VKORC1 Mutations Research Articles

Distribution of anticoagulant resistance in the brown rat in Belgium

Anticoagulant resistance is known as one of the major factors interfering with rodent control. Within this context we investigated the distribution of anticoagulant resistance in Flanders, northern Belgium. From 2003 to 2005, we tested 691 rats from different locations with blood clotting response tests for their susceptibility to the anticoagulant compounds warfarin, bromadiolone and difenacoum. Of these, 119 were also screened for a mutation in the VKORC1 gene that is suspected to be responsible for anticoagulant resistance. Warfarin resistant rats were found in the western and eastern parts of Flanders. The same distribution pattern was found for bromadiolone with the exception of the south-eastern area, where this form of resistance was largely absent. We detected difenacoum resistance in only six rats and did not observe any resistant rats in the central part of Flanders. Susceptible rats were found all over Flanders. Genetic analyses showed that anticoagulant
 resistance in Belgium was related to two different mutations in VKORC1, namely Y139F and L120Q. Our results indicate that rodent control should be regionally tailored to be most effective.

Sensitivity assessment of diphacinone by pharmacokinetic analysis in invasive black rats in the Bonin (Ogasawara) Archipelago, Japan

The Bonin Archipelago is a United Nations Educational, Scientific and Cultural Organization's World Natural Heritage Site in Japan with a unique ecosystem; however, the invasive rodents preying on endemic species have been a significant concern. The anticoagulant rodenticide, diphacinone, sprayed by the Ministry of the Environment, has succeeded; however, its repeated use leads to rodenticide resistance. This study evaluated the sensitivity by in vivo pharmacokinetics/pharmacodynamics (PK/PD) analysis and physiologically-based pharmacokinetic modeling to diphacinone in black rats (Rattus rattus) captured on the Bonin Archipelago in February 2022. The Bonin rats exhibited prolonged coagulation time after diphacinone administration. They recovered earlier than susceptible black rats, indicating that Bonin rats were less susceptible, though there were no genetic mutations in Vkorc1, the target enzyme of diphacinone. After the administration of diphacinone, hepatic expression levels of Fsp1, identified as the vitamin K reductase, was decreased, however, the Bonin rats exhibited the most minor suppression. The PK analysis showed that the excretion capacity of the Bonin rats was lower than that of the resistant black rats. In the PBPK modeling, the resistant black rats showed higher clearance than the Bonin and susceptible black rats due to high hepatic metabolic capacity. The Bonin rats demonstrated slow absorption and relatively low clearance. This study highlighted the reduced rodenticide-sensitive tendency of wild black rats in the Bonin Archipelago at an in vivo phenotype level. At the same time, they do not have known rodenticide resistance mechanisms, such as hepatic metabolic enhancement or Vkorc1 mutations. It is crucial to monitor the biological levels to evaluate rodenticide sensitivity accurately.

Acute kidney injury interacts with VKORC1 genotype on initiative warfarin dose among heart surgery recipients: a real-world research

Patients who receive heart valve surgery need anticoagulation prophylaxis to reduce the risk of thrombosis. Warfarin often is a choice but its dosage varies due to gene and clinical factors. We aim to study, among them, if there is an interaction between acute kidney injury and two gene polymorphisms from this study. We extracted data of heart valve surgery recipients from the electronic health record (EHR) system of a medical center. The primary outcome is about the average daily dose of warfarin, measured as an additive interaction effect (INTadd) between acute kidney injury (AKI) and warfarin-related gene polymorphisms. The confounders, including age, sex, body surface area (BSA), comorbidities (i.e., atrial fibrillation [AF], hypertension [HTN], congestive heart failure [CHF]), serum albumin level, warfarin-relevant gene polymorphism (i.e., CYP2C9, VKORC1), prosthetic valve type (i.e., metal, bio), and warfarin history were controlled via a multivariate-linear regression model. The study included 200 patients, among whom 108 (54.00%) are female. Further, the mean age is 54.45 years, 31 (15.50%) have CHF, and 40 (20.00%) patients were prescribed concomitant amiodarone, which potentially overlays with the warfarin prophylaxis period. During the follow-up, AKI occurred in 30 (15.00%) patients. VKORC1 mutation (1639G>A) occurred in 25 (12.50%) patients and CYPC29 *2 or *3 mutations presented in 20 patients (10.00%). We found a significant additive interaction effect between AKI and VKORC1 (− 1.17, 95% CI − 1.82 to − 0.53, p = 0.0004). This result suggests it is probable that there is an interaction between acute kidney injury and the VKORC1 polymorphism for the warfarin dose during the initial period of anticoagulation prophylaxis.

