Overexpression of protein disulfide isomerase enhances vitamin K epoxide reductase activity

Vitamin K epoxide reductase (VKOR) activity is catalyzed by the VKORC1 enzyme. It is the target of vitamin K antagonists (VKA). Numerous mutations of VKORC1 have been reported and have been suspected to confer resistance to VKA and/or affect its velocity. Nevertheless, the results between studies have been conflicting, the functional characterization of these mutations in a cell system being complex due to the interweaving of VKOR activity in the vitamin K cycle. In this study, a new cellular approach was implemented to globally evaluate the vitamin K cycle in the HEK293 cells. This global approach was based on the vitamin K quinone/vitamin K epoxide (K/KO) balance. In the presence of VKA or when the VKORC1/VKORC1L1 were knocked out, the K/KO balance decreased significantly due to an accumulation of vitamin KO. On the contrary, when VKORC1 was overexpressed, the balance remained unchanged, demonstrating a limitation of the VKOR activity. This limitation was shown to be due to an insufficient expression of the activation partner of VKORC1, as overexpressing the protein disulfide isomerase (PDI) overcomes the limitation. This study is the first to demonstrate a functional interaction between VKORC1 and the PDI enzyme.

The catalytic mechanism of vitamin K epoxide reduction in a cellular environment

Vitamin K epoxide reductases (VKOR) constitute a major family of integral membrane thiol oxidoreductases. In humans, VKOR sustains blood coagulation and bone mineralization through the vitamin K cycle. Previous chemical models assumed that the catalysis of human VKOR (hVKOR) starts from a fully reduced active site. This state, however, constitutes only a minor cellular fraction (5.6%). Thus, the mechanism whereby hVKOR catalysis is carried out in the cellular environment remains largely unknown. Here we use quantitative mass spectrometry (MS) and electrophoretic mobility analyses to show that KO likely forms a covalent complex with a cysteine mutant mimicking hVKOR in a partially oxidized state. Trapping of this potential reaction intermediate suggests that the partially oxidized state is catalytically active in cells. To investigate this activity, we analyze the correlation between the cellular activity and the cellular cysteine status of hVKOR. We find that the partially oxidized hVKOR has considerably lower activity than hVKOR with a fully reduced active site. Although there are more partially oxidized hVKOR than fully reduced hVKOR in cells, these two reactive states contribute about equally to the overall hVKOR activity, and hVKOR catalysis can initiate from either of these states. Overall, the combination of MS quantification and biochemical analyses reveal the catalytic mechanism of this integral membrane enzyme in a cellular environment. Furthermore, these results implicate how hVKOR is inhibited by warfarin, one of the most commonly prescribed drugs.

Multiplexed measurement of variant abundance and activity reveals VKOR topology, active site and human variant impact

Vitamin K epoxide reductase (VKOR) drives the vitamin K cycle, activating vitamin K-dependent blood clotting factors. VKOR is also the target of the widely used anticoagulant drug, warfarin. Despite VKOR’s pivotal role in coagulation, its structure and active site remain poorly understood. In addition, VKOR variants can cause vitamin K-dependent clotting factor deficiency or alter warfarin response. Here, we used multiplexed, sequencing-based assays to measure the effects of 2,695 VKOR missense variants on abundance and 697 variants on activity in cultured human cells. The large-scale functional data, along with an evolutionary coupling analysis, supports a four transmembrane domain topology, with variants in transmembrane domains exhibiting strongly deleterious effects on abundance and activity. Functionally constrained regions of the protein define the active site, and we find that, of four conserved cysteines putatively critical for function, only three are absolutely required. Finally, 25% of human VKOR missense variants show reduced abundance or activity, possibly conferring warfarin sensitivity or causing disease.

A cell-based high-throughput screen identifies drugs that cause bleeding disorders by off-targeting the vitamin K cycle

Drug-induced bleeding disorders contribute to substantial morbidity and mortality. Antithrombotic agents that cause unintended bleeding of obvious cause are relatively easy to control. However, the mechanisms of most drug-induced bleeding disorders are poorly understood, which makes intervention more difficult. As most bleeding disorders are associated with the dysfunction of coagulation factors, we adapted our recently established cell-based assay to identify drugs that affect the biosynthesis of active vitamin K–dependent (VKD) coagulation factors with possible adverse off-target results. The National Institutes of Health (NIH) Clinical Collection (NCC) library containing 727 drugs was screened, and 9 drugs were identified, including the most commonly prescribed anticoagulant warfarin. Bleeding complications associated with most of these drugs have been clinically reported, but the pathogenic mechanisms remain unclear. Further characterization of the 9 top-hit drugs on the inhibition of VKD carboxylation suggests that warfarin, lansoprazole, and nitazoxanide mainly target vitamin K epoxide reductase (VKOR), whereas idebenone, clofazimine, and AM404 mainly target vitamin K reductase (VKR) in vitamin K redox cycling. The other 3 drugs mainly affect vitamin K availability within the cells. The molecular mechanisms underlying the inactivation of VKOR and VKR by these drugs are clarified. Results from both cell-based and animal model studies suggest that the anticoagulation effect of drugs that target VKOR, but not VKR, can be rescued by the administration of vitamin K. These findings provide insights into the prevention and management of drug-induced bleeding disorders. The established cell-based, high-throughput screening approach provides a powerful tool for identifying new vitamin K antagonists that function as anticoagulants.

