Vitamin K Epoxide Reductase Research Articles

Target Discovery for New Antitubercular Drugs Using a Large Dataset of Growth Inhibitors from PubChem.

The number of drugs available for treatment of active tuberculosis is diminishing due to increased multidrug resistance selection in Mycobacterium tuberculosis leading to multiple (MDR) and extensively (XDR) resistant strains. Also, TB is treated with multiple drugs to minimize further resistance development, mandating a sustained effort to identify new lead compounds for treating drug-resistant TB and shortening time to cure for all TB infections. High throughput screening, a well-known approach to discovery of new leads, is conducted in two basic modes 1) using whole cells and screening for inhibition of growth, or whole cell reporter cells that signal when a specific pathway is perturbed, and 2) in vitro non-cell based enzyme or other functional assays for direct ligand-target binding. Combining high throughput screening for inhibitors of growth (to identify and chemically assess inhibitors active on whole cells), followed by target identification abrogates the problem of discovering new leads in non-cell based systems that are inactive on whole cells due to issues with target access (e.g., uptake). High throughput screening of 341,778 compounds by the National Institutes of Health identified 8,950 primary hit, growth inhibitors of M. tuberculosis. Final evaluation based on reproducibility, potency, medicinal chemistry inspection, and cytotoxicity on tissue culture cells identified 1,113 priority compounds. These data were deposited in PubChem, making data available to TB research labs for follow up studies on target identification. This effort led to the identification of compounds targeting Pks13, MmpL3, DprE1, AspS, EthA, GuaB2, nonreplicating cells, and VKOR (Vitamin K epoxide reductase).

Characterization of Warfarin Inhibition Kinetics Requires Stabilization of Intramembrane Vitamin K Epoxide Reductases

Intramembrane enzymes are often difficult for biochemical characterization. Human vitamin K epoxide reductase (VKOR) is the target of warfarin. However, this intramembrane enzyme becomes insensitive to warfarin inhibition in vitro, preventing the characterization of inhibition kinetics for decades. Here we employ structural biology methods to identify stable VKOR and VKOR-like proteins and purify them to near homogeneity. We find that the key to maintain their warfarin sensitivity is to stabilize their native protein conformation in vitro. Reduced glutathione drastically increases the warfarin sensitivity of a VKOR-like protein from Takifugu rubripes, presumably through maintaining a disulfide-bonded conformation. Effective inhibition of human VKOR-like requires also the use of LMNG, a mild detergent developed for crystallography to increase membrane protein stability. Human VKOR needs to be preserved in ER-enriched microsomes to exhibit warfarin sensitivity, whereas human VKOR purified in LMNG is stable only with pre-bound warfarin. Under these optimal conditions, warfarin inhibits with tight-binding kinetics. Overall, our studies show that structural biology methods are ideal for stabilizing intramembrane enzymes. Optimizing toward their inhibitor-binding conformation enables the characterization of enzyme kinetics in difficult cases.

Comparison of two reducing agents dithiothreitol and tris(3-hydroxypropyl)phosphine for in vitro kinetic assay of vitamin K epoxide reductase

Vitamin K epoxide reductase (VKOR) is a target enzyme for anticoagulants, such as warfarin, that are used as medicines or rodenticides. Assessing VKOR activity is required to ensure the proper usage of these drugs. Dithiothreitol (DTT) is a typical disulfide reductant that is used as a substrate for in vitro VKOR assays. However, DTT is considered problematic because of its side effects. Tris(3-hydroxypropyl)phosphine (THP) has been found to be a reliable alternative to DTT, as shown by kinetic analyses of the VKOR with them. THP showed significantly lower Vmax and Km values than those of DTT; however, there was no significant difference in their Vmax/Km and IC50 for warfarin.

