the activities of the two pathways that are known to produce active reduced vitamin K1 cofactor for the carboxylation reaction were measured. Also the ability of high concentrations of vitamin K1 to overcome inhibition of clotting factor synthesis was studied. In the systems prepared from livers of warfarin and difenacoum intoxicated rats, pathway I was inactive. Vitamin K epoxide reductase was also inactive which strongly suggests that this enzyme catalyzes the activity of pathway I in vivo. Reduction of vitamin K1 by pathway II bypassed the inactive pathway I and resulted in carboxylation activity. This pathway therefore mediates the antidotic effect of vitamin K1 in the coumarin intoxicated liver. In the in vitro system prepared from dicumarol intoxicated livers the activity of pathway I was not significantly affected. Dicumarol however was a strong inhibitor when added to liver microsomes in vitro.

• Uitendaal MP, Thijssen HH, Drittij-Reijnders MJ, Hoeijmakers MJ
• B16 tumor cells contain a warfarin sensitive vitamin K1 2,3 epoxide reductase.
• Biochem Biophys Res Commun. 1986; 137: 1015-20
• Display abstract

• Using an adapted assay that requires an enzyme aliquot that forms only 5 pmoles vitamin K, we were able to demonstrate vitamin K1 2,3 epoxide reductase activity in cultured B16 mouse melanoma cells. The enzyme uses dithiothreitol, but not NADH as a reducing cofactor and is sensitive to inhibition by warfarin (2% residual activity at 10 micrograms/ml warfarin). Incubation of B16 cells in culture with 30 micrograms/ml warfarin leads to an 45% residual reductase as compared to normally cultured B16 cells. Combined with the reported presence of vitamin K dependent carboxylase in B16 cells and the cytotoxicity of warfarin towards B16 cells this suggests an active vitamin K cycle in these melanoma cells that may be essential for survival.

• Suttie JW, Preusch PC
• Studies of the vitamin K-dependent carboxylase and vitamin K epoxide reductase in rat liver.
• Haemostasis. 1986; 16: 193-215
• Display abstract

• Vitamin K is required as a cofactor for a microsomal enzyme that converts glutamyl residues in precursor proteins to gamma-carboxyglutamyl residues in completed proteins. These residues are essential for the biological function of prothrombin, factors VII, IX, and X, protein C, and protein S. Current data suggest that recognition of protein substrates by the carboxylase requires an unidentified protein-protein interaction in addition to the Glu substrate binding site. The primary vitamin K-dependent event has now been shown to be the abstraction of the gamma-hydrogen of the substrate Glu residue with the concurrent formation of vitamin K 2,3-epoxide. Coumarin anticoagulants appear to inhibit the microsomal vitamin K epoxide reductase and one of a number of microsomal quinone reductases. They therefore block vitamin K action by preventing the recycling of vitamin K epoxide to the quinone and to the active cofactor form, the hydroquinone. Excess vitamin K can reverse a coumarin anticoagulant effect as the nonsensitive quinone reductase can continue to furnish the active coenzyme.

• de Boer-van den Berg MA, Thijssen HH, Vermeer C
• The in vivo effects of acenocoumarol, phenprocoumon and warfarin on vitamin K epoxide reductase and vitamin K-dependent carboxylase in various tissues of the rat.
• Biochim Biophys Acta. 1986; 884: 150-7
• Display abstract

• In rats the in vivo effects of a chronic low-dose treatment (+/- 60 micrograms/rat per day) with different coumarins (acenocoumarol, phenprocoumon and warfarin) on hepatic and non-hepatic vitamin K-dependent enzyme systems were compared. The plasma concentrations of the three coumarins differed largely but these differences were not reflected in the microsomal coumarin contents. The non-hepatic microsomes contained less than 20% of the coumarins found in liver microsomes. No substantial differences were observed between the following effects of the three anticoagulant treatments. The blood coagulation factor activities were about 10% of normal. The hepatic microsomal vitamin K epoxide reductase activity was diminished to about 35% of control values. The vitamin K epoxide reductase activities present in kidney, lung, spleen, testis and brain microsomes were less influenced by the coumarin treatments; activities ranged between 45 and 65% of normal. In the liver microsomes a 15-fold accumulation of non-carboxylated precursor proteins was found; in the non-hepatic microsomes this effect was less pronounced but still present. The hepatic vitamin K-dependent carboxylase activity was enhanced but the corresponding non-hepatic enzyme activities were slightly or not affected. In addition, the effects of a chronic low-dose warfarin treatment were compared with those after an acute high dose of the drug.

