Vitamin K epoxide reductase significantly improves carboxylation in a cell line overexpressing factor X

Previously we reported that we could increase the fraction of carboxylated factor X by reducing the affinity of the propeptide for its binding site on human gamma glutamyl carboxylase. We attributed this to an increased turnover rate. However, even with the reduced affinity propeptide, when sufficient overproduction of factor X is achieved, there is still a significant fraction of uncarboxylated recombinant factor X. We report here that the factor X of such a cell line was only 52% carboxylated but that the fraction of carboxylated factor X could be increased to 92% by coexpressing the recently identified gene for vitamin K epoxide reductase. Because vitamin K is in excess in both the untransfected and vitamin K epoxide reductase (VKOR)-transfected cells, the simplest explanation for this result is that VKOR catalyzes both the reduction of vitamin K epoxide to vitamin K and the conversion of vitamin K to vitamin K hydroquinone. In addition to its mechanistic relevance, this observation has practical implications for overproducing recombinant vitamin K-dependent proteins for therapeutic use.     

The carboxylation efficiency of the vitamin K‐dependent clotting factors: studies with factor IX

Summary. Haemophilia B is characterized by a deficiency of the γ‐carboxylated protein, factor IX (FIX). As a first step to optimize a gene therapy strategy to treat haemophilia B, we employed a previously described approach (Biochemistry 2000;39: 14322) of altering the propeptide of vitamin K‐dependent proteins in vitro, to improve the carboxylation efficiency of FIX. Both native FIX and FIX with a prothrombin propeptide (proPT‐FIX) produced recombinant FIX in vitro following transfection of their cDNAs into human embryonic kidney (HEK) 293 cells. Using hydroxyapatite chromatography to separate carboxylated from uncarboxylated FIX, we are able to show that >90% of FIX is γ‐carboxylated and that substituting the propeptide of prothrombin into FIX does not further increase the relative amounts of carboxylated material. These results demonstrate that the nature of the propeptide, per se is not the sole determinant of optimal carboxylation of FIX in our expression system in HEK 293 cells.     

Vitamin K epoxide reductase: Fresh blood for oral anticoagulant therapies

INTRODUCTION: Vitamin K epoxide reductase complex subunit I (VKORC1) is a key enzyme in the vitamin K cycle, cofactor required for the activation of vitamin K-dependent clotting factors. EXEGESIS: VKORC1 recycles vitamin K 2,3 epoxide back to active vitamin K hydroquinone, an important factor for the carboxylation step of clotting factors. VKORC1 is the target enzyme of inhibition by oral anticoagulants or anti-vitamin K (warfarin, acenocoumarol). CONCLUSION: We show here the clinical consequences of genetic variations of VKORC1 during VKA therapy.     

Inhibition of bacterial disulfide bond formation by the anticoagulant warfarin

Blood coagulation in humans requires the activity of vitamin K epoxide reductase (VKOR), the target of the anticoagulant warfarin (Coumadin). Bacterial homologs of VKOR were recently found to participate in a pathway leading to disulfide bond formation in secreted proteins of many bacteria. Here we show that the VKOR homolog from the bacterium Mycobacterium tuberculosis, the causative agent of human tuberculosis, is inhibited by warfarin and that warfarin-resistant mutations of mycobacterial VKOR appear in similar locations to mutations found in human patients who require higher doses of warfarin. Deletion of VKOR results in a severe growth defect in mycobacteria, and the growth of M. tuberculosis is inhibited by warfarin. The bacterial VKOR homolog may represent a target for antibiotics and a model for genetic studies of human VKOR. We present a simple assay in Escherichia coli, based on a disulfide-sensitive β-galactosidase, which can be used to screen for stronger inhibitors of the M. tuberculosis VKOR homolog.     