Design of a structure-activity relationship model of vitamin K epoxide reductase (VKORC1) inhibitors combining chemical synthesis of new compounds, enzymatic assays and molecular modelling

Vitamin K antagonists (VKAs) anticoagulants have been used since the 1950s as medicines and rodenticides. These molecules are mainly 4-hydroxycoumarin derivatives and act by inhibiting the vitamin K epoxide reductase (VKORC1), an endoplasmic reticulum membrane resident enzyme. However, many VKORC1 mutations have been reported over the last decade, inducing VKAs resistances and thus treatments failures. Although studies have reported experimental and computational investigations of VKAs based on VKORC1 structural homology models, the development of new effective anticoagulants has been quite complex due to the lack of structural data and reliable structure–activity relationships. However, the recent publication of VKORC1 crystal structure provides new information for further studies. Based on these findings, we combined chemical synthesis, enzymatic assays and molecular modelling methods to design a structure–activity relationship (SAR) model. Our results proved that the lipophilicity, the membrane permeability of inhibitors and their affinity towards human VKORC1 enzyme are the main characteristics for potent anticoagulants. Our SAR model managed to rank compounds according to their ability to inhibit the human VKORC1. Such a tool might constitute an alternative to evaluate new molecules potency before their chemical synthesis and biological assessment and might assist the development of new VKAs.

Investigation of anticoagulant rodenticide resistance induced by Vkorc1 mutations in rodents in Lebanon

Anticoagulant rodenticides (AR) remain the most effective chemical substances used to control rodents in order to limit their agricultural and public health damage in both rural and urban environments. The emergence of genetically based resistance to AR worldwide has threatened effective rodent control. This study gives a first overview of the distribution and frequency of single nucleotide polymorphism in the vitamin K epoxide reductase subcomponent 1 (Vkorc1) gene in rodents in Lebanon. In the Mus genus, we detected two missense mutations Leu128Ser and Tyr139Cys, that confer resistance to anticoagulant rodenticides in house mice and a new missense mutation Ala72Val in the Mus macedonicus species, not previously described. In the Rattus genus, we found one missense mutation Leu90Ile in the roof rat and one missense mutation Ser149Ile in the Norway rat. This is the first study to demonstrate potential resistance to AR in Lebanese rodents and therefore it provides data to pest control practitioners to choose the most suitable AR to control rodents in order to keep their efficacy.

VKORC1 mutations in rodent populations of a tropical city-state as an indicator of anticoagulant rodenticide resistance

Anticoagulant rodenticides are commonly used in rodent control because they are economical and have great deployment versatility. However, rodents with Single Nucleotide Polymorphism (SNP) mutations within the Vkorc1 gene are resistant to the effects of anticoagulant rodenticide use and this influences the effectiveness of control strategies that rely on such rodenticides. This study examined the prevalence of rat SNP mutations in Singapore to inform the effectiveness of anticoagulant rodenticide use. A total of 130 rat tail samples, comprising 83 Rattus norvegicus (63.8%) and 47 Rattus rattus complex (36.2%) were conveniently sampled from November 2016 to December 2019 from urban settings and sequenced at exon 3 of Vkorc1. Sequencing analysis revealed 4 synonymous and 1 non-synonymous mutations in Rattus rattus complex samples. A novel synonymous mutation of L108L was identified and not previously reported in other studies. Non-synonymous SNPs were not detected in the notable codons of 120, 128 and 139 in R. norvegicus, where these regions are internationally recognised to be associated with resistance from prior studies. Our findings suggest that the prevalence of anticoagulant rodenticide resistance in Singapore is low. Continued monitoring of rodenticide resistance is important for informing rodent control strategies aimed at reducing rodent-borne disease transmission.