The Reductive Activity of Human Liver Microsomes for Vitamin K Epoxides

Vitamin K (VK) is oxidized to vitamin K epoxide (VK-O) during the production of VK-dependent blood clotting factors. Thereafter, VK-O is reduced to VK by vitamin K epoxide reductase (VKOR) in the liver and reused. This reductive reaction is inhibited by warfarin, an oral anticoagulant. VK in nature is roughly divided into two types, VK1 (phylloquinone) and VK2 (menaquinone). Although their bioavailabilities and elimination half-lives from human blood differ, information on the influence of each VK on the effectiveness of warfarin is limited. In this study, the difference in the metabolism of VK1-O and MK4-O by VKOR was evaluated in an in vitro study using human liver microsomes. The results showed that the substrate affinity (1/Km), and the maximum reaction rate (Vmax) of the VKOR reduction was around 7 and 4 times higher for MK4-O than for VK1-O, respectively. The intrinsic clearance of MK4-O, obtained by dividing the Vmax value by the Km value, was about 30 times greater than that of VK1-O. According to these data, the production of VK-dependent blood coagulation factors can be considered to be dominated mainly by MK4-O, at least under normal conditions. We may thus have to be more careful about controlling the intake of MK4 than VK1 in patients receiving warfarin therapy.Keywords: vitamin K1 (phylloquinone), vitamin K2 (menaquinone), VKOR, warfarin

Enhanced functional recombinant factor IX production by human embryonic kidney cells engineered to overexpress VKORC1.

Replacement therapy with recombinant drugs is the main therapeutic strategy for hemophilia B patients. To reduce the production costs of recombinant coagulation factors, improvement of their expression and activity by enhancement of γ-carboxylation might be of interest. The expression and functional activity of vitamin K-dependent (VKD) coagulation proteins rely, in part, on the VKD process of γ-carboxylation that is mediated by the enzymes γ-carboxylase and vitamin K epoxide reductase (VKOR). Since the recombinant production of VKD proteins is hampered by the inefficiency of this enzymatic process, we specifically have examined the stable expression of functional blood coagulation factor IX (FIX) in HEK293 cells following transient overexpression of VKORC1 as an important part of VKOR component. Recombinant hFIX-producing human embryonic kidney (HEK) cells were transfected to overexpress VKORC1. Following reverse transcription polymerase chain reaction (RT-PCR) analysis, expression efficiency of the active hFIX was analyzed by performing enzyme-linked immunosorbent assay and coagulation test. In addition, to quantify γ-carboxylated recombinant FIX, the barium citrate method was used. Overexpression of VKORC1 in FIX-producing HEK cells, resulting in a 3.2-fold higher expression of functional FIX, which displayed a 1.4-fold enhanced specific activity. Moreover, a 3.9-fold enhanced recovery of fully γ-carboxylated FIX following barium citrate adsorption was achieved. Collectively, these findings indicate that the overexpression of VKORC1 results in the production of higher levels of functional hFIX in HEK293 cells. The increase of the VKORC1 as a supplier of γ-carboxylase seems to play a significant role in increasing the amount and efficiency of recombinant FIX production, thereby reducing the production costs.