Vitamin K epoxide reductase prefers ER membrane-anchored thioredoxin-like redox partners

Vitamin K epoxide reductase (VKOR) sustains blood coagulation by reducing vitamin K epoxide to the hydroquinone, an essential cofactor for the γ-glutamyl carboxylation of many clotting factors. The physiological redox partner of VKOR remains uncertain, but is likely a thioredoxin-like protein. Here, we demonstrate that human VKOR has the same membrane topology as the enzyme from Synechococcus sp., whose crystal structure was recently determined. Our results suggest that, during the redox reaction, Cys43 in a luminal loop of human VKOR forms a transient disulfide bond with a thioredoxin (Trx)-like protein located in the lumen of the endoplasmic reticulum (ER). We screened for redox partners of VKOR among the large number of mammalian Trx-like ER proteins by testing a panel of these candidates for their ability to form this specific disulfide bond with human VKOR. Our results show that VKOR interacts strongly with TMX, an ER membrane-anchored Trx-like protein with a unique CPAC active site. Weaker interactions were observed with TMX4, a close relative of TMX, and ERp18, the smallest Trx-like protein of the ER. We performed a similar screen with Ero1-α, an ER-luminal protein that oxidizes the Trx-like protein disulfide isomerase. We found that Ero1-α interacts with most of the tested Trx-like proteins, although only poorly with the membrane-anchored members of the family. Taken together, our results demonstrate that human VKOR employs the same electron transfer pathway as its bacterial homologs and that VKORs generally prefer membrane-bound Trx-like redox partners.     

A fibroblast cell culture model to study vitamin K metabolism and the inhibition of vitamin K epoxide reductase by known and suspected antagonists

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.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Structural features of the kringle domain determine the intracellular degradation of under-γ-carboxylated prothrombin: Studies of chimeric rat/human prothrombin

Vitamin K antagonists such as warfarin inhibit the vitamin K-dependent γ-glutamyl carboxylation during protein processing and block the secretion of under-γ-carboxylated prothrombin (FII) in the rat but not in the human or bovine. Under-γ-carboxylated prothrombin is also secreted from warfarin-treated human (HepG2) cell cultures but is degraded in the endoplasmic reticulum in warfarin-treated rat (H-35) cell cultures. This differential response to warfarin has been shown to be determined by the structural difference in the proteins rather than by the origin of the cell line. When recombinant rat prothrombin (rFII) and human prothrombin (hFII) were expressed in a transformed human kidney cell line (HEK293), secretion of rFII but not hFII was drastically decreased in response to warfarin. To determine the structural signal required for this differential response, chimeric cDNAs with the propeptide/Gla domains, kringle domain, and serine protease domain exchanged between rFII and hFII were generated (FII_RHH and FII_HRR, FII_RRH and FII_HHR, FII_RHR and FII_HRH) and expressed in both warfarin-treated HEK293 cells and HepG2 cells. The presence of the hFII kringle domain changed the stability of rFII to that of hFII, and the rFII kringle domain changed the stability of hFII to that of rFII. The kringle domain therefore is critical in determining the metabolic fate of under-γ-carboxylated prothrombin precursors during processing. Prothrombin contains two kringle structures, and expression of additional rFII/hFII chimeras (FII_HrhH and FII_HhrH, FII_RrhR, and FII_RhrR) was used to determine that the first of the two kringles plays a more important role in the recognition process.     

Combination index of the concentration and in vivo antagonism activity of racemic warfarin and its metabolites to assess individual drug responses

Journal of Thrombosis and Thrombolysis - The present study was undertaken to examine whether in vivo vitamin K epoxide reductase complex 1 (VKOR) “actual” antagonism activity,...     

扩张型心肌病致病基因LMNA新突变E82K对细胞周期影响的实验研究

目的探讨国人家族性扩张型心肌病LMNA致病基因中发现的新突变E82K位点对细胞周期的影响。方法构建野生型及突变型LMNA基因的真核表达载体并与空载体分别转染HEK293细胞,抗生素筛选得到稳定转染的细胞株,用0．8mmoL／L过氧化氢诱导对照组以及分别转染空载体、野生型LMNA基因和突变型LMNA基因的细胞组24h,流式细胞仪检测各组细胞的细胞周期。结果转染突变型LMNA基因的细胞细胞周期被阻滞于G0／G1期,而其他3组均被阻滞于G2／M期。结论LMNA基因E82K突变可以阻止过氧化氢诱导的细胞周期向G2／M期积聚。     