Multiplexed measurement of variant abundance and activity reveals VKOR topology, active site and human variant impact

ABSTRACTVitamin K epoxide reductase (VKOR) drives the vitamin K cycle, activating vitamin K-dependent blood clotting factors. VKOR is also the target of the widely used anticoagulant drug, warfarin Despite VKOR’s pivotal role in coagulation, its structure and active site remain poorly understood. In addition, VKOR variants can cause vitamin K-dependent clotting factor deficiency 2 or alter warfarin response. Here, we used multiplexed, sequencing-based assays to measure the effects of 2,695 VKOR missense variants on abundance and 697 variants on activity in cultured human cells. The large-scale functional data, along with an evolutionary coupling analysis, supports a four transmembrane domain topology, with variants in transmembrane domains exhibiting strongly deleterious effects on abundance and activity. Functionally constrained regions of the protein define the active site, and we find that, of four conserved cysteines putatively critical for function, only three are absolutely required. Finally, 25% of human VKOR missense variants show reduced abundance or activity, possibly conferring warfarin sensitivity or causing disease.

Association between polymorphisms in microRNA seed region and warfarin stable dose

BackgroundThe optimal dose of anticoagulant warfarin varies among patients to achieve the target international normalised ratio. Although genetic variations related to warfarin pharmacokinetics and vitamin K cycle are important factors associated with warfarin dose requirements, these variations do not completely explain the large interindividual variability observed in the most populations, suggesting that additional factors may contribute to this variability. microRNAs have recently been introduced as regulators of drug function genes, and therefore, may be involved in drug responses. In this study, we aimed to evaluate the possible association between variants in the seed region of microRNAs, which target the genes involved in the action of warfarin and warfarin dose requirement.Methods526 samples were collected from Iranian patients. Four selected polymorphisms in the seed region of microRNAs (rs2910164, rs66683138, rs12416605 and rs35770269 in miR-146a, miR-3622a, miR-938 and miR-449c, respectively) were genotyped by PCR-restriction fragment length polymorphism method.Resultsrs2910164 C/G in the seed region of miR-146a was associated with warfarin dose requirement (p<0.001); the patients with GG genotype had the higher mean dose of warfarin (40.6 mg/week, compared with 33.9 and 31.8 mg/week for GC and CC genotypes, respectively). The association of other polymorphisms with warfarin dose requirement was not statistically significant.Conclusionrs2910164 C/G in the seed region of miR-146a is associated with warfarin maintenance dose, likely by disrupting interaction between miR-146a and ATP-binding cassette subfamily B member 1 gene, ABCB1. Therefore, this polymorphism may possibly be a potential factor for assessment of warfarin dose requirements.

Аnti-inflammatory effects of vitamin K on women's health

This article presents the latest research data on the role of vitamin K in the implementation of its multiple non-classical extra-coagulation effects associated with the regulation of a number of physiological and pathological processes in the human body. In recent years, numerous studies have been performed on vitamin K function to better understand the effects of this micronutrient and its significance in various biological reactions. Vitamin K is well known to be a cofactor of the -carboxylation of a number of proteins, which is necessary for their activation and is part of the so-called vitamin K cycle. The cycle enzymes, metabolites and vitamin K-dependent proteins are identified and expressed in many cells and tissues of the human body: skin, lungs, liver, kidneys, vascular endothelium, nervous and bone tissues, reproductive (endometrium, ovaries, placenta) and immune systems. There were analyzed the main mechanisms of vitamin K action through vitamin K-dependent proteins. The results of epidemiological and experimental studies prove the association of reduced vitamin K levels with the increased risk of cardiovascular diseases, overall mortality, insulin resistance, metabolic syndrome, type 2 diabetes mellitus, progression of rheumatoid arthritis and osteoporosis. On the contrary, vitamin K increased intake has a positive effect on the immune and nervous systems, as well as on a number of other somatic pathologies, including breakdowns in the reproductive sphere. These data confirm the multifunctional role of vitamin K in various organs and systems of organism, presenting as high potential further studies in the field of determining vitamin K levels.

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.

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.

Use of Vitamin K Antagonists and Brain Morphological Changes in Older Adults: An Exposed/Unexposed Voxel-Based Morphometric Study

Background: Vitamin K antagonists (VKAs) are commonly used for their role in haemostasis by interfering with the vitamin K cycle. Since vitamin K also participates in brain physiology, this voxel-based morphometric study aimed to determine whether the duration of exposure to VKAs correlated with focal brain volume reduction in older adults. Methods: In this exposed/unexposed (1: 2) study nested within the GAIT (Gait and Alzheimer Interactions Tracking) cohort, 18 participants exposed to VKA (mean age 75 ± 5 years; 33.3% female; mean exposure 2,122 ± 1,799 days) and 36 matched participants using no VKA (mean age 75 ± 5 years; 33.3% female) underwent MRI scanning of the brain. Cortical grey and white matter volumes were automatically segmented using statistical parametric mapping. Age, gender, educational level, history of atrial fibrillation, type of MRI, and total intracranial volume were included as covariables. Results: The duration of exposure to VKA correlated inversely across the whole brain with the subvolumes of two clusters in the grey matter (right frontal inferior operculum and right precuneus) and one cluster in the white matter (left middle frontal gyrus). In contrast, the grade of white matter hyperintensities did not differ according to the use of VKA. Conclusion: We found focal atrophies in older adults exposed to VKA. These findings provide new insights elucidating the effects of VKAs on brain health and function in older adults.