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

International audienceVitamin 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

Résistance de cible aux antivitamines KR : analyse des conséquences catalytiques de différentes mutations de VKORC1 et, : étude du rôle d’une nouvelle enzyme, la VKORC1L1

Anticoagulant vitamin K antagonists (VKA) are molecules designed to prevent or delay blood clotting. They cause bleeding by slowing the recycling of vitamin K, an essential micronutrient for posttranslational modification of specific proteins (VKDP). It has been shown that VKA specifically inhibit VKORC1 enzyme which catalyze the VKOR reaction. VKA are used as rodenticides to control the proliferation of populations of pest rodents. In humans, they are used in the treatment and prevention of the occurrence of thromboembolic events. Due to the widespread use of these VKA, it was observed a phenomenon of resistance which is essential to better understand for economic, ecological or public health interests. In humans, 25 of 26 mutations were characterized. While these changes have been observed in patients resistant to VKA, the causality of these mutations has been demonstrated for 6 mutations. The ability to detect these changes before the start of treatment will allow the future implementation of the much faster and less expensive. Other mutations are not responsible for the observed phenotype.Moreover, VKORC1L1 has been described as an enzyme whose function is to act against oxidative stress. This study confirms that the enzyme catalyzes the VKOR reaction. If it appears that the liver in its participation in the reduction of vitamin K epoxide is insignificant, it is quite different in other tissues tested. In addition, VKORC1L1 appears more resistant to VKA over the VKORC1. Finally, directed mutagenesis of these residues lead to the decrease or the increase of VKORC1L1 sensitivity to VKA. These data result to the implication of residues in their interaction with VKALes anticoagulants antivitamine K (AVK) sont destinées à limiter la coagulation du sang. Ils sont donc susceptibles de provoquer des saignements. Les AVK ralentissent le cycle de la vitamine K qui est indispensable à la gamma-carboxylation de certaines protéines (PVKD). Les AVK inhibent l'activité vitamine K époxide reductase (VKOR), principalement catalysée par VKORC1. Ce sont des médicaments anticoagulants utilisés chez l'homme. Chez les rongeurs, ils servent de rodonticides. Une résistance aux AVK est observé tant chez l'homme que chez le rongeur.Chez des patients résistants aux AVK, 26 mutations ont été décrites dans la zone codante de VKORC1. L'expression hétérologue de ces enzymes mutées n'a permis de trouver que 6 mutations impliquées dans la résistance. Repérer ces mutations avant le début d'un traitement permettra une mise en place du traitement plus rapide. Les autres mutations ne seraient pas responsables du phénotype observé.La VKORC1L1 a été décrite comme une protéine agissant contre le stress oxydatif. Notre travail confirme que l'enzyme catalyse la réaction VKOR. Si sa participation dans la réduction de la vitamine K époxide est insignifiante dans le foie, il en est tout autrement dans les autres tissus testés. De plus, la VKORC1L1 apparait plus résistante aux AVK par rapport à la VKORC1. Ces propriétés catalytiques de la VKORC1L1 permettent d'expliquer l'absence d'effets des AVK sur les PVKD d'origine extra-hépatiques.Enfin, un travail de mutagénèse dirigée a permis d'abaisser ou d'augmenter considérablement la sensibilité de VKORC1L1 aux AVK. Ces résultats nous permettent de décrire l'implication de différents acides aminés dans l'interaction avec les AV

Target resistance to vitamin K antagonists : analysis of the catalytic consequences of different mutations of VKORC1 and study of the role of a new enzyme, VKORC1L1