The association of vitamin K status with warfarin sensitivity at the onset of treatment

We investigated the association of vitamin K status with warfarin sensitivity among 40 orthopaedic patients beginning perioperative algorithm-dosed warfarin. Baseline vitamin K status was assessed using plasma vitamin K-1 and vitamin K-1 2,3 epoxide concentrations, and a questionnaire-based estimation of usual vitamin K intake. Warfarin sensitivity was assessed as the increase in the International Normalized Ratio (INR) after two doses of 5 mg of warfarin and as the 4-d accumulation of under-gamma-carboxylated prothrombin (PIVKA-II), adjusted for warfarin dose requirement. Multivariate models were used to assess vitamin K variables as predictors of warfarin sensitivity. The mean INR increase was 0.53 U and the mean PIVKA-II increase was 771 ng/ml/mg warfarin. Demographic factors were not associated with warfarin response. For each 1 standard deviation (SD) lower value of plasma vitamin K-1, but not the other vitamin K variables, the INR rose 0.24 U (P < or = 0.01). A higher usual vitamin K intake and plasma vitamin K-1, and lower plasma vitamin K-1 2,3 epoxide, were all associated with a lower PIVKA-II increase over 4 d. Respective differences in PIVKA-II accumulation per SD increase of each variable were -165, -218 and 236 ng/ml/mg warfarin (all P < or = 0.05). We concluded that dietary and biochemical measures of vitamin K status were associated with early warfarin sensitivity.     

Impaired carboxylation of osteocalcin in warfarin-treated patients

The total circulating osteocalcin and ratio of inactive (noncarboxylated; GLU) to active (carboxylated; GLA) form of circulating osteocalcin were measured in patients receiving long term warfarin treatment (n = 20), age-matched control patients not receiving warfarin treatment (n = 10), and normal subjects before and after the administration of 30 mg warfarin (n = 7). There was no significant difference in the total osteocalcin concentrations between the control patients and the patients receiving long term warfarin treatment, and it did not significantly change after warfarin ingestion in the normal subjects. The GLU/GLA ratio was significantly increased (P less than 0.002) in the patients receiving long term warfarin treatment compared with that in the control patients. There was a significant increase (P less than 0.01) in the GLU/GLA ratio after warfarin ingestion in the normal subjects. This study demonstrates that osteocalcin carboxylation in humans is a vitamin K-dependent process and that circulating osteocalcin is structurally altered by warfarin administration. This finding has pathophysiological implications for the fetal warfarin embryopathy syndrome, bone disease associated with chronic liver diseases, and possibly for osteoporosis, in which vitamin K deficiency has been implicated.     

Prothrombin synthesis and degradation in rat hepatoma (H-35) cells: effects of warfarin

Vitamin K is a substrate for the enzyme catalyzing the carboxylation of specific glutamyl residues to gamma-carboxyglutamyl residues in hepatic precursors of a limited number of plasma proteins, including prothrombin. The gamma-carboxylation of these proteins can be blocked by the anticoagulant warfarin; and in the bovine and human, warfarin treatment results in the secretion of under-gamma-carboxylated forms of prothrombin into plasma. In the rat, this response is not seen, but plasma prothrombin concentrations are drastically decreased. This response has now been studied in rat hepatoma (H-35) cells in which prothrombin secretion is decreased 90% by incubation in the presence of warfarin. Neither prothrombin mRNA levels nor the apparent rate of prothrombin message translation were decreased when cells were cultured in the presence of warfarin rather than of vitamin K. The pool of intracellular prothrombin precursors is increased threefold by warfarin treatment, and this pool is rapidly secreted when vitamin K is administered. In contrast, continued incubation in the presence of warfarin resulted in the degradation of 60% of this pool in 24 hours. When transport of secretory proteins to the golgi apparatus was blocked with Brefeldin A, this precursor pool was gamma-carboxylated in the presence of vitamin K and no degradation occurred. Lysosomal enzyme inhibitors did not block the degradation, and the data suggest that, in rat hepatocytes, under-gamma-carboxylated prothrombin is specifically targeted to a pathway of protein degradation located in the endoplasmic reticulum.     

