Project

Inflammation; vitamin K-dependent protein; Gas6; Protein S; Hemostasis

Lead
Notre projet est d’étudier le rôle de deux molécules de la signalisation cellulaire dans les interactions entre le sang et les vaisseaux et celles entre les cellules du sang dans des processus vitaux comme la production des globules rouges, l’inflammation, les thromboses et les phénomènes permettant de faire cesser des hémorragies.
Lay summary
Contenu et objectifs du travail de recherche Les molécules de la signalisation qui nous intéressent sont Growth arrest-specific gene 6 (Gas6) et la protéine S. Gas6 a été décrit à l’origine comme faisant partie d’un groupe de gènes dont l’expression augmente dans les cellules en arrêt de croissance. Depuis, de nombreuses fonctions ont été attribuées à Gas6, la protéine qui est le produit du gène Gas6. Considéré initialement comme un facteur de survie et de croissance, il participe à de nombreuses fonctions cellulaires. Par exemple, il est impliqué dans la production et la différentiation des cellules sanguines, la biologie vasculaire, la fonction cérébrale, la fertilité et la thrombose. La protéine S est un anticoagulant naturel. Les patients qui ont un déficit en protéine S ont un risque augmenté de présenter des thromboses et l’absence complète de protéine S est incompatible avec la vie en l’absence de traitement. La compréhension des fonctions biologiques des ces deux molécules est cependant actuellement limitée. Le but de ce projet est de mieux comprendre le rôle de Gas6 et de la protéine S en utilisant le modèle de la souris avec la perspective de découvrir de nouvelles cibles pour le traitement de l’anémie, des saignements, des thromboses et des réactions exagérées à l’infection. Contexte scientifique et social du projet de recherche Gas6 pourrait être utile comme hémostatique et pour traiter l’anémie. De plus, Gas6, tout comme la protéine S, pourrait empêcher les réactions exagérées qui peuvent entraîner la mort dans le cadre d’une infection très grave.  En outre, des inhibiteurs de Gas6 pourraient prévenir et traiter les thromboses. Les résultats de ce travail pourraient ouvrir des perspectives pour des études cliniques impliquant Gas6, des inhibiteurs de Gas6 ou la protéine S.