Structural Basis of Vitamin K Antagonism

The vitamin K cycle supports blood coagulation, bone mineralization, and vascular calcium homeostasis. A key enzyme in this cycle, vitamin K epoxide reductase (VKOR), is the target of vitamin K antagonists (VKAs). Despite their extensive clinical use, the dose of VKAs (e.g., warfarin) is hard to regulate and overdose can lead to fatal bleeding. Improving the dose regulation requires understanding how VKAs inhibit VKOR, which is a membrane-embedded enzyme difficult to characterize with structural and biochemical studies. Here we achieve a long-standing goal of obtaining crystal structures of human VKOR with warfarin, which represents coumarin-based VKAs; with phenindione, which represents indandione-based VKAs; with superwarfarins, the most commonly used rodenticides; and with vitamin K epoxide in a reaction intermediate state. We have also solved structures of a VKOR-like homolog with warfarin, with vitamin K substrates, and without ligand. These structures show that human VKOR adopts an overall fold with four transmembrane helices (TM) and a large ER-luminal region. VKAs are bound at the active site of HsVKOR, which is formed by the surrounding four-TM bundle and a cap domain on top. The cap domain is stabilized by a linked anchor domain that interacts with the membrane surface. VKOR binds specifically to VKAs through hydrogen bonding to their diketone groups. Mutating VKOR residues recognizing the diketones render strong warfarin resistance. Except the hydrogen bonds, the binding pocket is largely hydrophobic. This pocket is incompatible with warfarin metabolite, explaining the inactivation of warfarin through CYP2C9 metabolism; CYP2C9 and VKOR genotypes can explain 30-50% of the patient variability in warfarin dose. In addition, the high potency of superwarfarins is due to the interaction of their side group with a tunnel where the isoprenyl chain of vitamin K is bound. For VKOR catalysis, the same residues affording the VKA-binding specificity also facilitate substrate reduction Initiation of the catalysis requires a reactive cysteine to form a substrate adduct. Interactions from this stably bound adduct induces a closed conformation, thereby triggering electron transfer to reduce the substrate. Importantly, the open to closed conformational change during catalysis similar to that induced by the binding of VKAs. Taken together, VKAs achieve inhibition through mimicking key interactions and conformational changes required for VKOR catalytic cycle. Understanding of these mechanisms will enable improved strategy to regulate warfarin dose and have a broad impact on thromboembolic diseases and bone disorders. Disclosures No relevant conflicts of interest to declare.

Avian interspecific differences in VKOR activity and inhibition: Insights from amino acid sequence and mRNA expression ratio of VKORC1 and VKORC1L1

Worldwide use of anticoagulant rodenticides (ARs) for rodents control has frequently led to secondary poisoning of non-target animals, especially raptors. In order to suggest some factors that may help considering the mechanism of the incidents, this study focused on the avian vitamin K 2, 3-epoxide reductase (VKOR) that is the target protein of ARs. We addressed the interspecific differences in VKOR activity and inhibition related to amino acid sequence and mRNA expression of VKORC1 and VKORC1-like1 (VKORC1L1). Poultry have been considered to be more tolerant to ARs than mammals. However, VKOR activity of owls, hawks, falcon and surprisingly, canaries, was lower and inhibited by warfarin more easily than that of chickens and turkeys. The amino acid sequence of VKORC1 and VKORC1L1 implied that the value of Ki for VKOR activity to ARs could depend on the amino acid at position 140 in the TYX warfarin-binding motif in VKORC1, and other amino acid mutations in VKORC1L1. The mRNA expression ratio of VKORC1:VKORC1L1 differed between turkey (8:1) and chicken (2:3) liver. VKORC1L1 has been reported to be resistant to warfarin compared to VKORC1. Hence, both the Ki of specific VKORC1 and VKORC1L1, and the mRNA expression ratio would cause avian interspecific difference of the VKOR inhibition. Our study also suggested the high inhibition of VKOR activities in raptors and surprisingly that in canaries as well. These factors are the most likely to contribute to the high sensitivity to ARs found in raptors.

Validation of Pharmacogenetic Testing Before Initiation of Warfarin Therapy

Warfarin is an oral anticoagulant used to prevent or treat clotting disorders associated with venous thrombosis, pulmonary embolism, atrial fibrilation, cardiac valve replacement, stroke and acute myocardial infarction. It is a vitamin K antagonist composed of S- and R- isomers. The more potent S-warfarin is metabolized by cytochrome 450 isoenzyme 2C9 (CYP2C9), encoded by CYP2C9 gene. Warfarin exerts its anticoagulants effect by inhibitingits target enzyme vitamin K epoxide reductase (VKOR), encoded by vitamin K epoxide reductase subunit 1 (VKOR1) gene. Genetic variation in the CYP2C9 and VKOR1 gene can affect warfarin efficacy and dose required to achieve stable International Normalization Ratio (INR). Specifically two variants in the CYP2CP gene (CYP2C9*2 and CYP2C9*3) result in an enzyme with reduced activity, leading to increased active warfarin levels. A variant in the VKORC1 gene (VKORC1-1639 G>A) can lead to reduced gene expression resulting in decresed level of VKOR. Together these three variants can account for 40-70% of the variability of warfarin dose. Carriers of variant alleles are at higher risk for bleeding complications, particularly at the induction of warfarin therapy. So, genotype-guided dosing algorithms would be better approximate for maintenance of warfarin dose than fixed-dose algorithms.
 University Heart Journal Vol. 15, No. 2, Jul 2019; 74-78

НУЖЕН ЛИ ЦЕНТРАЛИЗОВАННЫЙ КОНТРОЛЬ АНТИКОАГУЛЯНТНОЙ ТЕРАПИИ В ДОНБАССЕ?