• Thijssen HH, Janssen CA, Drittij-Reijnders MJ
• The effect of S-warfarin administration on vitamin K 2,3-epoxide reductase activity in liver, kidney and testis of the rat.
• Biochem Pharmacol. 1986; 35: 3277-82
• Display abstract

• The dithiothreitol-dependent vitamin K 2,3-epoxide (vitamin KO) reductase activity was assayed in rat liver, kidney and testis microsomes. Rat kidney and testis showed vitamin KO reductase activity. The activity was about one tenth of the activity present in liver microsomes. The effect of in vivo S-warfarin was investigated after single doses, i.e. 0.2, 0.4 and 1 mg/kg, and after its chronic administration, i.e. 4.8 micrograms/kg/hr for 3 days. At 20 hr following the acute warfarin administration vitamin KO reductase in liver microsomes was depressed in a dose-dependent way, 50, 30 and 20% of control activity. Vitamin KO reductase in testis was not affected, and in kidney reductase activity was only reduced after the highest warfarin dose, 40% of control activity. Following chronic administration of warfarin, vitamin KO reductase activity was reduced in liver as well as in kidney and testis microsomes, 15-20, 40 and 60% of control activity in liver, kidney and testis, respectively. Blood clotting activity was about 14% of normal (thrombotest). Vitamin KO reductase activity in tissue microsomes was inhibited by warfarin added in vitro. Tissue and microsomal warfarin concentration were assayed. Following the acute administration, warfarin was poorly distributed into kidney and testis. Following the chronic administration, warfarin tissue to plasma ratio was about 3 for liver, but 0.5 for kidney and testis. The results indicate that during chronic therapy with oral anticoagulants vitamin K-dependent systems in non-hepatic tissues are reduced. However, this reduction is less than the reduction of the hepatic system. This is determined mainly by the pharmacokinetic behaviour of the 4-hydroxycoumarins.

• MacNicoll AD
• A comparison of warfarin resistance and liver microsomal vitamin K epoxide reductase activity in rats.
• Biochim Biophys Acta. 1985; 840: 13-20
• Display abstract

• Vitamin K-1 epoxide reductase activity was investigated in liver microsomal preparations from warfarin-resistant and -susceptible rats. One rat strain (TAS) is susceptible to the anticoagulant and lethal effects of warfarin and the other two strains are homozygous for warfarin resistance genes from either wild Welsh (HW) or Scottish (HS) rats. The enzyme in microsomal preparations from HW rat livers apparently has a reduced affinity for both warfarin and vitamin K-1 2,3-epoxide. The kinetic parameters for the enzyme activity in HS microsomal preparations indicated, however, that vitamin K-1 epoxide reductase in this warfarin-resistant strain was very similar, in respect of substrate and inhibitor affinities, to that prepared from susceptible (TAS) animals. Analysis of vitamin K-1 epoxide reductase activity in the livers of animals that had been orally treated with sodium warfarin (20 mg/kg body wt.) indicated that enzyme activity was inhibited in all three strains, although this dose is lethal only to animals of the TAS strain.

• Lee JJ, Principe LM, Fasco MJ
• Identification of a warfarin-sensitive protein component in a 200S rat liver microsomal fraction catalyzing vitamin K and vitamin K 2,3-epoxide reduction.
• Biochemistry. 1985; 24: 7063-70
• Display abstract

• A partially purified, 200S submicrosomal fraction exhibiting thiol-dependent vitamin K1 (vitamin K) and epoxide reductase activities has been isolated by partial solubilization of rat hepatic microsomes with sodium cholate and separation by centrifugation at 105 000 g into a discontinuous sucrose gradient. At pH 7.4, the rates of vitamin K and vitamin K 2,3-epoxide reduction per milligram of 200S fraction protein were equivalent and were 2.5-3.0 times faster than in microsomes. Reduction of vitamin K 2,3-epoxide occurred in a tightly coupled, two-step reaction initially to vitamin K and subsequently to vitamin K hydroquinone (vitamin KH2). Incorporation of glycerol or sucrose and of sodium cholate into reaction mixtures equivalently affected the rates of both vitamin K and vitamin K 2,3-epoxide reduction, but in the case of epoxide metabolism, the ratios of vitamin KH2/vitamin K were much lower, suggesting that the second reaction has been partially uncoupled from the first. A 14 000-17 000-dalton warfarin-sensitive protein (WSP) that participates in vitamin K and vitamin K 2,3-epoxide reduction in the 200S fraction was identified by incorporation of N-[3H]ethylmaleimide ([3H]NEM) into the catalytically active reduced form of one or more attached disulfides. Reduction of WSP with dithiothreitol was required for reaction with [3H]NEM, and the substrates vitamin K and vitamin K 2,3-epoxide and the inhibitor warfarin all effectively blocked the reaction. 2-Mercaptoethanol could not substitute for dithiothreitol.(ABSTRACT TRUNCATED AT 250 WORDS)