Distribution of non-synonymous Vkorc1 mutations in roof rats (Rattus rattus) in France and in Spain - consequences for management

Rodent control is mainly done using anticoagulant rodenticides leading to the death of rodents through internal bleeding by targeting the VKORC1 protein. However, mutations in VKORC1 can lead to resistance to anticoagulant rodenticides that can cause treatment failure in the field. This study provides the first insight into the distribution, frequency and characterization of Vkorc1 mutations in roof rats (Rattus rattus) in France and in three administrative areas of Spain.The roof rat is present in France while it was thought to have almost disappeared with the expansion of the brown rat. Nevertheless, it has been found mainly in maritime areas. 151 roof rats out of 219 tested presented at least one missense mutation in the coding sequences of Vkorc1 gene (i.e. 69.0% of the rat). Nine Vkorc1 genotypes were detected (Y25F, A26P, R40G, S57F, W59C, W59R, H68N, Y25F/K152T and Y25F/W59R. Biochemical characterization of the consequences of these different genotypes proved that these various genotypes did not induce severe resistance to anticoagulant rodenticides.Even if many mutations of the Vkorc1 gene are present in roof rat populations in France, their management may be based in a first approach, considering the low levels of resistance induced, on the use of first-generation anticoagulants less dangerous for wildlife. The use of second-generation may be considered when treatment failure is observed or when bait consumption is limited.

Frequency Distribution of CYP2C9 and VKORC1 Mutations among Bulgarian Patients and their Importance for Anticoagulant Therapy.

Genetic polymorphisms of CYP2C9 and VKORC1 play a major role in pharmacokinetics and pharmacodynamics of coumarin anticoagulants. The purpose of our study was to assess the relative frequency of the above mutations in Bulgarian population in order to predict bleeding tendencies and precisely manage the anticoagulant therapy during the postoperative period after cardiac surgery with extracorporeal circulation. Genomic DNA samples from 200 Bulgarian patients subjected to cardiac surgery with extracorporeal circulation were analyzed for VKORC1 1639G>A and CYP2C9*2&*3 polymorphisms by real-time polymerase chain reaction (PCR), then allele frequencies of various genotypes were calculated by Hardy-Weinberg Equilibrium. Median patients' age was 63.9 ± 10.8 years; 66.5% were male. Median BMI was 28.6 ± 5.4 kg/m2. Genotype distribution for CYP2C9 was *1/*1 - 51%, *1/*2 - 21%, *1/*3 - 13.5%, *2/*3 - 4%, *3/*3 - 2%, and *2/*2 - 1.5%. The calculated frequency of CYP2C9*1 allele was 74.25%, CYP2C9*2 allele was 13%, and CYP2C9*3 allele was 12.75%, and all allelic frequencies were in Hardy-Weinberg equilibrium (p-value = 0.358). The major VKORC1 genotype was G/A - 47%, followed by G/G - 35.5% and A/A - 17.5%). Based on Hardy-Weinberg Equilibrium, there was no significant difference between observed and expected frequencies (X - -3.779), presumably as a result of the homogeneity in the population. Analysis of the data obtained in the course of the study suggested that identification of homozygous carriers of VKORC1-1639 G>A (rs9923231) in Bulgarians may be useful in developing recommendations for personalized therapy. On the contrary, homozygous carriers of CYP2C9*2 or *3, included only 4.5% of the studied patients, thus indicating that this group would benefit less from dosing algorithms. Our results demonstrated good agreement with the results obtained in other studies conducted in the Caucasian population.

Sublethal dose of warfarin induction promotes the accumulation of warfarin resistance in susceptible Norway rats

Resistance to anticoagulant rodenticides in Norway rats has been primarily explained by selection on the mutations in the vitamin K epoxide reductase complex subunit 1 (Vkorc1) gene. Whether warfarin resistance can be induced independent of Vkorc1 mutations and its role in the acquirement of warfarin resistance is unclear. In this study, we screened susceptible Norway rats with a sublethal dose of warfarin from the generation F0–F5 and found the warfarin tolerance level of the screened rats was significantly improved. Further, lethal feeding period (LFP) tests indicated 5.7–7.1% of screened rats were resistant, while all unscreened rats were susceptible. Analysis of the Vkorc1 polymorphisms suggested none of the screened F5 rats carried any potential warfarin resistance Vkorc1 mutations. As a fitness cost, the litter size of the screened rats tended to be smaller over generations, implicating the reproductive success could be impacted by warfarin induction. Our findings reveal that susceptible Norway rats can actively acquire inheritable warfarin resistance under constant warfarin induction without changing the Vkorc1 genotype. Further exploration of epigenetic and cryptic genetic mechanisms of warfarin resistance in those resistant rats is essential to fully understand the 'missing' heritability of warfarin resistance.

