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

Study of antivitamin K teratogenicity : the importance to find new anticoagulant targets : on the way to proposed redox partner for VKORC1 activation step

Les anticoagulants antivitamine K (AVK) constituent l’une des thérapeutiques majeures dans le traitement et la prévention des affections thromboemboliques chez l’homme. Ce sont aussi, les principaux raticides utilisés pour de lutte contre les populations de rongeurs sauvages. Leurs utilisations sont associées à des effets secondaires : comme biocide, les AVK posent des problèmes d’écotoxicité et font face à l’apparition d’importantes résistances de cible chez les rongeurs. L’utilisation des AVK en thérapie semble augmenter le risque de calcification vasculaire et de fragilité osseuse. De même, l’utilisation de la warfarine (chef de file des AVK en thérapie), est associée à un syndrome malformatif : le « fetal warfarin syndrome » (FWS). En juin 2007, l’agence européenne des produits chimiques (ACHA) au travers de la nouvelle réglementation de l’utilisation des produits chimiques (REACH) a classé tous les raticides anticoagulants comme reprotoxiques (1A ou 1B) ((EU) 2016/1179). Ce classement est basé exclusivement sur la warfarine par « read across » (classement des biocides en fonction de similarités structurelles), alors que cette molécule n’est plus utilisée comme raticide. Cette règlementation modifie les autorisations d’utilisation de ces biocides. Dans le contexte d’émergence des résistances aux rodonticides, la nouvelle norme européenne pourrait aboutir à une gestion chaotique des populations de rongeurs sauvages. L’utilisation des AVK provoque l’arrêt du cycle de la vitamine K dans la cellule. Cette vitamine est essentielle pour l’hémostase car elle est notamment le cofacteur d’activation de certains facteurs de coagulation. Le mode d’action des AVK passe par l’inhibition de l’enzyme « Vitamine K epOxyde Reductase Complexe subunit 1 » (VKORC1). Cette enzyme à cystéines est totalement transmembranaire (membrane du réticulum endoplasmique) et fortement exprimée dans le foie. Étudiée depuis les années 1970, de nombreuses incertitudes subsistent quant à son fonctionnement catalytique. L’étape d’activation préliminaire de l’enzyme (avant son intervention dans le cycle de la vitamine K) est très mal caractérisée et constitue une zone d’ombre importante. Elle ferait intervenir un système redox cellulaire du lumen du réticulum endoplasmique de type PDI-like, dont l’identité reste à préciser. Ce travail de thèse présente pour la première fois, une étude de comparaison entre les potentiels tératogènes de la warfarine et d’autres AVK (notamment raticides) à l’aide de modèles murins. Alors que la warfarin montre des capacités tératogènes, il n’en est rien pour d’autres AVK. Dans la suite du travail, nous nous sommes intéressés à l’origine de ces différences : une étude pharmacocinétique conduite chez la ratte gestante, a démontré que les profils de distributions tissulaires des AVK sont à l’origine de ces différences. L’ensemble de nos résultats remet en cause la véracité du nouveau classement européen des AVK. Bien que l’utilisation des AVK peut être améliorée, pour réduire l’apparition des effets secondaires, la découverte de nouvelles cibles thérapeutiques antivitamine K, permettrait de proposer de nouveaux agents pharmacologiques plus efficace et moins toxiques. Ainsi, dans une deuxième phase, ce travail de thèse s’est orienté vers la découverte de nouvelles cibles thérapeutiques antivitamine K. Après la mise au point d’une méthode innovante de mesure d’activité enzymatique de VKORC1, méthode basée sur la quantification des formes intracellulaires de la vitamine K, nous avons pu identifier de potentiels activateurs redox de VKORC1. Ces résultats ont été complétés par deux études de la fonctionnalité des protéines appartenant à la famille des protéines candidates (PDI-like). Ces protéines d’activation redox pourraient constituer de nouvelles cibles anticoagulantes antivitamine K. L’ensemble de ces travaux participe à l’amélioration de l’utilisation des AVK et propose la piste de nouvelles cibles anticoagulantes antivitamine KVitamin K antagonists (VKA) are major drugs for the treatment and prevention of thromboembolic diseases. They are also the main biocides used to control wild rodent populations. As a biocide, their use is associated with some side effects: VKA lead to target resistance in rodents and appear to be highly ecotoxic. The use of VKA in therapy, seems to increase the risk of vascular calcification and bone fragility. Furthermore, the use of warfarin (leader of VKA in therapies) is associated with a malformative syndrome: the «fetal warfarin syndrome» (FWS). In June 2007, the European Chemicals Agency (ACHA) by the new regulation for the use of chemicals (REACH), classified all VKA as reprotoxic (1A or 1B) (EU) 2016/1179). This classification is based on warfarin through «read across» approach (classification of biocides according to structural similarities), even if warfarin is no longer used in rodent management. This new regulation has changed the way to use these biocides and could leads to chaotic management of wild rodent populations in a context of emerging target resistance. The use of VKA disrupt the vitamin K cycle in cells. This vitamin is essential for hemostasis through the activation of some clotting factors. The mode of action consists in the enzymatic inhibition of "vitamin K epoxide reductase complex subunit 1" (VKORC1). This enzyme is anchored in the endoplasmic reticulum transmembrane and strongly expressed in liver. Since the 1970s, many questions remained regarding its catalytic function. The initial activation step of the enzyme (before its intervention in the vitamin K cycle) is poorly characterized and constitutes an important topic. Redox system of PDI-like protein could facilitate this enzymatic activation. Unfortunately, the identity of the VKORC1 redox partner is unknow. This thesis work presents for the first time, a comparison study of teratogenic potentials between warfarin and other VKA (especially rodenticide), using a rat model. While warfarin presents teratogenic abilities, it was not for other rodenticides. In the following work, we focused on the origin of this difference. A pharmacokinetic study conducted in rat, demonstrated that the tissue distribution profiles of VKAs are the origin of these teratogenicity differences. All of results call into question the veracity of the new European biocides ranking of VKA. Althought the use of VKA could be improved to reduce the onset of side effects, the discovery of new antivitamin K targets could lead to new efficient and safe anticoagulant drugs. Thus, in a second phase, we focused on the discovery of new antivitamin K targets. After the development of an innovative cell-based assay to measure VKORC1 activity, we were able to point out potentials VKORC1 redox partners. Two complementary studies were conducted to described functionalities of PDI-like protein which are best candidates for VKORC1 redox partners. Theses redox activation proteins could be new anticoagulant targets. All this work participated on the improvement of the use of VKA and proposed new anticoagulant targets based on antivitamin K mechanis

