scholarly journals Functional Study of the Vitamin K Cycle Enzymes in Live Cells

2017 ◽  
pp. 349-394 ◽  
Author(s):  
J.-K. Tie ◽  
D.W. Stafford
Keyword(s):  
Vitamin K ◽  
Live Cells ◽  
Functional Study ◽  
Vitamin K Cycle

scholarly journals Functional study of the vitamin K cycle in mammalian cells

Blood ◽  
2011 ◽  
Vol 117 (10) ◽  
pp. 2967-2974 ◽  
Author(s):  
Jian-Ke Tie ◽  
Da-Yun Jin ◽  
David L. Straight ◽  
Darrel W. Stafford
Keyword(s):  
Cell Lines ◽  
Vitamin K ◽  
Mammalian Cells ◽  
Cell System ◽  
Factor Ix ◽  
Hek293 Cells ◽  
Functional Study ◽  
Reporter Protein ◽  
Quinone Oxidoreductase ◽  
Vitamin K Cycle

Abstract We describe a cell-based assay for studying vitamin K–cycle enzymes. A reporter protein consisting of the gla domain of factor IX (amino acids 1-46) and residues 47-420 of protein C was stably expressed in HEK293 and AV12 cells. Both cell lines secrete carboxylated reporter when fed vitamin K or vitamin K epoxide (KO). However, neither cell line carboxylated the reporter when fed KO in the presence of warfarin. In the presence of warfarin, vitamin K rescued carboxylation in HEK293 cells but not in AV12 cells. Dicoumarol, an NAD(P)H-dependent quinone oxidoreductase 1 (NQO1) inhibitor, behaved similarly to warfarin in both cell lines. Warfarin-resistant vitamin K epoxide reductase (VKOR-Y139F) supported carboxylation in HEK293 cells when fed KO in the presence of warfarin, but it did not in AV12 cells. These results suggest the following: (1) our cell system is a good model for studying the vitamin K cycle, (2) the warfarin-resistant enzyme reducing vitamin K to hydroquinone (KH2) is probably not NQO1, (3) there appears to be a warfarin-sensitive enzyme other than VKOR that reduces vitamin K to KH2, and (4) the primary function of VKOR is the reduction of KO to vitamin K.

scholarly journals Bioavailability and Chemical/Functional Aspects of Synthetic MK-7 vs Fermentation-Derived MK-7 in Randomised Controlled Trials

2017 ◽  
Vol 87 (5-6) ◽  
pp. 1-15 ◽  
Author(s):  
Mona Møller ◽  
Ingrid M. Fange Gjelstad ◽  
Ingebjørg Baksaas ◽  
Tone Grande ◽  
Inger Reidun Aukrust ◽  
...  
Keyword(s):  
Confidence Interval ◽  
Vitamin K ◽  
Healthy Subjects ◽  
Biological Effects ◽  
Daily Intake ◽  
Functional Study ◽  
Randomised Controlled ◽  
Double Blinded ◽  
Synthetic Form ◽  
K Activity

Abstract. We investigated the bioavailability of a synthetic form of the vitamin K2 molecule menaquinone-7 (MK-7) in a randomised single-blinded two-way cross-over study. Healthy subjects (20 – 66 years of age) took a single 180 μg dose of synthetic MK-7 or fermentation-derived MK-7, and serum MK-7 concentrations were monitored for 72 hours to calculate AUC(0 – 72 h) and Cmax. We also compared the biological effects of placebo, fermentation-derived MK-7 (90 μg) and 3 doses of synthetic MK-7(45, 90 and 180 μg) in a randomised double-blinded parallel study. Healthy subjects (20 – 60 years of age) took one of the supplements daily for 43 days, and the fraction of carboxylated osteocalcin (OC) was compared between day 1 and day 43 as a marker for vitamin K activity. In the bioavailability study, the 90 % confidence interval for the ratio of the AUC(0-72 h) values for synthetic and fermentationderived MK-7 was 83 – 111, indicating bioequivalence. The 90 % confidence interval for the Cmax ratio was 83 – 131. The serum concentrations of carboxylated OC and undercarboxylated OC were increased (p = 0.01) and reduced (p = 0.02), respectively, after daily intake of 180 μg of synthetic MK-7 for 43 days, indicating increased vitamin K activity. Across both studies, only 1 participant reported an adverse event (dry mouth; 180 μg synthetic MK-7 group, functional study) that was considered possibly related to synthetic MK-7 supplementation. Our findings provide evidence that the tested synthetic form of MK-7 is bioequivalent to fermentation-derived MK-7, exhibits vitamin K activity and is well tolerated in healthy subjects.