Mus musculus populations in Western Australia lack VKORC1 mutations conferring resistance to first generation anticoagulant rodenticides: Implications for conservation and biosecurity.

Humans routinely attempt to manage pest rodent populations with anticoagulant rodenticides (ARs). We require information on resistance to ARs within rodent populations to have effective eradication programs that minimise exposure in non-target species. Mutations to the VKORC1 gene have been shown to confer resistance in rodents with high proportions of resistance in mice found in all European populations tested. We screened mutations in Mus musculus within Western Australia, by sampling populations from the capital city (Perth) and a remote island (Browse Island). These are the first Australian mouse populations screened for resistance using this method. Additionally, the mitochondrial D-loop of house mice was sequenced to explore population genetic structure, identify the origin of Western Australian mice, and to elucidate whether resistance was linked to certain haplotypes. No resistance-related VKORC1 mutations were detected in either house mouse population. A genetic introgression in the intronic sequence of the VKORC1 gene of Browse Island house mouse was detected which is thought to have originated through hybridisation with the Algerian mouse (Mus spretus). Analysis of the mitochondrial D-loop reported two haplotypes in the house mouse population of Perth, and two haplotypes in the population of Browse Island. Both house mouse populations exhibited no genetic resistance to ARs, in spite of free use of ARs in Western Australia. Therefore weaker anticoagulant rodenticides can be employed in pest control and eradication attempts, which will result in reduced negative impacts on non-target species. Biosecurity measures must be in place to avoid introduction of resistant house mice, and new house mouse subspecies to Western Australia.

A VKORC1-based SNP survey of anticoagulant rodenticide resistance in the house mouse, Norway rat and roof rat in the USA.

We conducted a vitamin K epoxide reductase subcomponent 1 (Vkorc1)-based nonsynonymous Single Nucleotide Polymorphism (nsSNP) screen with focus on the house mouse (Mus musculus domesticus), but that also considered the Norway rat (Rattus norvegicus) and roof rat (R. rattus) in the USA. We detected six Vkorc1 nsSNPs underlying the amino-acid polymorphisms Ala21Thr, Trp59Leu, Ile104Val, Val118Leu, Leu128Ser and Tyr139Cys in house mice (average coverage/SNP; n = 182 individuals), two nsSNPs underlying Arg35Pro and Gly46Ser in the Norway rat (n = 93), with the notable absence of Tyr139Cys (n = 179), and one nsSNP underlying Tyr25Phe in the roof rat (n = 27). Inferred resistance frequency is 29.1% for mice (variability of states 0-98.8%), 6.5% (0-33.3%) for the Norway rat, and 39.3% (0-52.6%) for the roof rat based on Tyr25Phe frequencies. Resistance detected in the USA in the 1980s likely was the consequence of Vkorc1 mutations in mice (Leu128Ser and Tyr139Cys), Norway rats (Arg35Pro) and roof rats (Tyr25Phe). Patterns of variant sharing between the USA and Europe indicate the importance of convergent evolution and gene flow in spreading resistance. The spread of nsSNPs in mice between continents appears to have been more effective than in Norway rats. We hypothesize that Arg35Pro may have originated in Norway rats in the USA, whereas Tyr139Cys variants originated in Europe. Tyr25Phe is the likely cause for resistance in roof rats. Further genetic testing in the USA is required to close sampling gaps, and population genomic data are needed to study the origin and spread of this adaptive trait.

Structural Investigation of the Vitamin K Epoxide Reductase (VKORC1) Binding Site with Vitamin K.

The vitamin K epoxide reductase (VKORC1) enzyme is of primary importance in many physiological processes, i.e., blood coagulation, energy metabolism, and arterial calcification prevention, due to its role in the vitamin K cycle. Indeed, VKORC1 catalyzes reduction of vitamin K epoxide to quinone and then to hydroquinone. However, the three-dimensional VKORC1 structure remains experimentally undetermined, because of the endoplasmic reticulum membrane location of this enzyme. Here we present a molecular modeling investigation of the VKORC1 enzymatic site structure and function, supported by in vitro enzymatic assays. Four VKORC1 mutants were designed in silico (F55G, F55Y, N80G, and F83G) based on a previous study that identified residues F55, N80, and F83 as being crucial for vitamin K epoxide binding. F55G, N80G, and F83G nonconservative mutants were all predicted to be inactive by molecular modeling analyses. However, the F55Y conservative mutant was expected to be active compared to wild-type VKORC1. In vitro enzymatic assays performed on recombinant proteins assessed our molecular modeling hypotheses and led us to describe the role of accurate VKORC1 active site residues with respect to VKORC1. Residues F55, N80, and F83 appeared to act in a concerted manner to keep vitamin K epoxide close to the C135 catalytic residue. Residues F55 and N80 prevent naphthoquinone head rotation away from the active site, assisted by residue F83 that prevents vitamin K from sliding outside the enzymatic pocket, through hydrophobic tail stabilization. Our results thus highlighted the specific functions of VKORC1 catalytic pocket residues and evidenced the ability of our structural model to predict biological effects of VKORC1 mutations.