В представленном обзоре освещаются показания, побочные эффекты и возможные стратегии ведения пациентов, принимающих антикоагулянты. Показанием к длительной или пожизненной терапии антикоагулянтами являются тромботическое событие и профилактика тромботических событий у пациентов с высоким риском, включая послеоперационные состояния, фибрилляцию предсердий и пациентов с искусственными клапанами. В этой терапии наиболее распространены антагонисты витамина К, такие как варфарин, которые блокируют витамин К-эпоксидредуктазу, предотвращая тем самым образование активной формы витамин К-зависимых факторов свертывания крови. Наиболее значимым и опасным побочным эффектом, связанным с приемом варфарина, является кровотечение, развитие которого напрямую связано с уровнем МНО. Осложнениями такой терапии могут быть также и тромботические осложнения при недостаточной дозе препарата. Это и объясняет необходимость строгого контроля МНО у пациентов, принимающих препараты соответствующего действия. В статье проанализирован опыт работы антикоагулянтных кабинетов в некоторых регионах Российской Федерации. Количество пациентов, требующих проведения такого лечения в Донбассе, достаточно велико, что диктует необходимость создания антикоагулянтных кабинетов на территории нашего региона.

Determining the necessity of phenyl ring π-character in warfarin.

Despite the difficulty in administering a safe dose regimen and reports of emerging resistance, warfarin (1) remains the most widely-used oral anticoagulant for the prevention and treatment of thrombosis in humans globally. Systematic substitution of the warfarin phenyl ring with either 1,3,5,7-cyclooctatetraene (COT) (2), cubane (3), cyclohexane (4) or cyclooctane (5) and subsequent evaluation against the target enzyme, vitamin K epoxide reductase (VKOR), facilitated interrogation of both steric and electronic properties of the phenyl pharmacophore. The tolerance of VKOR to further functional group modification (carboxylate 14, PTAD adduct 15) was also investigated. The results demonstrate the importance of both annulene conferred π-interactions and ring size in the activity of warfarin.

Evaluation of oral anticoagulants with vitamin K epoxide reductase in its native milieu

AbstractWarfarin, acenocoumarol, phenprocoumon, and fluindione are commonly prescribed oral anticoagulants for the prevention and treatment of thromboembolic disorders. These anticoagulants function by impairing the biosynthesis of active vitamin K–dependent coagulation factors through the inhibition of vitamin K epoxide reductase (VKOR). Genetic variations in VKOR have been closely associated with the resistant phenotype of oral anticoagulation therapy. However, the relative efficacy of these anticoagulants, their mechanisms of action, and their resistance variations among naturally occurring VKOR mutations remain elusive. Here, we explored these questions using our recently established cell-based VKOR activity assay with the endogenous VKOR function ablated. Our results show that the efficacy of these anticoagulants on VKOR inactivation, from most to least, is: acenocoumarol > phenprocoumon > warfarin > fluindione. This is consistent with their effective clinical dosages for stable anticoagulation control. Cell-based functional studies of how each of the 27 naturally occurring VKOR mutations responds to these 4 oral anticoagulants indicate that phenprocoumon has the largest resistance variation (up to 199-fold), whereas the resistance of acenocoumarol varies the least (<14-fold). Cell-based kinetics studies show that fluindione appears to be a competitive inhibitor of VKOR, whereas warfarin is likely to be a mixed-type inhibitor of VKOR. The anticoagulation effect of these oral anticoagulants can be reversed by the administration of a high dose of vitamin K, apparently due to the existence of a different enzyme that can directly reduce vitamin K. These findings provide new insights into the selection of oral anticoagulants, their effective dosage management, and their mechanisms of anticoagulation.

