Molecular genetics of ocular diseases

The eye is a complex organ, which provides one of our most important senses, sight. The retina is the neuronal component of the eye and represents the connection with the central nervous system for the transmission of the information that leads to image processing. Retinitis pigmentosa (RP) is one of the most common forms of inherited retinal degeneration, in which the primary death of rods, resulting in night blindness, is always followed by the loss of cones, which leads to legal blindness. Clinical and genetic heterogeneity in retinitis pigmentosa is not only due to different mutations in different genes, but also to different effects of the same mutation in different individuals, sometimes even within the same family. My thesis work has been mainly focused on an autosomal dominant form of RP linked to mutations in the PRPF31 gene, which often shows reduced penetrance. Our study has led to the identification of the major regulator of the penetrance of PRPF31 mutations, the CNOT3 protein, and to the characterization of its mechanism of action. Following the same rationale of investigating molecular mechanisms that are responsible for clinical and genetic heterogeneity of retinitis pigmentosa, we studied a recessive form of the disease associated with mutations in the recently-identified gene FAMI61 A, where mutations in the same gene give rise to variable clinical manifestations. Our data have increased the knowledge of the relationship between genotype and phenotype in this form of the disease. Whole genome sequencing technique was also tested as a strategy for disease gene identification in unrelated patients with recessive retinitis pigmentosa and proved to be effective in identifying disease-causing variants that might have otherwise failed to be detected with other screening methods. Finally, for the first time we reported a choroidal tumor among the clinical manifestations of PTEN hamartoma tumor syndrome, a genetic disorder caused by germline mutations of the tumor suppressor gene PTEN. Our study has highlighted the heterogeneity of this choroidal tumor, showing that genetic and/or epigenetic alterations in different genes may contribute to the tumor development and growth. - L'oeil est un organe complexe, à l'origine d'un de nos sens les plus importants, la vue. La rétine est la composante neuronale de l'oeil qui constitue la connexion avec le système nerveux central pour la transmission de l'information et qui conduit à la formation des images. La rétinite pigmentaire (RP) est une des formes les plus courantes de dégénérescence rétinienne héréditaire, dans laquelle la mort primaire de bâtonnets, entraînant la cécité nocturne, est toujours suivie par la perte de cônes qui conduit à la cécité complète. L'hétérogénéité clinique et génétique dans la rétinite pigmentaire n'est pas seulement due aux différentes mutations dans des gènes différents, mais aussi à des effets différents de la même mutation chez des individus différents, parfois même dans la même famille. Mon travail de thèse s'est principalement axé sur une forme autosomique dominante de RP liée à des mutations dans le gène PRPF31, associées souvent à une pénétrance réduite, me conduisant à l'identification et à la caractérisation du mécanisme d'action du régulateur principal de la pénétrance des mutations: la protéine CNOT3. Dans la même logique d'étude des mécanismes moléculaires responsables de l'hétérogénéité clinique et génétique de la RP, nous avons étudié une forme récessive de la maladie associée à des mutations dans le gène récemment identifié FAMI61 A, dont les mutations dans le même gène donnent lieu à des manifestations cliniques différentes. Nos données ont ainsi accru la connaissance de la relation entre le génotype et le phénotype dans cette forme de maladie. La technique de séquençage du génome entier a été ensuite testée en tant que stratégie pour l'identification du gène de la maladie chez les patients atteints de RP récessive. Cette approche a montré son efficacité dans l'identification de variantes pathologiques qui n'auraient pu être détectées avec d'autres méthodes de dépistage. Enfin, pour la première fois, nous avons identifié une tumeur choroïdienne parmi les manifestations cliniques du PTEN hamartoma tumor syndrome, une maladie génétique causée par des mutations germinales du gène suppresseur de tumeur PTEN. Notre étude a mis en évidence l'hétérogénéité de cette tumeur choroïdienne, montrant que les altérations génétiques et/ou épigénétiques dans les différents gènes peuvent contribuer au développement et à la croissance tumorale.