Positive Association of Mutations in VKORC1 and CYP2C9 Genes with Venous Thrombo-Embolism (VTE) in Indian Population: A Case Control Study

Background: Venous thrombo-embolism (VTE) refers to a blood clot that starts in vein. It is the third most common vascular disease in the world, after myocardial infarction (MI) and stroke, affecting millions of individuals every year. Two main clinical manifestations of VTE include deep vein thrombosis (DVT) and pulmonary embolism (PE), the later one being potentially fatal. Objective: The aim of this study is to investigate whether mutations in two genes VKORC1 and CYP2C9 and subsequent changes in their plasma levels can be used to predict the risk for venous thromboembolism (VTE). Method: A total of five polymorphisms in two genes VKORC1 and CYP2C9 were genotyped in approximately 145 VTE patients and 229 control subjects. The genotyping was done through PCR-RFLP method. Plasma concentrations of VKORC1 and CYP2C9 were estimated using ELISA technique. Results: Genotypic analysis of common polymorphisms in VKORC1 gene showed significantly higher percentage of GG genotype of -1639G>A, CC genotype of 1173 C>T and TT genotype of -497T>G polymorphism in control subjects compared to VTE patients. Also, significantly higher percentage of CC genotype in 416C>T and AA genotype in 1061 A>C polymorphism of CYP2C9*2 and CYP2C9*3 genes respectively was observed in VTE patients in comparison to healthy controls. In addition to genotypic significance, plasma concentrations of both VKORC1 and CYP2C9 were significantly higher in patients group. Conclusion: All the three polymorphisms in VKORC1 gene; -1639G>A, 1173 C>T and -497T>G and two polymorphism 416C>T and 1061A>C of CYP2C9 gene showed significant association with VTE pathophysiology

Establishment of the Variation of Vitamin K Status According to Vkorc1 Point Mutations Using Rat Models.

Vitamin K is crucial for many physiological processes such as coagulation, energy metabolism, and arterial calcification prevention due to its involvement in the activation of several vitamin K-dependent proteins. During this activation, vitamin K is converted into vitamin K epoxide, which must be re-reduced by the VKORC1 enzyme. Various VKORC1 mutations have been described in humans. While these mutations have been widely associated with anticoagulant resistance, their association with a modification of vitamin K status due to a modification of the enzyme efficiency has never been considered. Using animal models with different Vkorc1 mutations receiving a standard diet or a menadione-deficient diet, we investigated this association by measuring different markers of the vitamin K status. Each mutation dramatically affected vitamin K recycling efficiency. This decrease in recycling was associated with a significant alteration of the vitamin K status, even when animals were fed a menadione-enriched diet suggesting a loss of vitamin K from the cycle due to the presence of the Vkorc1 mutation. This change in vitamin K status resulted in clinical modifications in mutated rats only when animals receive a limited vitamin K intake totally consistent with the capacity of each strain to recycle vitamin K.

House Mice as a Real Sanitary Threat of Human and Animal Leptospirosis: Proposal for Integrated Management