Overexpression of protein disulfide isomerase enhances vitamin K epoxide reductase activity

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

Differences in teratogenicity of some vitamin K antagonist substances used as human therapeutic or rodenticide are due to major differences in their fate after an oral administration

International audienceAll vitamin K antagonist active substances used as rodenticides were reclassified in 2016 by the European authorities as active substances "toxic for reproduction", using a "read-across" alternative method based on warfarin, a human vitamin K antagonist drug. Recent study suggested that all vitamin K antagonist active substances are not all teratogenic. Using a neonatal exposure protocol, warfarin evokes skeletal deformities in rats , while bromadiolone, a widely used second-generation anticoagulant rodenticide, failed to cause such effects. Herein, using a rat model we investigated the mechanisms that may explain teratogenicity differences between warfarin and bromadiolone, despite their similar vitamin K antagonist mechanism of action. This study also included coumatetralyl, a first-generation active substance rodenticide. Pharmacokinetic studies were conducted in rats to evaluate a potential difference in the transfer of vitamin K antagonists from mother to fetus. The data clearly demonstrate that warfarin is highly transferred from the mother to the fetus during gestation or lactation. In contrast, bromadiolone transfer from dam to the fetus is modest (5% compared to warfarin). This difference appears to be associated to almost complete uptake of bromadiolone by mother's liver, resulting in very low exposure in plasma and eventually in other peripheric tissues. This study suggests that the pharmacokinetic properties of vitamin K antagonists are not identical and could challenge the classification of such active substances as "toxic for reproduction"

Self-assembly of achiral building blocks into chiral cyclophanes using non-directional interactions

The transfer of stereo-electronic information from achiral building blocks and templates to chiral cyclophanes is rationalized and exploited to produce self-assembled macrocyclic species carrying up to 16 stereogenic elements