Vitamin K-dependent Proteins,Warfarin, and Vascular Calcification

Vitamin K-dependent proteins (VKDPs) require carboxylation to become biologically active. Although the coagulant factors are the most well-known VKDPs, there are many others with important physiologic roles. Matrix Gla Protein (MGP) and Growth Arrest Specific Gene 6 (Gas-6) are two particularly important VKDPs, and their roles in vascular biology are just beginning to be understood. Both function to protect the vasculature; MGP prevents vascular calcification and Gas-6 affects vascular smooth muscle cell apoptosis and movement. Unlike the coagulant factors, which undergo hepatic carboxylation, MGP and Gas-6 are carboxylated within the vasculature. This peripheral carboxylation process is distinct from hepatic carboxylation, yet both are inhibited by warfarin administration. Warfarin prevents the activation of MGP and Gas-6, and in animals, induces vascular calcification. The relationship of warfarin to vascular calcification in humans is not fully known, yet observational data suggest an association. Given the high risk of vascular calcification in those patients with chronic kidney disease, the importance of understanding warfarin''s effect on VKDPs is paramount. Furthermore, recognizing the importance of VKDPs in vascular biology will stimulate new areas of research and offer potential therapeutic interventions.The most well-known vitamin K-dependent proteins (VKDPs) are the coagulant factors II,VII, IX, and X. Produced by the liver, they are converted into their biologically active forms by the carboxylation of glutamic acid residues, a process requiring vitamin K as a cofactor. By interfering with this carboxylation process, warfarin has become the mainstay of anticoagulant therapy. However, beyond these coagulant factors, there are other VKDPs with widespread physiologic activities. Recent studies have focused on two particularly important VKDPs, Matrix Gla protein (MGP) and Growth Arrest Specific gene 6 (Gas-6) protein. These proteins have many diverse biologic functions, yet with the recognition that they are produced by vascular smooth muscle cells, their roles in vascular biology are being increasingly explored. MGP functions primarily as a vascular calcification inhibitor. Gas-6 affects vascular smooth muscle cell movement and apoptosis. Together, these proteins constitute a new mechanism of local vascular regulation, where the blood vessel defends itself against injury and participates in self-repair. A failure of these local mechanisms might be an important first step in a cascade of events culminating in vascular calcification, and supports the notion that vascular calcification is an active, regulated process.To become biologically active, both MGP and Gas-6 undergo carboxylation, a process that occurs at the blood vessel level. Like hepatic carboxylation, this peripheral carboxylation is inhibited by the administration of warfarin, yet whereas warfarin''s anticoagulant effect is well known, its effect on the vasculature is less certain. Emerging in vitro and whole animal data suggest that warfarin may induce vascular calcification, a potential relationship that has not yet been well studied in humans.Given the widespread use of warfarin, understanding the full spectrum of its biologic effects is important. This article reviews the general physiology of VKDPs and explores a potential relationship between warfarin and vascular calcification in susceptible individuals.     

Familial deficiency of vitamin K‐dependent clotting factors

Summary. Combined deficiency of vitamin K‐dependent clotting factors II, VII, IX and X (and proteins C, S, and Z) is usually an acquired clinical problem, often resulting from liver disease, malabsorption, or warfarin overdose. A rare inherited form of defective γ‐carboxylation resulting in early onset of bleeding was first described by McMillan and Roberts in 1966 and subsequently has been termed ‘vitamin K‐dependent clotting factor deficiency’ (VKCFD). Biochemical and molecular studies identify two variants of this autosomal recessive disorder: VKCFD1, which is associated with point mutations in the γ‐glutamylcarboxylase gene (GGCX), and VKCFD2, which results from point mutations in the vitamin K epoxide reductase gene (VKOR). Bleeding ranges in severity from mild to severe. Therapy includes high oral doses of vitamin K for prophylaxis, usually resulting in partial correction of factor deficiency, and episodic use of plasma infusions or prothrombin complex concentrate. Recent molecular studies have the potential to further our understanding of vitamin K metabolism, γ‐carboxylation, and the functional role this post‐translational modification has for other proteins. The results may also provide potential targets for molecular therapeutics and pharmacogenetics.     

