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

Wu, Sangwook; Chen, Xuejie; Jin, Da Yun; Stafford, Darrel W.; Pedersen, Lee G.; Tie, Jian Ke

doi:10.1182/blood-2018-01-830901

Cited by 42 publications

(38 citation statements)

References 56 publications

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“…The subcellular localization of reporter-proteins were examined by immunofluorescence confocal imaging, as previously described 22 . For examining the effect of the propeptide on reporter-protein carboxylation, different propeptide attached reporter-protein fusions were transiently expressed in COS-7 cells on coverslips.…”

Section: Immunofluorescence Confocal Imagingmentioning

confidence: 99%

Vitamin K-dependent carboxylation of coagulation factors: insights from a cell-based functional study

et al. 2019

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Ke Tie. Vitamin K-dependent carboxylation of coagulation factors: insights from a cell-based functional study. AbstractVitamin K-dependent carboxylation is a posttranslational modification essential for the biological function of coagulation factors. Defects in carboxylation are mainly associated with bleeding disorders. With the discovery of new vitamin K-dependent proteins, the importance of carboxylation now encompasses vascular calcification, bone metabolism, and other important physiological processes. Our current knowledge of carboxylation, however, comes mainly from in vitro studies carried out under artificial conditions, which have a limited usefulness in understanding the carboxylation of vitamin K-dependent proteins in native conditions. Using a recently established mammalian cell-based assay, we studied the carboxylation of coagulation factors in a cellular environment. Our results show that the coagulation factor's propeptide controls substrate binding and product releasing during carboxylation, and factor IX's propeptide appears to have the optimal affinity for efficient carboxylation. Additionally, non-conserved residues in the propeptide play an important role in carboxylation. A cell-based functional study of naturally occurring mutations in the propeptide successfully interpreted the clinical phenotype of warfarin's hypersensitivity during anticoagulation therapy in patients with these mutations. Unlike results obtained from in vitro studies, results from our cell-based study indicate that although the propeptide of osteocalcin cannot direct the carboxylation of the coagulation factor, it is required for osteocalcin's efficient carboxylation. This suggests that the coagulation factors may have a different mechanism of carboxylation from osteocalcin. Together, results from this study provide insight into efficiently controlling one physiological process, such as coagulation without affecting the other, like bone metabolism.

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Section: Immunofluorescence Confocal Imagingmentioning

confidence: 99%

Vitamin K-dependent carboxylation of coagulation factors: insights from a cell-based functional study

et al. 2019

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“…Whether human VKORC1 is organized in a four-TM structure, like the bacterial VKOR homologue, has been controversial, since the biochemical analysis of human VKORC1 topology generated conflicting conclusions, supporting either a four-TM [ 28 , 29 , 30 , 31 , 32 , 33 ] or a three-TM model [ 17 , 27 , 34 ]. Indeed, another model has been proposed for human VKORC1, in which the protein contains only three transmembrane helixes and where Cys43 and Cys51 are localized in the cytosol.…”

Section: Structure Of Vkorc1 and Vkorc1l1mentioning

confidence: 99%

VKORC1L1, An Enzyme Mediating the Effect of Vitamin K in Liver and Extrahepatic Tissues

Lacombe

Ferron

2018

Nutrients

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Vitamin K is an essential nutrient involved in the regulation of blood clotting and tissue mineralization. Vitamin K oxidoreductase (VKORC1) converts vitamin K epoxide into reduced vitamin K, which acts as the co-factor for the γ-carboxylation of several proteins, including coagulation factors produced by the liver. VKORC1 is also the pharmacological target of warfarin, a widely used anticoagulant. Vertebrates possess a VKORC1 paralog, VKORC1-like 1 (VKORC1L1), but until very recently, the importance of VKORC1L1 for protein γ-carboxylation and hemostasis in vivo was not clear. Here, we first review the current knowledge on the structure, function and expression pattern of VKORC1L1, including recent data establishing that, in the absence of VKORC1, VKORC1L1 can support vitamin K-dependent carboxylation in the liver during the pre- and perinatal periods in vivo. We then provide original data showing that the partial redundancy between VKORC1 and VKORC1L1 also exists in bone around birth. Recent studies indicate that, in vitro and in cell culture models, VKORC1L1 is less sensitive to warfarin than VKORC1. Genetic evidence is presented here, which supports the notion that VKORC1L1 is not the warfarin-resistant vitamin K quinone reductase present in the liver. In summary, although the exact physiological function of VKORC1L1 remains elusive, the latest findings clearly established that this enzyme is a vitamin K oxidoreductase, which can support γ-carboxylation in vivo.