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Last update: 25.08.2014

Active participation

Title	Year

Number	Title	Start	Funding scheme

We intend to further study the role of growth arrest-specific gene 6 (Gas6) and protein S (Pros), their mechanism of action and the possible therapeutic use of Gas6 agonists or antagonists in medically important -often lifethreatening- conditions such as bleeding, thrombosis and inflammation. Gas6 belongs to the vitamin K-dependent protein family. Apart from a ?-carboxyglutamic acid-domain interaction with phospholipid membranes, Gas6 also binds as a ligand to the receptor tyrosine kinases Tyro3, Axl and Mer (or TAM receptors) by its carboxy-terminal globular domains. We became interested in Gas6 because of its sequence homology with the anticoagulant Pros. Pros is well recognized as an important natural anticoagulant. This is evidenced by the increased risk of thromboembolic events (recurrent deep vein thrombosis at adult age) in heterozygous patients deficient in Pros. Notably homozygous Pros deficiency is associated with dramatic clinical manifestations (disseminated intravascular coagulation, purpura fulminans) and, if untreated, is incompatible with life. Current knowledge on the biological functions of Pros is only limited, although a number of in vitro studies have helped elucidate the structure/function relationships of Pros.Even though our initial studies of the Gas6 gene inactivation did not reveal any “spontaneous” phenotype, detailed analyses of these mice have unveiled that Gas6 plays an “amplifier” role in hemostasis where it is redundant for baseline hemostasis but critical for effective platelet aggregation upon vascular injury -precisely by amplifying the platelet aggregation activity of the classical agonists. Similarly, we found very recently that Gas6 amplifies erythropoietin responses during compensatory erythropoietic response to anemia. Thus, Gas6 belongs to a novel class of molecules, which are redundant for normal homeostasis but critical for stress-responses. Not surprisingly, therefore, inactivation of Gas6 does not cause life-threatening developmental defects, but may importantly modulate the severity of disease related phenotypes.The first part of the project aims at a better understanding of the in vivo role of Pros in mice. We recently described the lethal prothrombic phenotype of Pros-/- mice. To circumvent the problem of life incompatibility and thereby extend the field of our investigations, mice with very low levels of Pros will be generated. Mice with consistently lower Pros levels would be viable while presenting a much stronger phenotype than that of Pros+/- mice. Such mice would be very helpful to investigate new biological functions for Pros. Two experimental approaches have been chosen: 1) the selective repression of Pros expression in the liver parenchymal cells which is expected to result in dramatic reduction of circulating Pros levels, as Pros is mainly expressed in the liver; 2) the repression of Pros expression in adult mice. Another elegant way to circumvent the embryonically lethal phenotype of Pros-/- mice would be to generate mice with total deficiency in Pros but expressing high levels of activated protein C (APC) or low tissue factor levels. Mice expressing high plasma levels of APC (APChigh) will be therefore crossed with Pros+/- mice to generate Pros+/--APChigh mice, which will be in turn intercrossed to generate Pros-/--APChigh mice. The viability and prothrombic phenotype of the latter mice will be investigated. Conditional Pros knockout mice will also allow us to specifically repress Pros expression in various tissues. Indeed, Pros is not only expressed in the liver but is expressed by endothelial cells, megakaryocytes and contained at high levels in circulating platelets. The contribution of platelet and endothelial Pros to thrombosis and inflammation will be investigated by crossing ProsLox/Lox mice to mice where Cre expression is under the control of platelet- and endothelium-specific promoter, respectively. In the longer run, generation of other tissue-specific Pros knockout mice will allow us to investigate the ability of Pros to function as a ligand of receptors tyrosine kinase of the Tyro3 family (TAM) in various cell types and biological processes.The second part of the project aims at a better understanding of the in vivo role of Gas6 and Pros pathways in platelet function in mice lacking Gas6 or any TAM receptors. In addition, as we found that the lack of one TAM receptor is responsible for a more severe phenotype than the lack of Gas6 itself, we hypothesize that a second ligand might be present in platelets. Pros is present in platelet ? granules from which it is secreted upon platelet activation. However, its role in platelet function is unknown. Therefore, we will study the role of Pros in platelet function. For this investigation, we have chosen to generate mice deficient in Pros only in platelets and study their platelet function in vitro and in vivo. If mice lacking Pros in their platelets have a platelet dysfunction, we will intercross them with Gas6 deficient mice or mice lacking Gas6 only in platelets to see if the platelet defect intensity correspond to that of mice lacking Gas6 alone or one or more of TAM receptors. We will also study the Gas6/Pros pathway in patients suffering from platelet dysfunction or thrombotic disorders.The third part of the project will be dedicated to the study of the role of Gas6/Pros pathway in inflammation in relationship with hemostasis. We found that Gas6, when secreted by monocytic cells in response to endotoxin dampens cytokine release from these cells. In sharp contrast, Gas6 deficient endothelial cells respond to TNF-? without increases in adhesion molecules and tissue factor exposure (the primary initiator of coagulation) and leukocytes transmigrate less extensively through them than through wild-type endothelial cells. Based on these observations, we will complete our studies on the role of Gas6 in inflammation by investigating the influence of Gas6/Pros pathway on coagulation parameters during endotoxemia and infection by using conditional tissue specific mice lacking Gas6, Pros, or one or more of the TAM receptors and by investigating Gas6/Pros pathway in the Lausanne sepsis cohort. Besides, we will further investigate another complication of inflammation involving Gas6, the anemia of chronic diseases and the potential role of Gas6/Pros in iron metabolism.In conclusion, our proposal aims at a better understanding of Gas6/Pros pathway in mechanisms of hemostasis, thrombosis and inflammation. Perspectives of this proposal include clinical trials implying Gas6 agonists or antagonists to treat human diseases such as bleeding disorders, thrombosis or sepsis.