• Wallin R, Martin LF
• Vitamin K-dependent carboxylation and vitamin K metabolism in liver. Effects of warfarin.
• J Clin Invest. 1985; 76: 1879-84
• Display abstract

• The systems involved in vitamin K-dependent carboxylation and vitamin K metabolism have been extensively studied in rat liver. To determine how clinically applicable this information is, similar in vitro studies were completed using human liver. One major difference exists in the pathways that provide reduced vitamin K1 cofactor for the carboxylation reaction. The coumarin-sensitive DT-diaphorase (EC.1.6.99.2) in human liver appears to play a relatively minor role in the dehydrogenase pathway. However, similar to rat liver, the human liver contains a warfarin-insensitive enzyme in this dehydrogenase pathway. The data suggest that this enzyme is responsible for the antidotic effect of vitamin K1 in cases of coumarin intoxication. Human vitamin K epoxide reductase, which constitutes the other pathway for vitamin K1 reduction, has kinetic and enzymological characteristics that are very similar to the rat enzyme. This enzyme exhibited similar activity in rat and human microsomes. Initial velocities for vitamin K1 epoxide reduction in rat and human microsomes were 20 and 32 pmol/mg X min, respectively. The human enzyme is highly sensitive to warfarin inhibition. The mechanism for this inhibition appears to be similar to what has been proposed for the rat enzyme. Also, a vitamin K-dependent carboxylation system is described that allows both pathways to support the carboxylation reaction with reduced vitamin K1 cofactor. The effect of warfarin on this in vitro system is consistent with the current model for the mechanism of action of coumarin anticoagulant drugs in the rat.

• Mukharji I, Silverman RB
• Purification of a vitamin K epoxide reductase that catalyzes conversion of vitamin K 2,3-epoxide to 3-hydroxy-2-methyl-3-phytyl-2,3-dihydronaphthoquinone.
• Proc Natl Acad Sci U S A. 1985; 82: 2713-7
• Display abstract

• An enzyme from bovine liver microsomes that catalyzes the reduction of vitamin K 2,3-epoxide to 2- and 3-hydroxy-2-methyl-3-phytyl-2,3-dihydronaphthoquinone was purified 1152-fold to apparent homogeneity. Microsomes were solubilized with 3-[3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and the enzyme was purified by chromatography on PBE-94 ion exchanger, hydroxylapatite, and DEAE-cellulose, and then gel filtration on Sephacryl S-200. The homogeneity of the final preparation was established by polyacrylamide slab gel electrophoresis in the presence of sodium dodecyl sulfate. The molecular weight of the native enzyme is 25,000 and that of denatured enzyme is 12,400, which suggests that the enzyme is a dimer with identical subunits. No chromophoric cofactors are associated with the enzyme. Dithiothreitol and CHAPS are essential for activity, but high concentrations of glycerol reduces the activity. The enzyme is not inhibited by warfarin, a potent inhibitor of the vitamin K epoxide reductase, which catalyzes the conversion of vitamin K 2,3-epoxide to vitamin K. Evidence is presented indicating that the purified enzyme is not simply a fragment of the warfarin-sensitive vitamin K epoxide reductase.