Warfarin Resistance Is Associated with a Protein Component of the Vitamin K 2,3-Epoxide Reductase Enzyme Complex in Rat Liver

1998 ◽  
Vol 80 (07) ◽  
pp. 128-133 ◽  
Author(s):  
Dean Cain ◽  
Susan Hutson ◽  
Reidar Wallin
Keyword(s):  
Vitamin K ◽  
Genetic Resistance ◽  
The United States ◽  
Enzyme Complex ◽  
Laboratory Rats ◽  
Vitamin K Cycle ◽  
Epoxide Reductase ◽  
Warfarin Resistance ◽  
Gst Activity

SummaryWarfarin, 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.

scholarly journals Warfarin and vitamin K epoxide reductase: a molecular accounting for observed inhibition

Blood ◽  
2018 ◽  
Vol 132 (6) ◽  
pp. 647-657 ◽  
Author(s):  
Sangwook Wu ◽  
Xuejie Chen ◽  
Da-Yun Jin ◽  
Darrel W. Stafford ◽  
Lee G. Pedersen ◽  
...  
Keyword(s):  
Vitamin K ◽  
Md Simulation ◽  
Functional Study ◽  
Binding Motif ◽  
Vitamin K Epoxide Reductase ◽  
Stacking Interaction ◽  
Key Points ◽  
Epoxide Reductase

Key Points Warfarin reversibly inhibits VKOR by forming a T-shaped stacking interaction with residue Y139 of the proposed TYA warfarin-binding motif. Warfarin-resistant nonbleeding phenotype for patients bearing VKOR mutations explained by MD simulation and cell-based functional study.

VKORC1 deficiency in mice causes early postnatal lethality due to severe bleeding

2009 ◽  
Vol 101 (06) ◽  
pp. 1044-1050 ◽  
Author(s):  
Gabriele Spohn ◽  
Andre Kleinridders ◽  
F. Thomas Wunderlich ◽  
Matthias Watzka ◽  
Frank Zaucke ◽  
...  
Keyword(s):  
Vitamin K ◽  
Knockout Mice ◽  
Severe Bleeding ◽  
Clotting Factors ◽  
Vitamin K Cycle ◽  
Severe Deficiency ◽  
Epoxide Reductase ◽  
Bone Calcification

SummaryVitamin K hydroquinone is oxidised to the epoxide form (K>O) during vitamin K-dependent posttranslational γ-glutamyl carboxylation resulting in biological active so called vitamin K-dependent proteins. In turn, K>O is reduced by the enzyme VKORC1 (vitamin K epoxide reductase complex component 1) to complete the vitamin K cycle. To investigate the biological role of VKORC1 in vivo, we generated VKORC1 knockout mice. Homozygous VKORC1-deficient mice developed normally until birth. Within 2–20 days after birth, the knockout mice died due to extensive, predominantly intracerebral haemorrhage. Bleeding resulted from a severe deficiency of γ-carboxylated clotting factors. This lethal phenotype could be rescued by oral administration of vitamin K. Additionally, morphometric analysis of the limbs in VKORC1-deficient animals revealed reduced length of bone calcification relative to wild-type control mice. The observed phenotype of VKORC1 knockout mice excludes the existence of other enzymes with VKOR activity that can substitute to supply vitamin K hydroquinone required for maturation of blood clotting factors. Thus, our study underscores the essential role of VKORC1 in vitamin K-dependent γ-glutamyl carboxylation.