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“…A homology model based on bacterial VKOR has four transmembrane 46 domains, but the quality of the homology model is unclear, as human VKOR has only 12% 47 sequence identity to bacterial VKOR. Moreover, experimental validation of VKOR topology 48 yielded mixed results: similar biochemical assays suggested either three-or four-49 transmembrane-domain topologies Tie et al, 2012;Wu et al, 2018). 50…”

Section: Introduction 31mentioning

confidence: 99%

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

Chiasson

Rollins

Stephany

et al. 2020

Preprint

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18Vitamin K epoxide reductase (VKOR) drives the vitamin K cycle, activating vitamin K-dependent 19 blood clotting factors. VKOR is also the target of the widely used anticoagulant drug, warfarin 20Despite VKOR's pivotal role in coagulation, its structure and active site remain poorly understood. 21In addition, VKOR variants can cause vitamin K-dependent clotting factor deficiency 2 or alter 22 on abundance and activity. While most variants show wild type-like activity, 25% show low 83 abundance or activity, which could confer warfarin sensitivity or cause disease in a homozygous 84 context. Finally, we analyzed warfarin resistance variants and found that they span a range of 85 abundances, indicating that increased abundance is an uncommon mechanism of warfarin 86 resistance. 87 88 RESULTS 89 Multiplexed measurement of VKOR variant abundance using VAMP-seq 90To measure the abundance of VKOR variants, we applied Variant Abundance by 91Massively Parallel sequencing (VAMP-seq), an assay we recently developed (Matreyek et al., 92 2018). In VAMP-seq, a protein variant is fused to eGFP with a short amino acid linker. If the 93 variant is stable and properly folded, then the eGFP fusion will not be degraded, and cells will 94 have high eGFP fluorescence. In contrast, if the variant causes the protein to misfold, protein 95 quality control machinery will detect and degrade the eGFP fusion, leading to a decrease in eGFP 96 signal (Fig. 1a). mCherry is also expressed from an internal ribosomal entry site (IRES) to control 97 for expression. Differences in abundance are measured on a flow cytometer using the ratio of 98 eGFP to mCherry signal. To determine whether VAMP-seq could be applied to VKOR, we fused 99 eGFP to VKOR N-or C-terminally and found that both orientations had high eGFP signal ( Figure 100 1-figure supplement 1). We compared N-terminally tagged wild type (WT) VKOR to R98W, a 101 variant that ablates a putative ER retention motif and reduces abundance (Czogalla et al., 2014), 102 and to TMD1Δ, a deletion of residues 10-30 which comprise the putative first transmembrane 103 domain (TMD1; Fig. 1b). Both reduced-abundance variants exhibited much lower eGFP:mCherry 104 ratios than WT, demonstrating that VAMP-seq could be applied to VKOR. 105We constructed a barcoded site-saturation mutagenesis VKOR library that covered 92.5% 106 of all 3,240 possible missense variants. To express this library in HEK293T cells we used a Bxb1 107 recombinase landing pad system we previously developed (Matreyek et al., 2017). In this system,

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

Cited by 42 publications

References 56 publications

Vitamin K-dependent carboxylation of coagulation factors: insights from a cell-based functional study

Vitamin K-dependent carboxylation of coagulation factors: insights from a cell-based functional study

VKORC1L1, An Enzyme Mediating the Effect of Vitamin K in Liver and Extrahepatic Tissues

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

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