• MacNicoll AD, Nadian AK, Townsend MG
• Inhibition by warfarin of liver microsomal vitamin K-reductase in warfarin-resistant and susceptible rats.
• Biochem Pharmacol. 1984; 33: 1331-6
• Display abstract

• The NADH-dependent vitamin K-reductase activity of liver microsomes from three closely related rat strains has been studied. One strain (TAS) is susceptible and two strains (HW and HS) resistant to the anticoagulant and lethal effects of warfarin. The effects of cofactors, temperature, detergent and dithiothreitol on vitamin K1 reduction and solvent extraction of substrate and product have been investigated. Vitamin K-reductase activity was inhibited by approximately 13 and 8% respectively when microsomal preparations from TAS and HW animals were incubated with 50 microM vitamin K1 and 10 microM warfarin. In HS rat liver microsomes the enzyme was highly resistant to inhibition by warfarin. Evidence is presented and discussed that suggests that NADH-dependent vitamin K-reductase may be inhibited in the anticoagulant effect of warfarin and may be altered as a result of expression of the warfarin-resistance gene in HS rats. The enzyme activity studied was probably not a DT-diaphorase although both NADH and NADPH acted as cofactors for the reaction.

• Hildebrandt EF, Preusch PC, Patterson JL, Suttie JW
• Solubilization and characterization of vitamin K epoxide reductase from normal and warfarin-resistant rat liver microsomes.
• Arch Biochem Biophys. 1984; 228: 480-92
• Display abstract

• Two procedures have been developed for the solubilization of vitamin K epoxide reductase from rat liver microsomal membranes using the detergent Deriphat 160 at pH 10.8. The methods are applicable to both normal and Warfarin-resistant-strain rat liver microsomes and yield material suitable for further purification. The preparations retain dithiothreitol-dependent vitamin K quinone reductase activity as well as vitamin K epoxide reductase and are free of vitamin K-dependent carboxylase and epoxidase activities. Optimal epoxide reductase activity is obtained at 0.1 M KCl and pH 9 in the presence of sodium cholate. Artifactual formation of vitamin K metabolites was eliminated through the use of mercuric chloride to remove excess dithiothreitol prior to extraction and metabolite assay. Using the solubilized enzyme, valid initial velocities were measured, and reproducible kinetic data was obtained. The substrate initial velocity patterns were determined and are consistent with a ping-pong kinetic mechanism. The kinetic parameters obtained are a function of the cholate concentration, but do not vary drastically from those obtained using intact microsomal membranes. At 0.8% cholate, the enzymes solubilized from normal Warfarin-sensitive- and Warfarin-resistant-strain rat livers exhibit respective values of Vmax = 3 and 0.75 nmol/min/g liver; Km for vitamin K epoxide = 9 and 4 microM; and Km for dithiothreitol of 0.6 and 0.16 mM.

• Preusch PC, Suttie JW
• Formation of 3-hydroxy-2,3-dihydrovitamin K1 in vivo: relationship to vitamin K epoxide reductase and warfarin resistance.
• J Nutr. 1984; 114: 902-10
• Display abstract

• Hydroxy vitamin K [3(2)-hydroxy-2,3- dihydrovitamin K1] has been identified as a quantitatively important metabolite of injected vitamin K epoxide in vivo. The metabolite has been isolated and identified by comparison of its UV, mass spectra and high-performance liquid chromatography (HPLC) retention times with those of synthetic standards, and by its characteristic conversion to vitamin K quinone on treatment with the base triethylamine. This metabolite is formed from the vitamin K epoxide, not from the vitamin K quinone and can represent up to 3.5% of dose and 13% of hexane-extractable metabolites present in liver 1 hour after injection of 330 micrograms vitamin K1 epoxide per kilogram body weight. It is formed in both normal and warfarin-resistant rat strains, but to a significantly greater extent in the latter. Unlike the hydroxy vitamin K formed by warfarin-resistant rat liver microsomes in vitro, the metabolite formed from racemic vitamin K epoxide in vivo was not optically active, nor was its formation inhibited by coumarin anticoagulants under conditions that completely blocked vitamin K epoxide reduction in vivo. On this basis, hydroxy vitamin K formation in vivo differs from its formation in vitro; it is not a product of vitamin K epoxide reductase in vivo, but of some other possibly non-enzymatic reaction.

• Preusch PC, Suttie JW
• Lapachol inhibition of vitamin K epoxide reductase and vitamin K quinone reductase.
• Arch Biochem Biophys. 1984; 234: 405-12
• Display abstract