Homozygosity mapping of a second gene locus for hereditary combined deficiency of vitamin K-dependent clotting factors to the centromeric region of chromosome 16

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.     

Vitamin K-dependent carboxylation of the carboxylase

Vitamin K-dependent (VKD) proteins require modification by the VKD-γ-glutamyl carboxylase, an enzyme that converts clusters of glus to glas in a reaction that requires vitamin K hydroquinone, for their activity. We have discovered that the carboxylase also carboxylates itself in a reaction dependent on vitamin K. When pure human recombinant carboxylase was incubated in vitro with ¹⁴CO₂ and then analyzed after SDS/PAGE, a radiolabeled band corresponding to the size of the carboxylase was observed. Subsequent gla analysis of in vitro-modified carboxylase by base hydrolysis and HPLC showed that all of the radioactivity could be attributed to gla residues. Quantitation of gla, asp, and glu residues indicated 3 mol gla/mol carboxylase. Radiolabeled gla was acid-labile, confirming its identity, and was not observed if vitamin K was not included in the in vitro reaction. Carboxylase carboxylation also was detected in baculovirus(carboxylase)-infected insect cells but not in mock-infected insect cells, which do not express endogenous VKD proteins or carboxylase. Finally, we showed that the carboxylase was carboxylated in vivo. Carboxylase was purified from recombinant carboxylase BHK cells cultured in the presence or absence of vitamin K and analyzed for gla residues. Carboxylation of the carboxylase only was observed with carboxylase isolated from BHK cells cultured in vitamin K, and 3 mol gla/mol carboxylase were detected. Analyses of carboxylase and factor IX carboxylation in vitro suggest a possible role for carboxylase carboxylation in factor IX turnover, and in vivo studies suggest a potential role in carboxylase stability. The discovery of carboxylase carboxylation has broad implications for the mechanism of VKD protein carboxylation and Warfarin-based anti-coagulant therapies that need to be considered both retrospectively and in the future.     

Vitamin K metabolism and vitamin K1 status in human liver samples: a search for inter-individual differences in warfarin sensitivity

Summary. 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, 150 about 0·1 μ m . 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.     

Discrimination of normal and abnormal prothrombin and protein C in plasma using a calcium ion-inhibited monoclonal antibody to a common epitope on several vitamin K-dependent proteins

Vitamin K deficiency or administration of vitamin K antagonists results in the biosynthesis of abnormal des-gamma-carboxy forms of the vitamin K-dependent proteins. Monoclonal antibody H-11 binds several vitamin K- dependent proteins at a determinant that includes the first two residues of gamma-carboxyglutamic acid. Antibody H-11 binds fully carboxylated prothrombin and protein C in the presence of EDTA but binding is inhibited by the divalent metal ions, calcium, magnesium, and manganese. By contrast, des-gamma-carboxy prothrombin and protein C bind antibody H-11 the same in the presence of EDTA or calcium ion. Antibody H-11 thus appears to bind a conserved antigenic site containing gamma-carboxyglutamic acid that in the presence of divalent metal ion undergoes a conformational transition. This ability of antibody H-11 to bind des-gamma-carboxy prothrombin and protein C in the presence of calcium ion allowed the development of an immunoassay for these proteins in plasma. Prothrombin and protein C from stably anticoagulated individuals receiving warfarin were characterized by their ability to bind antibody H-11 in the presence of calcium ion. Binding of prothrombin and protein C to antibody H-11 in the presence of calcium correlated temporally with warfarin administration. The inability of calcium ion to inhibit binding of antibody H-11 to abnormal prothrombin and protein C in plasma suggests that the circulating forms of both proteins following warfarin administration cannot undergo the metal ion-dependent conformational transition that includes sequence residues 1 through 12.