Leptospirosis is a reemerging zoonosis and ranges in severity from benign to sometimes fatal. In cattle, infection may be responsible for abortion and infertility cases causing economic losses. Humans may be contaminated through direct contact with urine of infected animals or indirectly though interaction with urine-contaminated environment. Many wildlife species living close to cattle, especially commensal rodents may play a role in the transmission of leptospires. Because little is known on the epidemiology of nonmaintenance Leptospira serovars, appropriate management is still limited. On a French farm where human and cattle leptospirosis were detected, the transmission cycle was explored to propose appropriate mitigation measures. For that, commensal rodents present on the farm were trapped and their leptospires carriage was studied by molecular methods. Trapped mice were shown to carry two pathogenic Leptospira species (L. interrogans and L. kirschneri). Since these 2 serogroups were simultaneously detected in the trapped mice and in the cows of this farm, we suspected an initial Leptospira transmission from mice to cows requiring an effective management of mice on this farm. Because resistance to anticoagulant rodenticides due to Vkorc1 mutations has been largely described in rodents and first-generation anticoagulant rodenticides seemed to be inefficient in controlling mice on this farm, susceptibility of these mice to anticoagulants has been characterized by Vkorc1 sequencing. 50% of the trapped mice carried mutations in the Vkorc1 gene leading to severe resistance to first-generation anticoagulants. The management of such mice that are a real sanitary threat can be achieved only by using the most toxic second-generation anticoagulants or nonanticoagulant solutions.

VKORC1 mutation in European populations of Rattus norvegicus with first data for Italy and the report of a new amino acid substitution.

VKORC1 mutation in European populations of Rattus norvegicus with first data for Italy and the report of a new amino acid substitution.

Height, VKORC1 1173, and CYP2C9 Genotypes Determine Warfarin Dose for Pediatric Patients with Kawasaki Disease in Southwest China

Long-term oral warfarin is recommended in pediatric Kawasaki disease patients with large coronary artery aneurysms; however, heterogeneity is considerable. This study aimed to determine variables affecting warfarin dosage in Kawasaki disease. The enrolled individuals (194 children) were divided into four groups: (1) Cases with severe coronary artery lesions (CAL) of IV to V degrees or thrombogenesis treated with oral warfarin were assigned to Group A; (2) Group B, CAL of I degrees; (3) Group C, CAL of II and III degrees cases with small or medium-sized CAL not treated with warfarin; (4) Group D, normal children without Kawasaki disease. The relevant genotypes of CYP2C9, VKORC1 (1173, − 1639, and 3730), and CYP4F2 were assessed. There were no statistically significant differences in CYP2C9, VKORC1, and CYP4F2 mutation frequencies among the 4 groups. In the 44 Group A patients, demographic features, clinical characteristics, and genotypes were recorded, and their associations with warfarin dose variability were assessed. Multivariate linear regression analysis revealed that height, VKORC1 1173, and CYP2C9 accounted for 61.2%, 7.9%, and 4.3% of dosing variability, respectively. Conclusions: Patient height is the main factor determining warfarin dosage, while genotype effects on warfarin dosage vary among studies. New formula should be defined using data obtained from children in cases with demonstrated efficacy.

Abstract 790: Vitamin K pathway functions in triple-negative breast cancer

Abstract The vitamin K pathway comprises three major enzymes: gamma-glutamyl carboxylase (GGCX) and the vitamin K oxidoreductases VKORC1 and VKORC1L1. GGCX requires the reduced form of vitamin K as a co-factor to post-translationally generate γ-carboxylated (GLA) residues in proteins. Either VKORC1 or VKORC1L1 can reduce the oxidized form of vitamin K to maintain pathway activity. Interrogation of TCGA data indicates a subset of invasive breast cancer tumors harbor mutations in GGCX (8%), VKORC1 (9%), and/or VKORC1L1 (9%), with nearly all alterations being amplifications. As these data suggest upregulation of these genes is associated with aggressive breast cancer, we measured their expression in a variety of established breast cancer cell lines. qPCR results correlated with the TCGA data indicating the highest expression of GGCX, VKORC1 and VKORC1L1 expression in the two triple-negative breast cancer (TNBC) cell lines, Hs578T and SUM159PT. Since the function of these genes is to generate GLA residues in proteins, we used Western blotting to detect GLA-modified proteins in TNBC cells cultured in the presence or absence of vitamin K. In lysates from cells maintained in vitamin K, multiple bands representing GLA-modified proteins were observed between 40-60 kDa and 80 kDa. These bands were not present in lysates of cells grown in standard media that are typically devoid of vitamin K. Treatment with the VKOR antagonist, warfarin, decreased expression of these GLA-modified proteins in cells cultured in vitamin K. Thus, our data suggest that vitamin K acts in TNBC cells to produce GLA-modified proteins, and that these effects are abrogated by warfarin. To confirm the presence of a functional vitamin K pathway, we used a vitamin K-dependent reporter protein (FIXgla-PC) to measure γ-carboxylation activity. TNBC cells stably transfected with the FIXgla-PC reporter were cultured in the presence and absence of vitamin K, with or without warfarin, and secretion of γ-carboxylated FIX-gla was measured by ELISA. FIX-gla γ-carboxylation was enhanced in the presence of vitamin K and blocked by warfarin in both Hs578T and SUM159PT cell lines. These data confirm that the classical vitamin K pathway is activated in these representative TNBC cell lines when cultured in vitamin K-supplemented media. To assess the biologic relevance of this pathway in TNBC, mammosphere assays were performed with Hs578T and SUM159PT cells. Both cell lines formed significantly more mammospheres when cultured in the presence of vitamin K, suggesting that γ-carboxylation may enhance stemness. Further studies to identify the GLA-modified proteins generated in TNBC cells treated with vitamin K and their mechanisms of action may lead to new therapeutic targets for aggressive TNBC. Citation Format: Sarah Beaudin, Leila Kokabee, JoEllen Welsh. Vitamin K pathway functions in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 790.