The pediatric acenocoumarol dosing algorithm: the Children Anticoagulation and Pharmacogenetics Study

Background The large variability in dose requirement of vitaminK antagonists is well known. For warfarin, pediatric dosing algorithms have been developed to predict the correct dose for a patient;however,this is not the case for acenocoumarol. Objectives To develop dosing algorithms for pediatric patients receiving acenocoumarol with and without genetic information. Methods The Children Anticoagulation and Pharmacogenetics Study was designed as a multicenter retrospective follow-up study in Dutch anticoagulation clinics and children's hospitals. Pediatric patients who used acenocoumarol between 1995 and 2014 were selected for inclusion. Clinical information and saliva samples for genotyping of the genes encoding cytochromeP450 (CYP)2C9, vitaminK epoxide reductase complex subunit1 (VKORC1), CYP4F2, CYP2C18 and CYP3A4 were collected. Linear regression was used to analyze their association with the log mean stable dose. A stable period was defined as three or more consecutive International Normalized Ratio measurements within the therapeutic range over a period of ≥3weeks. Results In total, 175 patients were included in the study, of whom 86 had a stable period and no missing clinical information (clinical cohort; median age 8.9years, and 49% female). For 80 of these 86 patients, genetic information was also available (genetic cohort). The clinical algorithm, containing body surface area and indication, explained 45.0% of the variability in dose requirement of acenocoumarol. After addition of the VKORC1, CYP2C9, and CYP2C18 genotypes to the algorithm, this increased to 61.8%. Conclusions These findings show that clinical factors had the largest impact on the required dose of acenocoumarol in pediatric patients. Nevertheless, genetic factors, and especially VKORC1, also explained a significant part of the variability.

Identification of the Thioredoxin Partner of Vitamin K Epoxide Reductase in Mycobacterial Disulfide Bond Formation.

Disulfide bonds influence the stability and activity of many proteins. In Escherichia coli, the DsbA and DsbB enzymes promote disulfide bond formation. Other bacteria, including the Actinobacteria, use instead of DsbB the enzyme vitamin K epoxide reductase (VKOR), whose gene is found either fused to or in the same operon as a dsbA-like gene. Mycobacterium tuberculosis and other Gram-positive actinobacteria secrete many proteins with even numbers of cysteines to the cell envelope. These organisms have predicted oxidoreductases and VKOR orthologs. These findings indicate that such bacteria likely form disulfide bonds in the cell envelope. The M. tuberculosisvkor gene complements an E. colidsbB deletion strain, restoring the oxidation of E. coli DsbA. While we have suggested that the dsbA gene linked to the vkor gene may express VKOR's partner in mycobacteria, others have suggested that two other extracytoplasmic oxidoreductases (DsbE or DsbF) may be catalysts of protein disulfide bond formation. However, there is no direct evidence for interactions of VKOR with either DsbA, DsbE, or DsbF. To identify the actual substrate of VKOR, we identified two additional predicted extracytoplasmic DsbA-like proteins using bioinformatics analysis of the M. tuberculosis genome. Using the five potential DsbAs, we attempted to reconstitute disulfide bond pathways in E. coli and in Mycobacterium smegmatis, a close relative of M. tuberculosis Our results show that only M. tuberculosis DsbA is oxidized by VKOR. Comparison of the properties of dsbA- and vkor-null mutants in M. smegmatis shows parallels to the properties of dsb mutations in E. coliIMPORTANCE Disulfide bond formation has a great impact on bacterial pathogenicity. Thus, disulfide-bond-forming proteins represent new targets for the development of antibacterials, since the inhibition of disulfide bond formation would result in the simultaneous loss of the activity of several classes of virulence factors. Here, we identified five candidate proteins encoded by the M. tuberculosis genome as possible substrates of the M. tuberculosis VKOR protein involved in disulfide bond formation. We then reconstituted the mycobacterial disulfide bond formation pathway in E. coli and showed that of the five candidates, only M. tuberculosis DsbA is efficiently oxidized by VKOR in E. coli We also present evidence for the involvement of VKOR in DsbA oxidation in M. smegmatis.