• Lapachol [2-hydroxy-3-(3-methyl-2-butenyl)-1,4-naphthoquinone] has been shown to be a potent inhibitor of both vitamin K epoxide reductase and the dithiothreitol-dependent vitamin K quinone reductase of rat liver microsomes in vitro. These observations explain the anticoagulant activity of lapachol previously observed in both rats and humans. Lapachol inhibition of the vitamin K epoxide and quinone reductases resembled coumarin anticoagulant inhibition, and was observed in normal strain but not in warfarin-resistant strain rat liver microsomes. This similarity of action suggests that the lactone functionality of the coumarins is not critical for their activity. The initial-velocity steady-state inhibition patterns for lapachol inhibition of the solubilized vitamin K epoxide reductase were consistent with tight binding of lapachol to the oxidized form of the enzyme, and somewhat lower affinity for the reduced form. It is proposed that lapachol assumes a 4-enol tautomeric structure similar to that of the 4-hydroxy coumarins. These structures are analogs of the postulated hydroxyvitamin K enolate intermediate bound to the oxidized form of the enzyme in the chemical reaction mechanism of vitamin K epoxide reductase, thus explaining their high affinity.

• Fasco MJ, Preusch PC, Hildebrandt E, Suttie JW
• Formation of hydroxyvitamin K by vitamin K epoxide reductase of warfarin-resistant rats.
• J Biol Chem. 1983; 258: 4372-80
• Display abstract

• A new metabolite of vitamin K, 2(3)-hydroxy-2,3-dihydro-2-methyl,3-phytyl-1,4-naphthoquinone (hydroxyvitamin K), has been identified as a product of vitamin K epoxide metabolism in hepatic microsomes from warfarin-resistant rats, but not in those derived from normal rats. The structure was determined by comparison of the high performance liquid chromatography retention times, UV, IR, CD, and mass spectra of the unknown with chemically synthesized standards. Alterations in the formation of hydroxyvitamin K occur in parallel with alterations in total vitamin K epoxide conversion with respect to reaction time, extent of reaction, detergent stimulation, and inhibition by warfarin. Thus, hydroxyvitamin K appears to be a product of the warfarin-resistant vitamin K epoxide reductase. It is neither a substrate nor an inhibitor of epoxide reduction. Hydroxyvitamin K is formed from both enantiomers of racemic vitamin K epoxide with little stereoselectivity for the configuration of either the oxirane ring or the phytyl side chain. The reaction is stereospecific; however, the biologically formed (+)-vitamin K epoxide yields exclusively (+)-3-hydroxyvitamin K. Observation of this product is discussed as a key to understanding the normal reaction mechanism of the enzyme.

• Fasco MJ, Principe LM, Walsh WA, Friedman PA
• Warfarin inhibition of vitamin K 2,3-epoxide reductase in rat liver microsomes.
• Biochemistry. 1983; 22: 5655-60
• Display abstract

• Warfarin is a potent inhibitor of vitamin K 2,3-epoxide reduction to vitamin K in vitro and in vivo. Dithiothreitol, an in vitro reductant for the vitamin K 2,3-epoxide reductase, antagonizes inhibition of the reductase by warfarin via mechanisms that have not been determined [Zimmermann, A., & Matschiner, J. T. (1974) Biochem. Pharmacol. 23, 1033-1040]. Experiments with rat hepatic microsomes were undertaken to characterize the interactions that exist between vitamin K 2,3-epoxide, warfarin, and dithiothreitol. Increasing concentrations of dithiothreitol decreased inhibition of the reductase by warfarin. When dithiothreitol was present prior to exposure of the reductase to warfarin, there was less inhibition than when the same concentration of dithiothreitol was present after its exposure to warfarin. Moreover, maximum inhibition of the reductase by warfarin occurred at a much slower rate when dithiothreitol was present initially. Inhibition of the reductase by warfarin was greater when the substrate concentration was 100 microM vitamin K 2,3-epoxide than when it was 10 microM epoxide. On the basis of these data, we conclude that (i) dithiothreitol reduces either directly or indirectly a critical disulfide within the reductase that it reoxidized during reduction of the epoxide substrate, (ii) warfarin and vitamin K 2,3-epoxide are not competitive with respect to one another, and (iii) warfarin binding, which produces inhibition, occurs solely to the disulfide form of the reductase. Once it is bound, warfarin inhibits further reduction of the critical disulfide by dithiothreitol. Dithiothreitol therefore antagonizes warfarin by maintaining the reductase in the reduced state.