Les anticoagulants antivitamine K (AVK) sont destinées à limiter la coagulation du sang. Ils sont donc susceptibles de provoquer des saignements. Les AVK ralentissent le cycle de la vitamine K qui est indispensable à la gamma-carboxylation de certaines protéines (PVKD). Les AVK inhibent l'activité vitamine K époxide reductase (VKOR), principalement catalysée par VKORC1. Ce sont des médicaments anticoagulants utilisés chez l'homme. Chez les rongeurs, ils servent de rodonticides. Une résistance aux AVK est observé tant chez l'homme que chez le rongeur.Chez des patients résistants aux AVK, 26 mutations ont été décrites dans la zone codante de VKORC1. L'expression hétérologue de ces enzymes mutées n'a permis de trouver que 6 mutations impliquées dans la résistance. Repérer ces mutations avant le début d'un traitement permettra une mise en place du traitement plus rapide. Les autres mutations ne seraient pas responsables du phénotype observé.La VKORC1L1 a été décrite comme une protéine agissant contre le stress oxydatif. Notre travail confirme que l'enzyme catalyse la réaction VKOR. Si sa participation dans la réduction de la vitamine K époxide est insignifiante dans le foie, il en est tout autrement dans les autres tissus testés. De plus, la VKORC1L1 apparait plus résistante aux AVK par rapport à la VKORC1. Ces propriétés catalytiques de la VKORC1L1 permettent d'expliquer l'absence d'effets des AVK sur les PVKD d'origine extra-hépatiques.Enfin, un travail de mutagénèse dirigée a permis d'abaisser ou d'augmenter considérablement la sensibilité de VKORC1L1 aux AVK. Ces résultats nous permettent de décrire l'implication de différents acides aminés dans l'interaction avec les AVKAnticoagulant vitamin K antagonists (VKA) are molecules designed to prevent or delay blood clotting. They cause bleeding by slowing the recycling of vitamin K, an essential micronutrient for posttranslational modification of specific proteins (VKDP). It has been shown that VKA specifically inhibit VKORC1 enzyme which catalyze the VKOR reaction. VKA are used as rodenticides to control the proliferation of populations of pest rodents. In humans, they are used in the treatment and prevention of the occurrence of thromboembolic events. Due to the widespread use of these VKA, it was observed a phenomenon of resistance which is essential to better understand for economic, ecological or public health interests. In humans, 25 of 26 mutations were characterized. While these changes have been observed in patients resistant to VKA, the causality of these mutations has been demonstrated for 6 mutations. The ability to detect these changes before the start of treatment will allow the future implementation of the much faster and less expensive. Other mutations are not responsible for the observed phenotype.Moreover, VKORC1L1 has been described as an enzyme whose function is to act against oxidative stress. This study confirms that the enzyme catalyzes the VKOR reaction. If it appears that the liver in its participation in the reduction of vitamin K epoxide is insignificant, it is quite different in other tissues tested. In addition, VKORC1L1 appears more resistant to VKA over the VKORC1. Finally, directed mutagenesis of these residues lead to the decrease or the increase of VKORC1L1 sensitivity to VKA. These data result to the implication of residues in their interaction with VK

Structural Insights into Phylloquinone (Vitamin K1), Menaquinone (MK4, MK7), and Menadione (Vitamin K3) Binding to VKORC1

Vitamin K family molecules—phylloquinone (K1), menaquinone (K2), and menadione (K3)—act as γ-glutamyl carboxylase (GGCX)-exclusive cofactors in their hydroquinone state, activating proteins of main importance for blood coagulation in the liver and for arterial calcification prevention and energy metabolism in extrahepatic tissues. Once GGCX is activated, vitamin K is found in the epoxide state, which is then recycled to quinone and hydroquinone states by vitamin K epoxide reductase (VKORC1). Nevertheless, little information is available concerning vitamin K1, K2, or K3 tissue distribution and preferential interactions towards VKORC1. Here we present a molecular modeling study of vitamin K1, menaquinones 4, 7 (MK4, MK7), and K3 structural interactions with VKORC1. VKORC1 was shown to tightly bind vitamins K1 and MK4 in the epoxide and quinone states, but not in the hydroquinone state; five VKORC1 residues were identified as crucial for vitamin K stabilization, and two other ones were essential for hydrogen bond formation. However, vitamin MK7 revealed shaky binding towards VKORC1, induced by hydrophobic tail interactions with the membrane. Vitamin K3 exhibited the lowest affinity with VKORC1 because of the absence of a hydrophobic tail, preventing structural stabilization by the enzyme. Enzymatic activity towards vitamins K1, MK4, MK7, and K3 was also evaluated by in vitro assays, validating our in silico predictions: VKORC1 presented equivalent activities towards vitamins K1 and MK4, but much lower activity with respect to vitamin MK7, and no activity towards vitamin K3. Our results revealed VKORC1’s ability to recycle both phylloquinone and some menaquinones, and also highlighted the importance of vitamin K’s hydrophobic tail size and membrane interactions

VKORC1L1, an enzyme rescuing the vitamin K 2,3-epoxide reductase activity in some extrahepatic tissues during anticoagulation therapy