Stabilization of warfarin‐binding pocket of VKORC1 and VKORL1 by a peripheral region determines their different sensitivity to warfarin inhibition

Background The human genome encodes two paralogs of vitamin-K-epoxide reductase, VKORC1 and VKORL1, that support blood coagulation and other vitamin-K-dependent processes. Warfarin inhibits both enzymes, but VKORL1 is relatively resistant to warfarin. Objectives To understand the difference between VKORL1 and VKORC1, and the cause of warfarin-resistant (WR) mutations in VKORC1. Methods We performed systematic mutagenesis and analyzed warfarin responses with a cell-based activity assay. Mass spectrometry analyses were used to detect cellular redox state. Results VKORC1 and VKORL1 adopt a similar intracellular redox state with four-transmembrane-helix topology. Most WR mutations identified in VKORC1 also confer resistance in VKORL1, indicating that warfarin inhibits these paralogs at a common binding site. A group of WR mutations, distant from the warfarin-binding site, show significantly less resistance in VKORL1 than in VKORC1, implying that their different warfarin responses are determined by peripheral interactions. Remarkably, we identify a critical peripheral region in which single mutations, Glu37Lys or His46Tyr, drastically increase the warfarin sensitivity of VKORL1. In the background of these warfarin-sensitive VKORL1 mutants, WR mutations showing relative less resistance in wild-type VKORL1 become much more resistant, suggesting a structural conversion to resemble VKORC1. At this peripheral region, we also identified a human single nucleotide polymorphism that confers warfarin sensitivity of VKORL1. Conclusions Peripheral regions of VKORC1 and VKORL1 primarily maintain the stability of their common warfarin-binding pocket, and differences of such interactions determine their relative sensitivity to warfarin inhibition. This new model also explains most WR mutations located at the peripheral regions of VKORC1.

Adaptative evolution of the Vkorc1 gene in Mus musculus domesticus is influenced by the selective pressure of anticoagulant rodenticides.

Anticoagulant rodenticides are commonly used to control rodent pests worldwide. They specifically inhibit the vitamin K epoxide reductase (VKORC1), which is an enzyme encoded by the Vkorc1 gene, involved in the recycling of vitamin K. Therefore, they prevent blood clotting. Numerous mutations of Vkorc1 gene were reported in rodents, and some are involved in the resistant to rodenticides phenotype. Two hundred and sixty‐six mice tails were received from 65 different locations in France. Coding sequences of Vkorc1 gene were sequenced in order to detect mutations. Consequences of the observed mutations were evaluated by the use of recombinant VKORC1. More than 70% of mice presented Vkorc1 mutations. Among these mice, 80% were homozygous. Contrary to brown rats for which only one predominant Vkorc1 genotype was found in France, nine missense single mutations and four double mutations were observed in house mice. The single mutations lead to resistance to first‐generation antivitamin K (AVKs) only and are certainly associated with the use of these first‐generation molecules by nonprofessionals for the control of mice populations. The double mutations, probably obtained by genetic recombination, lead to in vitro resistance to all AVKs. They must be regarded as an adaptive evolution to the current use of second‐generation AVKs. The intensive use of first‐generation anticoagulants probably allowed the selection of a high diversity of mutations, which makes possible the genetic recombination and consequently provokes the emergence of the more resistant mutated Vkorc1 described to date.