• Hildebrandt EF, Suttie JW
• Mechanism of coumarin action: sensitivity of vitamin K metabolizing enzymes of normal and warfarin-resistant rat liver.
• Biochemistry. 1982; 21: 2406-11
• Display abstract

• The in vitro effects of two coumarin anticoagulants, warfarin and difenacoum, on rat liver microsomal vitamin K dependent carboxylase, vitamin K epoxidase, vitamin K epoxide reductase, and cytosolic vitamin K reductase (DT-diaphorase) from the livers of normal and a warfarin-resistant strain of rats have been determined. Millimolar concentrations of both coumarins are required to inhibit the carboxylase and epoxidase activities in both strains of rats. Sensitivity of DT-diaphorase to coumarin inhibition differs when a soluble or liposomal-associated substrate is used, but the diaphorases isolated from both strains of rats have comparable sensitivity. The anticoagulant difenacoum is an effective rodenticide in the warfarin-resistant strain of rats, and the only enzyme studied from warfarin-resistant rat liver that demonstrated a significant differential inhibition by the two coumarins used was the vitamin K epoxide reductase. This enzyme also showed the greatest sensitivity to coumarin inhibition among the enzymes studied. These results support the hypothesis that the physiologically important site of action of coumarin anticoagulants is the vitamin K epoxide reductase.

• Fasco MJ, Hildebrandt EF, Suttie JW
• Evidence that warfarin anticoagulant action involves two distinct reductase activities.
• J Biol Chem. 1982; 257: 11210-2
• Display abstract

• The dithiothreitol-dependent vitamin K and vitamin K 2,3-epoxide hepatic microsomal reductase activities of warfarin-susceptible and warfarin-resistant rats were compared to gain insight into the role(s) of these activities in vitamin K metabolism and function. In microsomes from resistant rats, 3- to 4-fold more warfarin was required to produce 50% inhibition (I50) of vitamin K reduction to vitamin K hydroquinone than in microsomes from susceptible rats. For the reduction of vitamin K 2,3-epoxide to vitamin K a 6-fold higher warfarin concentration was required. In microsomes from resistant rats, the I50 warfarin concentration required to inhibit gamma-carboxylation of microsomal precursor protein was also 4-fold higher with vitamin K as substrate and was 6-fold higher with the epoxide as substrate than in microsomes from susceptible rats. Collectively, these data suggest that the vitamin K reductase contributes to the metabolism of vitamin K in intact rats and that warfarin inhibition of both the vitamin K and vitamin K 2,3-epoxide reductases is involved in its anticoagulant effect.

• Fasco MJ, Principe LM
• R- and S-Warfarin inhibition of vitamin K and vitamin K 2,3-epoxide reductase activities in the rat.
• J Biol Chem. 1982; 257: 4894-901
• Display abstract

• Reduction of vitamin K 2,3-epoxide and vitamin K catalyzed by hepatic microsomal enzymes is required for normal, postribosomal, gamma-carboxyglutamate formation in the prothrombin complex Factors II, VII, IX, and X. The R- and S-warfarin enantiomers differentially inhibit (S-warfarin is 2 to 5 times more active) vitamin K function by mechanisms which have not been unambiguously determined. As a step toward determining the physiologically relevant site(s) of warfarin-antivitamin K activity we investigated in Wistar rats the effects of R- and S-warfarin on vitamin K 2,3-epoxide and vitamin K reductase activities and correlated them with effects on plasma concentrations of the Factors II, VII, and X. Based on the results of these studies we conclude that: 1) warfarin inhibition of the vitamin K 2,3-epoxide and vitamin K reductases is essentially irreversible; 2) S-warfarin stereoselectively inhibits both reductases in vivo but not in vitro; 3) the vitamin K reductase which utilizes dithiothreitol as cofactor in vitro is primarily responsible for vitamin K reduction to vitamin K hydroquinone under physiological conditions; 4) warfarin initially inhibits gamma-carboxyglutamate formation by inhibiting simultaneously the vitamin K 2,3-epoxide and vitamin K reductases; and 5) following enantiomer administration there is an apparent lack of correlation between the restoration of the reductase activities and the reinitiation of coagulation factor synthesis.