The role of WNT and mTOR in human memory T cell formation

Cancer is one of the world's leading causes of death with a rising trend in incidence. These epidemiologic observations underline the need for novel treatment strategies. In this regard, a promising approach takes advantage of the adaptive effector mechanisms of the immune system, using T lymphocytes to specifically target and destroy tumour cells. However, whereas current approaches mainly depend on short-lived, terminally differentiated effector T cells, increasing evidence suggests that long lasting and maximum efficient immune responses are mediated by low differentiated memory T cells. These memory T cells should display characteristics of stem cells, such as longevity, self-renewal capacity and the ability to continuously give rise to further differentiated effectors. These stem celllike memory T (TSCM) cells are thought to be of key therapeutic value as they might not only attack differentiated tumour cells, but also eradicate the root cause of cancer, the cancer stem cells themselves. Thus, efforts are made to characterize TSCM cells and to identify the signalling pathways which mediate their induction. Recently, a human TSCM cell subset was described and the activation of the Wnt-ß-catenin signalling pathway by the drug TWS119 during naive CD8+ T (TN) cell priming was suggested to mediate their induction. However, a precise deciphering of the signalling pathways leading to TSCM cell induction and an in-depth characterization of in vitro induced and in vivo occurring TSCM cells remain to be performed. Here, evidence is presented that the induction of human and mouse CD8+ and CD4+ TSCM cells may be triggered by inhibition of mechanistic/mammalian target of rapamycin (mTOR) complex 1 with simultaneously active mTOR complex 2. This molecular mechanism arrests a fraction of activated TN cells in a stem cell-like differentiation state independently of the Wnt-ß-catenin signalling pathway. Of note, TWS119 was found to also inhibit mTORCl, thereby mediating the induction of TSCM cells. Suggesting an immunostimulatory effect, the acquired data broaden the therapeutic range of mTORCl inhibitors like rapamycin, which are, at present, exclusively used due to their immunosuppressive function. Furthermore, by performing broad metabolic analyses, a well-orchestrated interplay between intracellular signalling pathways and the T cells' metabolic programmes could be identified as important regulator of the T cells' differentiation fate. Moreover, in vitro induced CD4+ TSCM cells possess superior functional capacities and share fate-determining key factors with their naturally occurring counterparts, assessed by a first-time full transcriptome analysis of in vivo occurring CD4+ TN cell, TSCM cells and central memory (TCM) cells and in vitro induced CD4+ TSCM cells. Of interest, a group of 56 genes, with a unique expression profile in TSCM cells could be identified. Thus, a pharmacological mechanism allowing to confer sternness to activated TN cells has been found which might be highly relevant for the design of novel T cell-based cancer immunotherapies.

Role of the GNOM gene in Arabidopsis apical-basal patterning--From mutant phenotype to cellular mechanism of protein action.

How the apical-basal axis of polarity is established in embryogenesis is still a mystery in plant development. This axis appeared specifically compromised by mutations in the Arabidopsis GNOM gene. Surprisingly, GNOM encodes an ARF guanine-nucleotide exchange factor (ARF-GEF) that regulates the formation of vesicles in membrane trafficking. In-depth functional analysis of GNOM and its closest relative, GNOM-LIKE 1 (GNL1), has provided a mechanistic explanation for the development-specific role of a seemingly mundane trafficking regulator. The current model proposes that GNOM is specifically involved in the endosomal recycling of the auxin-efflux carrier PIN1 to the basal plasma membrane in provascular cells, which in turn is required for the accumulation of the plant hormone auxin at the future root pole through polar auxin transport. Thus, the analysis of GNOM highlights the importance of cell-biological processes for a mechanistic understanding of development.

Mapping, organic matter mass and water volume of a peatland in Serra do Espinhaço Meridional

Peatlands form in areas where net primary of organic matter production exceeds losses due to the decomposition, leaching or disturbance. Due to their chemical and physical characteristics, bogs can influence water dynamics because they can store large volumes of water in the rainy season and gradually release this water during the other months of the year. In Diamantina, Minas Gerais, Brazil, a peatland in the environmental protection area of Pau-de-Fruta ensures the water supply of 40,000 inhabitants. The hypothesis of this study is that the peat bogs in Pau-de-Fruta act as an environment for carbon storage and a regulator of water flow in the Córrego das Pedras basin. The objective of this study was to estimate the water volume and organic matter mass in this peatland and to study the influence of this environment on the water flow in the Córrego das Pedras basin. The peatland was mapped using 57 transects, at intervals of 100 m. Along all transects, the depth of the peat bog, the Universal Transverse Mercator (UTM) coordinates and altitude were recorded every 20 m and used to calculate the area and volume of the peatland. The water volume was estimated, using a method developed in this study, and the mass of organic matter based on samples from 106 profiles. The peatland covered 81.7 hectares (ha), and stored 497,767 m³ of water, representing 83.7 % of the total volume of the peat bog. The total amount of organic matter (OM) was 45,148 t, corresponding to 552 t ha-1 of OM. The peat bog occupies 11.9 % of the area covered by the Córrego das Pedras basin and stores 77.6 % of the annual water surplus, thus controlling the water flow in the basin and consequently regulating the water course.