Key Pathways and Regulators of Vitamin K Function and Intermediary Metabolism.

Vitamin K (VK) is an essential cofactor for the post-translational conversion of peptide-bound glutamate to γ-carboxyglutamate. The resultant vitamin K-dependent proteins are known or postulated to possess a variety of biological functions, chiefly in the maintenance of hemostasis. The vitamin K cycle is a cellular pathway that drives γ-carboxylation and recycling of VK via γ-carboxyglutamyl carboxylase (GGCX) and vitamin K epoxide reductase (VKOR), respectively. In this review, we show how novel molecular biological approaches are providing new insights into the pathophysiological mechanisms caused by rare mutations of both GGCX and VKOR. We also discuss how other protein regulators influence the intermediary metabolism of VK, first through intestinal absorption and second through a pathway that converts some dietary phylloquinone to menadione, which is prenylated to menaquinone-4 (MK-4) in target tissues by UBIAD1. The contribution of MK-4 synthesis to VK functions is yet to be revealed.

Investigation of hepatic warfarin metabolism activity in rodenticide-resistant black rats (Rattus rattus) in Tokyo by in situ liver perfusion

Anti-blood coagulation rodenticides, such as warfarin, have been used all over the world. They inhibit vitamin K epoxide reductase (VKOR), which is necessary for producing several blood clotting factors. This inhibition by rodenticides results in lethal hemorrhage in rodents. However, heavy usage of these agents has led to the appearance of rodenticide-resistant rats. There are two major mechanisms underlying this resistance, i.e., mutation of the target enzyme of warfarin, VKOR, and enhanced metabolism of warfarin. However, there have been few studies regarding the hepatic metabolism of warfarin, which should be related to resistance. To investigate warfarin metabolism in resistant rats, in situ liver perfusion of warfarin was performed with resistant black rats (Rattus rattus) from Tokyo, Japan. Liver perfusion is an in situ methodology that can reveal hepatic function specifically with natural composition of the liver. The results indicated enhanced hepatic warfarin hydroxylation activity compared with sensitive black rats. On the other hand, in an in vitro microsomal warfarin metabolism assay to investigate kinetic parameters of cytochrome P450, which plays a major role in warfarin hydroxylation, the Vmax of resistant rats was slightly but significantly higher compared to the results obtained in the in situ study. These results indicated that another factor like electron donators may also contribute to the enhanced metabolism in addition to high expression of cytochrome P450.

Uticaj genetičkog polimorfizma citohroma R-450 izoformi 2S9 i 1A2 na metabolizam varfarina

The influence of genetic polymorphism on the scope of drug metabolism is now well-known, since the metabolism of drugs depends on the amount of active enzyme in human tissues, primarily in the liver. The aim of the paper is to process data related to genetic polymorphism of isoforms of cytochrome enzymes that are important for metabolism and dosing of warfarin, considering that serious adverse effects can sometimes end with a fatal outcome. In addition, the systemic review article pointed to the importance of pharmacogenomic studies prior to the introduction of warfarin in therapy. A systematic research of literature using the medical databases 'Pub Med' and ‘Google Scholar’ found a total of 251 publications. The results included eight publications that met the criteria for inclusion. The extraction of the most important data from all works necessary for an adequate evaluation was performed and presented in seven tables containing general data of studies and pharmacokinetic parameters related to the metabolism of warfarin. The therapeutic effect of coumarin derivatives such as warfarin is influenced by genetic factors, but also other factors related to the patient. The frequent mutations of the gene encoding the cytochrome P450 isoform CYP2C9, but also the vitamin K-epoxide reductase with one or more polymorphic combinations, are responsible for reducing the required dose of warfarin or even for resistance to warfarin. Although genotyping data improves the accuracy of dose prediction, the improvement in controlling the anticoagulant effect and warfarin metabolism has yet to be proven in terms of effectiveness and cost-effectiveness in future prospective clinical studies.