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

Blood ◽  
2020 ◽  
Vol 136 (7) ◽  
pp. 898-908
Author(s):  
Xuejie Chen ◽  
Caihong Li ◽  
Da-Yun Jin ◽  
Brian Ingram ◽  
Zhenyu Hao ◽  
...  
Keyword(s):  
Vitamin K ◽  
High Throughput ◽  
High Throughput Screening ◽  
Molecular Mechanisms ◽  
Coagulation Factors ◽  
Bleeding Complications ◽  
Bleeding Disorders ◽  
Drug Induced ◽  
Vitamin K Cycle ◽  
Established Cell

Abstract 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.

scholarly journals A cellular system for quantitation of vitamin K cycle activity: structure-activity effects on vitamin K antagonism by warfarin metabolites

Blood ◽  
2014 ◽  
Vol 123 (4) ◽  
pp. 582-589 ◽  
Author(s):  
Jamil A. Haque ◽  
Matthew G. McDonald ◽  
John D. Kulman ◽  
Allan E. Rettie
Keyword(s):  
Vitamin K ◽  
Activity Analysis ◽  
Factor Ix ◽  
Hek 293 ◽  
Vitamin K Cycle ◽  
Structure Activity ◽  
Activity Structure ◽  
293 Cells ◽  
Key Points

Key Points Factor IX glutamyl carboxylation in engineered HEK 293 cells recapitulates in vivo anticoagulant inhibition of vitamin K cycle activity. Warfarin metabolite structure-activity analysis on vitamin K cycle antagonism determines their contributions to in vivo anticoagulation.

VKORc1 Is Under-Expressed in Skeletal Muscle of Humans, Dogs and Mice: Potential Implications for Ectopic Coagulation Factor Expression in Pre-Clinical and Therapeutic Applications

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1477-1477
Author(s):  
Courtney T Connolly ◽  
Armida Faella ◽  
Timothy C. Nichols ◽  
Katherine A. High ◽  
Valder R. Arruda ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Vitamin K ◽  
Specific Activity ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Biologically Active ◽  
Transcript Levels ◽  
Vitamin K Cycle

Abstract Post-translational modifications of coagulation factors in the liver are essential for function. The vitamin K dependent coagulation proteins (VKCPs) require vitamin K to undergo gamma carboxylation of the glutamic residues in their Gla domain by gamma-glutamyl carboxylase [GGCX]. The vitamin K is then recycled by the action of epoxide reductase [VKORc1] and/or quinone reductase [NQO1]. The hemostatic importance of the vitamin K “cycle” is evidenced by patients who may suffer bleeding complications when anticoagulated with warfarin, which targets the vitamin K cycle. Moreover, the ability of a variety of VKCPs to secrete a biologically active product depends on the removal of their propeptide by the action of the intracellular endoprotease furin [FURIN gene]. Previous in vitro work on recombinant coagulation Factor IX, which is used for hemophilia B treatment, has connected these two processing steps by showing that endogenous VKORc1 as well as FURIN can be limiting factors in high-yield expression systems. In vivo, skeletal muscle (in contrast to liver) has been utilized to express low levels of coagulation Factor IX in the first hemophilia B gene therapy clinical trial. However, our experiments in mice demonstrated that the specific activity of muscle-synthesized Factor IX via gene transfer decreased at the high levels of FIX expression by a limited muscle area (Schuettrumpf J. et al., Blood 2005). These results suggest that in vitro and in vivo expression of biologically-active VKCPs outside the liver may be limited by the host cell post-translational modification machinery. Here, we performed a systematic study to determine the expression profiles of the vitamin K cycle and furin endoprotease genes in human liver and muscle, compared to the mouse. We also established these profiles in two hemophilic dogs, given the extensive use of this animal model in gene-based hemophilia therapies. RNA from liver and skeletal muscle was used as a template for reverse transcription and the subsequent relative quantification of the GGCX, VKORc1, NQO1, and FURIN genes by qPCR in each tissue using a housekeeping reporter gene. For this, a variety of housekeeping genes were investigated in all three species to identify ones with similar transcript levels in both liver and muscle tissue. We identified the housekeeping genes HPRT1, beta actin, and 18s rRNA as equivalently expressed in the liver and skeletal muscle of human, mouse, and dog, respectively. The relative mRNA transcript quantification of the vitamin K cycle genes in humans showed that the transcript levels of GGCX were similar in liver and muscle. In contrast, both VKORc1 and NQO1 were under-expressed in muscle vs. liver (69.5 ± 4.9% and 67.8 ± 12.5%, respectively, P<0.01). In the mouse, VKORc1 transcript levels in the muscle were reduced to 73.8 ± 9.9% vs. liver (P<0.05), while GGCX and NQO1 exhibited similar transcript levels in both tissues. In the dog, we observed a dramatic reduction in VKORc1 and GGCX transcript levels in the muscle vs. liver (11.8 ± 4.2% and 29.5 ± 15.8%, respectively, P<0.01). Surprisingly, NQO1 transcript levels were 253.8 ± 156.7% higher in muscle than liver (P<0.05). Lastly, in all three species tested, transcript levels for FURIN were similar in both muscle and liver. Our results indicate that VKORc1, a key enzyme in the vitamin K cycle, is consistently under-expressed in the skeletal muscle of humans as well as in mice and hemophilic dogs. In contrast, FURIN transcripts are similarly abundant in the liver and muscle of all three species tested. These suggest that the vitamin K cycle but not propeptide processing by furin can be a limiting factor in the secretion of biologically active muscle-expressed VKCPs. As a result, our observations provide (1) a plausible explanation for the inverse relationship between specific activity and Factor IX expression levels in mice following Factor IX gene transfer, and (2) further support for the mouse and dog as useful models for therapies that depend on the muscle-derived expression of VKCPs. Disclosures No relevant conflicts of interest to declare.