Vitamin K is involved in the -carboxylation of the vitamin K-dependent proteins, and vitamin K epoxide is a by-product of this reaction. Due to the limited intake of vitamin K, its regeneration is necessary and involves vitamin K 2,3-epoxide reductase (VKOR) activity. This activity is known to be supported by VKORC1 protein, but recently a second gene, VKORC1L1, appears to be able to support this activity when the encoded protein is expressed in HEK293T cells. Nevertheless, this protein was described as being responsible for driving the vitamin K-mediated antioxidation pathways. In this paper we precisely analyzed the catalytic properties of VKORC1L1 when expressed in Pichia pastoris and more particularly its susceptibility to vitamin K antagonists. Vitamin K antagonists are also inhibitors of VKORC1L1, but this enzyme appears to be 50-fold more resistant to vitamin K antagonists than VKORC1. The expression of Vkorc1l1 mRNA was observed in all tissues assayed, i.e. in C57BL/6 wild type and VKORC1-deficient mouse liver, lung, and testis and rat liver, lung, brain, kidney, testis, and osteoblastic cells. The characterization of VKOR activity in extrahepatic tissues demonstrated that a part of the VKOR activity, more or less important according to the tissue, may be supported by VKORC1L1 enzyme especially in testis, lung, and osteoblasts. Therefore, the involvement of VKORC1L1 in VKOR activity partly explains the low susceptibility of some extrahepatic tissues to vitamin K antagonists and the lack of effects of vitamin K antagonists on the functionality of the vitamin K-dependent protein produced by extrahepatic tissues such as matrix Gla protein or osteocalcin

Identification of Key Functional Residues in the Active Site of Vitamin K Epoxide Reductase-like Protein (VKORC1L1)

International audienceBackground/Purpose: Vitamin K is involved in the gamma-carboxylation of the vitamin K dependent proteins. Due to the limited intake of vitamin K, its regeneration is necessary and involves the vitamin K 2,3-epoxide reductase (VKOR) activity. This activity is catalyzed by VKORC1 and/or VKORC1L1 proteins. Warfarin is able to inhibit both enzymes, but VKORC1L1 appears to be 30-fold more resistant to warfarin than VKORC1.Methods: To predict functional peptide regions or amino acid residues important for VKOR activity or resistance to vitamin K antagonists (VKA) of human VKORC1L1, we conducted a multiple alignment of VKORC1L1 and VKORC1 sequences. The role of conserved amino acid residues between VKORC1 and VKORC1L1, but also the role of conserved amino-acid residues in VKORC1L1 but not in VKORC1 were challenged by systematic engineering of point mutations combined with in vitro functional assays.Findings: Interestingly, engineering mutants at position 130 allowed us to obtain a VKORC1L1 as susceptible to VKA as wild type VKORC1. Our results also suggested the involvement of Cys-43(+7), Cys-51(+7), Cys-132(+7) and Cys-135(+7), in the transfer of the redox power to vitamin K epoxide.Conclusion: Altogether, this study provides novel insight into VKORC1L1 active site functional domains. Glu-130 is a key residue governing the natural resistance of VKORC1L1 to VKAs

Population genetics and genotyping as tools for planning rat management programmes

Brown rats are a prolific synanthropic pest species, but attempts to control their populations have had limited success. Rat population dynamics, dispersal patterns, and resistance to rodenticides are important parameters to consider when planning a control programme. We used population genetics and genotyping to investigate how these parameters vary in contrasting landscapes, namely one urban and two rural municipalities from eastern France. A total of 355 wild brown rats from 5 to 6 sites per municipality were genotyped for 13 microsatellite loci and tested for mutations in the Vkorc1 gene which confers resistance to some rodenticides. Results revealed a strong genetic structure of the sampled rat populations at both regional (between municipalities) and local (between sites within municipalities) levels. A pattern of isolation by distance was detected in the urban habitat and in one of the rural municipalities. GeneClass and DAPC analyses identified 25 (7%) and 36 (10%) migrants, respectively. Migrations occurred mostly between sites within each municipality. We deduced that rat dispersal is driven by both natural small-scale movements of individuals and longer-distance (human-assisted) movements. Mutation Y139F on gene Vkorc1 was significantly more prevalent in rural (frequency 0.26-0.96) than in urban sites (0.00-0.11), likely due to differences in selection pressures. Indeed, pest control is irregular and uncoordinated in rural areas, whereas it is better structured and strategically organised in cities. We conclude that simultaneous pest control actions between nearby farms in rural habitats are highly recommended in order to increase rat control success while limiting the spread of resistance to rodenticides

Synthesis and biological evaluation of C-3 aliphatic coumarins as vitamin K antagonists

Since the discovery of Warfarin in the 1940s, the design of new warfarin-derived anticoagulants for rodent management has been challenging, with mainly structural modifications performed on the C3 position of the coumarin skeleton. In order to better understand the pharmacomodulation of such derivatives, we have synthesized a family of C3 (linear and branched) alkyl-4-hydroxycoumarins, which led to the identification of compounds 5e and 5f as potential short-term active anticoagulants