• Friedman PA, Griep AE
• In vitro inhibition of vitamin K dependent carboxylation by tetrachloropyridinol and the imidazopyridines.
• Biochemistry. 1980; 19: 3381-6
• Display abstract

• The compounds 2,3,5,6-tetrachloro-4-pyridinol (TCP) and the structurally related imidazopyridines (IP) cause hemorrhage and lower the plasma prothrombin level in animals. In vitro, TCP and the IP are more potent inhibitors of both the vitamin K dependent carboxylase which catalyzes the posttranslational gamma-carboxylation of specific glutamyl residues in proteins and the related vitamin K epoxidase activity than they are either of vitamin K epoxide reductase or of NAD-(P)H-K oxidoreductase. TCP and IP, as is the case with the coumarin and indandione anticoagulants, are competitive inhibitors of NAD(P)H-K oxidoreductae with respect to NADH. The epoxide reductase from coumarin-resistant rats is quite resistant to inhibition not only by warfarin but also by the IP, and to a lesser extent by TCP. When interpreted in light of published in vivo experiments, the data suggest that the principal site of anticoagulant action of the IP, but not TCP, is the epoxide reductase. The anticoagulant effect of TCP may be inhibition of the carboxylase itself. TCP is a significantly more potent inhibitor of the carboxylase and epoxidase than the IP; it inhibits both the enzymatic activities to the same degree with 50% inhibition observed at about 10(-5) M. Inhibition of the carboxylase by TCP is not competitive with respect to the pentapeptide substrate phenylalanyl-leucylglutamylglutamylleucine nor with respect to the following components of the in vitro carboxylase assay: imidazole, pyridoxal 5'-phosphate, dithiothreitol, KCl, sodium bicarbonate, oxygen, and vitamin K. The order of addition of components of the assay relative to the addition of inhibitor did not affect the degree of inhibition. Inhibition is readily reversed in experiments designed to dissociate an enzyme-inhibitor complex. Analysis of double-inhibitor experiments suggests that TCP and IP have the same binding site on the carboxylase.

• Bjornsson TD, Meffin PJ, Swezey SE, Blaschke TF
• Effects of clofibrate and warfarin alone and in combination on the disposition of vitamin K1.
• J Pharmacol Exp Ther. 1979; 210: 322-6
• Display abstract

• Clofibrate may interact with warfarin by potentiating its effects on vitamin K disposition. To examine this possibility, specifically labeled [3H]vitamin K was given intravenously to four healthy volunteers under conditions of no drug administration, administration of warfarin or clofibrate alone, or co-administration of both drugs. Clofibrate alone did not affect the disposition of tritiated vitamin K. Warfarin alone produced an accumulation in plasma of substantial amounts of vitamin K epoxide, a metabolite of vitamin K which is reconverted to vitamin K by a specific reductase. Although reconversion is apparently blocked to a large extent by warfarin, the plasma disappearance of tritiated vitamin K in the presence of warfarin is almost superimpossible to that observed in the absence of drugs. Clofibrate coadministration did not result in greater accumulation of vitamin K epoxide in plasma. These results indicate that clofibrate does not enhance the inhibition of the reductase enzyme. Analysis of the tritiated vitamin K plasma disappearance data indicates that the pool size of vitamin K in the body is small, and is turned over almost 10 times daily. The vitamin K epoxide data suggest that, in the absence of drugs, a relatively small proportion of the epoxide is reconverted to the vitamin.

• Whitlon DS, Sadowski JA, Suttie JW
• Mechanism of coumarin action: significance of vitamin K epoxide reductase inhibition.
• Biochemistry. 1978; 17: 1371-7

• Bell RG
• Metabolism of vitamin K and prothrombin synthesis: anticoagulants and the vitamin K--epoxide cycle.
• Fed Proc. 1978; 37: 2599-604
• Display abstract

• Vitamin K is primarily located in hepatic microsomes, where the vitamin K-dependent carboxylation in prothrombin synthesis occurs. Recent evidence supports the idea that the carboxylation is linked to the metabolism of the vitamin--specifically the cyclic interconversion of vitamin K and vitamin K epoxide. The primary site of action of coumarin and indandione anticoagulants appears to be an inhibition of the epoxide-to-vitamin K conversion in this cycle. There is a correlation between the inhibition of prothrombin synthesis and the regeneration of vitamin K from the epoxide by anticoagulants. In hamsters and warfarin-resistant rats prothrombin synthesis and the epoxide-K conversion are less sensitive to warfarin than in the normal rat. The epoxide-K conversion is impaired in resistant rats, which may explain their high vitamin K requirement. There is also a correlation between vitamin K epoxidation and vitamin K-dependent carboxylation, but the apparent link may be because vitamin K hydroquinone is an intermediate in the formation of the epoxide and also the active form in carboxylation. The vitamin K-epoxide cycle is found in extrahepatic tissues such as kidney, spleen, and lung and is inhibited by warfarin.

© 2021 EMBL | Privacy Policy