Vitamin K epoxide reductase and its paralogous enzyme have different structures and functions

Vitamin K epoxide reductase (VKOR) is an essential enzyme for vitamin K-dependent carboxylation, while the physiological function of its paralogous enzyme VKOR-like (VKORL) is yet unknown. Although these two enzymes share approximately 50% protein sequence homology, the membrane topology of VKOR is still in debate. Here, we explored the differences in the membrane topology and disulfide-linked oligomerization of these two enzymes. Results from mutating the critical amino acid residues in the disputed transmembrane (TM) regions revealed that the second TM domain in the proposed 4-TM model of VKOR does not function as an authentic TM helix; supporting VKOR is a 3-TM protein, which is different from VKORL. Additionally, altering the loop sequence between the two conserved cysteine residues of VKORL affects its activity, supporting the notion that the conserved loop cysteines of VKORL are involved in its active site regeneration. However, a similar mutation in VKOR does not affect its enzymatic activity. Finally, our results show that although both VKOR and VKORL form disulfide-linked oligomers, the cysteine residues involved in the oligomerization appear to be different. Overall, the structural and functional differences between VKOR and VKORL shown here indicate that VKORL might have a different physiological function other than recycling vitamin K.

Long‐term outcomes of elderly patients with CYP2C9 and VKORC1 variants treated with vitamin K antagonists

Background The long-term effect of polymorphisms of the vitamin K-epoxide reductase (VKORC1) and the cytochrome P450 enzyme gene (CYP2C9) on clinical outcomes remains unclear. Objectives We examined the association between CYP2C9/VKORC1 variants and long-term clinical outcomes in a prospective cohort study of elderly patients treated with vitamin K antagonists for venous thromboembolism (VTE). Methods We followed 774 consecutive patients aged ≥ 65 years with acute VTE from nine Swiss hospitals for a median duration of 30 months. The median duration of initial anticoagulant treatment was 9.4 months. The primary outcome was the time to any clinical event (i.e. the composite endpoint of overall mortality, major and non-major bleeding, and recurrent VTE. Results Overall, 604 (78%) patients had a CYP2C9 or VKORC1 variant. Three hundred and thirty-four patients (43.2%) had any clinical event, 119 (15.4%) died, 100 (12.9%) had major and 167 (21.6%) non-major bleeding, and 100 had (12.9%) recurrent VTE. After adjustment, CYP2C9 (but not VKORC1) variants were associated with any clinical event (hazard ratio [HR], 1.34; 95% confidence interval [CI], 1.08-1.66), death (HR, 1.74; 95% CI, 1.19-2.52) and clinically relevant non-major bleeding (sub-hazard ratio [SHR], 1.39; 95% CI, 1.02-1.89), but not with major bleeding (SHR, 1.03; 95% CI, 0.69-1.55) or recurrent VTE (SHR, 0.95; 95% CI, 0.62-1.44). Patients with genetic variants had a slightly lower anticoagulation quality. Conclusions CYP2C9 was associated with long-term overall mortality and non-major bleeding. Although genetic variants were associated with a slightly lower anticoagulation quality, there was no relationship between genetic variants and major bleeding or VTE recurrence.

Reoxidation of the Thiol-Disulfide Oxidoreductase MdbA by a Bacterial Vitamin K Epoxide Reductase in the Biofilm-Forming Actinobacterium Actinomyces oris.