Retinal vascular occlusion and polymorphisms in genes coding for components of vitamin K cycle

2016 ◽  
Vol 132 (4) ◽  
pp. 94
Author(s):  
Sh. S. Kakhktsyan ◽  
L. K. Moshetova ◽  
K. I. Turkina ◽  
D. A. Sychev
Keyword(s):  
Vitamin K ◽  
Vascular Occlusion ◽  
Vitamin K Cycle ◽  
Retinal Vascular Occlusion

scholarly journals Stimulation of the dithiol-dependent reductases in the vitamin K cycle by the thioredoxin system. Strong synergistic effects with protein disulphide-isomerase

1992 ◽  
Vol 281 (1) ◽  
pp. 255-259 ◽  
Author(s):  
B A M Soute ◽  
M M C L Groenen-van Dooren ◽  
A Holmgren ◽  
J Lundström ◽  
C Vermeer
Keyword(s):  
Vitamin K ◽  
Synergistic Effects ◽  
Liver Microsomes ◽  
Bovine Liver ◽  
Secretory Cells ◽  
Protein Disulphide Isomerase ◽  
Vitamin K Cycle ◽  
Thioredoxin System

It has been shown previously that the thioredoxin system (thioredoxin + thioredoxin reductase + NADPH) may replace dithiothreitol (DTT) as a cofactor for vitamin KO and K reductase in salt-washed detergent-solubilized bovine liver microsomes. Here we demonstrate that the system can be improved further by adding protein disulphide-isomerase (PDI) to the components mentioned above. Moreover, NADPH may be replaced by reduced RNAase as a hydrogen donor. In our in vitro system the various protein cofactors were required at concentrations 2-5 orders of magnitude lower than that of DDT, whereas the maximal reaction rate was about 3-fold higher. PDI stimulated the thioredoxin-driven reaction about 10-fold, with an apparent Km value of 8 microM. These data suggest that in the vitro system the formation of disulphide bonds is somehow linked to the vitamin K-dependent carboxylation of glutamate residues. In vivo, both disulphide formation and vitamin K-dependent carboxylation are post-translational modifications taking place at the luminal side of the endoplasmic reticulum of mammalian secretory cells. The possibility that the reactions are also coupled in vivo is discussed.

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