Posttranslocational protein folding in the Gram-positive biofilm-forming actinobacterium Actinomyces oris is mediated by a membrane-bound thiol-disulfide oxidoreductase named MdbA, which catalyzes oxidative folding of nascent polypeptides transported by the Sec translocon. Reoxidation of MdbA involves a bacterial vitamin K epoxide reductase (VKOR)-like protein that contains four cysteine residues, C93/C101 and C175/C178, with the latter forming a canonical CXXC thioredoxin-like motif; however, the mechanism of VKOR-mediated reoxidation of MdbA is not known. We present here a topological view of the A. oris membrane-spanning protein VKOR with these four exoplasmic cysteine residues that participate in MdbA reoxidation. Like deletion of the VKOR gene, alanine replacement of individual cysteine residues abrogated polymicrobial interactions and biofilm formation, concomitant with the failure to form adhesive pili on the bacterial surface. Intriguingly, the mutation of the cysteine at position 101 to alanine (C101A mutation) resulted in a high-molecular-weight complex that was positive for MdbA and VKOR by immunoblotting and was absent in other alanine substitution mutants and the C93A C101A double mutation and after treatment with the reducing agent β-mercaptoethanol. Consistent with this observation, affinity purification followed by immunoblotting confirmed this MdbA-VKOR complex in the C101A mutant. Furthermore, ectopic expression of the Mycobacterium tuberculosis VKOR analog in the A. oris VKOR deletion (ΔVKOR) mutant rescued its defects, in contrast to the expression of M. tuberculosis VKOR variants known to be nonfunctional in the disulfide relay that mediates reoxidation of the disulfide bond-forming catalyst DsbA in Escherichia coli Altogether, the results support a model of a disulfide relay, from its start with the pair C93/C101 to the C175-X-X-C178 motif, that is required for MdbA reoxidation and appears to be conserved in members of the class ActinobacteriaIMPORTANCE It has recently been shown in the high-GC Gram-positive bacteria (or Actinobacteria) Actinomyces oris and Corynebacterium diphtheriae that oxidative folding of nascent polypeptides transported by the Sec machinery is catalyzed by a membrane-anchored oxidoreductase named MdbA. In A. oris, reoxidation of MdbA requires a bacterial VKOR-like protein, and yet, how VKOR mediates MdbA reoxidation is unknown. We show here that the A. oris membrane-spanning protein VKOR employs two pairs of exoplasmic cysteine residues, including the canonical CXXC thioredoxinlike motif, to oxidize MdbA via a disulfide relay mechanism. This mechanism of disulfide relay is essential for pilus assembly, polymicrobial interactions, and biofilm formation and appears to be conserved in members of the class Actinobacteria, including Mycobacterium tuberculosis.

VKORC1-1639 G&gt;A Polymorphism and the Risk of Non-Variceal Upper Gastrointestinal Bleeding

Background &amp; Aims: The mutations in the gene that encodes vitamin K epoxide reductase (VKOR) enzyme are responsible for low levels of vitamin K. The purpose of this study was to evaluate whether the presence of the VKORC1 -1639 G&gt; A polymorphism is a risk factor for non-variceal upper gastrointestinal bleeding (UGIB) in patients without concomitant therapy with vitamin K antagonists.Methods: This case-control study comprised 163 consecutive patients diagnosed with UGIB and 178 controls, in whom the diagnosis of UGIB was excluded. The following data were recorded: age, gender, alcohol consumption, smoking, history of UGIB, nonsteroidal anti-inflammatory drugs (NSAIDs) or low-dose aspirin consumption. Genetic analysis included genotyping for the VKORC1 -1639 G&gt;A polymorphism.Results: History of UGIB (OR 3.463, CI95% 1.463-8.198, p=0.005), smoking (OR 2.498, CI95% 1.358-4.597, p=0.003), alcohol consumption (OR 3.283, CI95% 1.796-6.000, p&lt;0.001), use of NSAIDs (OR 4.542, CI95% 2.502-8.247, p&lt;0.001) or of low-dose aspirin (OR 2.390, CI95% 1.326-4.310), and the VKORC1 -1639 G&gt; A AA genotype (OR 1.364, CI95% 0.998-1.863, p=0.05) were associated with an increased risk of UGIB. The risk of UGIB was analyzed in patients with genotype AA who used aspirin or NSAIDs. The genotype AA has not kept its status of independent risk factor (p=0.3). In subjects with NSAIDs/aspirin therapy and genotype AA there was a two times higher chance of UGIB compared to those under NSAIDs/aspirin therapy alone (OR 7.6 vs. 3.6, p&lt;0.001).Conclusion: Patients with non-variceal UGIB caused by the use of NSAIDs or low-dose aspirin are more frequent carriers of the VKORC1 -1639 G&gt;A AA genotype, as compared to those without UGIB.