Increased renal responsiveness to vasopressin and enhanced V2 receptor signaling in RGS2-/- mice.

The antidiuretic effect of vasopressin is mediated by V2 receptors (V2R) that are located in kidney connecting tubules and collecting ducts. This study provides evidence that V2R signaling is negatively regulated by regulator of G protein signaling 2 (RGS2), a member of the family of RGS proteins. This study demonstrates that (1) RGS2 expression in the kidney is restricted to the vasopressin-sensitive part of the nephron (thick ascending limb, connecting tubule, and collecting duct); (2) expression of RGS2 is rapidly upregulated by vasopressin; (3) the vasopressin-dependent accumulation of cAMP, the principal messenger of V2R signaling, is significantly higher in collecting ducts that are microdissected from the RGS2(-/-) mice compared with their wild-type littermates; and (4) analysis of urine output of mice that were exposed to water restriction followed by acute water loading revealed that RGS2(-/-) mice exhibit an increased renal responsiveness to vasopressin. It is proposed that RGS2 is involved in negative feedback regulation of V2R signaling.

Role of the ubiquitin system in regulating ion transport.

Ion channels and transporters play a critical role in ion and fluid homeostasis and thus in normal animal physiology and pathology. Tight regulation of these transmembrane proteins is therefore essential. In recent years, many studies have focused their attention on the role of the ubiquitin system in regulating ion channels and transporters, initialed by the discoveries of the role of this system in processing of Cystic Fibrosis Transmembrane Regulator (CFTR), and in regulating endocytosis of the epithelial Na(+) channel (ENaC) by the Nedd4 family of ubiquitin ligases (mainly Nedd4-2). In this review, we discuss the role of the ubiquitin system in ER Associated Degradation (ERAD) of ion channels, and in the regulation of endocytosis and lysosomal sorting of ion channels and transporters, focusing primarily in mammalian cells. We also briefly discuss the role of ubiquitin like molecules (such as SUMO) in such regulation, for which much less is known so far.

Merlin, a "Magic" Linker between Extracellular Cues and Intracellular Signaling Pathways that Regulate Cell Motility, Proliferation, and Survival.

Genetic alterations of neurofibromatosis type 2 (NF2) gene lead to the development of schwannomas, meningiomas, and ependymomas. Mutations of NF2 gene were also found in thyroid cancer, mesothelioma, and melanoma, suggesting that it functions as a tumor suppressor in a wide spectrum of cells. The product of NF2 gene is merlin (moesin-ezrin-radixin-like protein), a member of the Band 4.1 superfamily proteins. Merlin shares significant sequence homology with the ERM (Ezrin-Radixin-Moesin) family proteins and serves as a linker between transmembrane proteins and the actin-cytoskeleton. Merlin is a multifunctional protein and involved in integrating and regulating the extracellular cues and intracellular signaling pathways that control cell fate, shape, proliferation, survival, and motility. Recent studies showed that merlin regulates the cell-cell and cell-matrix adhesions and functions of the cell surface adhesion/extracellular matrix receptors including CD44 and that merlin and CD44 antagonize each other's function and work upstream of the mammalian Hippo signaling pathway. Furthermore, merlin plays important roles in stabilizing the contact inhibition of proliferation and in regulating activities of several receptor tyrosine kinases. Accumulating data also suggested an emerging role of merlin as a negative regulator of growth and progression of several non-NF2 associated cancer types. Together, these recent advances have improved our basic understanding about merlin function, its regulation, and the major signaling pathways regulated by merlin and provided the foundation for future translation of these findings into the clinic for patients bearing the cancers in which merlin function and/or its downstream signaling pathways are impaired or altered.

NLRC5, at the Heart of Antigen Presentation.

Nucleotide-binding domain and leucine-rich repeat containing receptors (NLRs) are intracellular proteins mainly involved in pathogen recognition, inflammatory responses, and cell death. Until recently, the function of the family member NLR caspase recruitment domain (CARD) containing 5 (NLRC5) has been a matter of debate. It is now clear that NLRC5 acts as a transcriptional regulator of the major-histocompatibility complex class I. In this review we detail the development of our understanding of NLRC5 function, discussing both the accepted and the controversial aspects of NLRC5 activity. We give insight into the molecular mechanisms, and the potential implications, of NLRC5 function in health and disease.

A Cell Permeable Rasgap-derived Peptide Sensitizes Cancer Cells To Radiotherapy

Purpose/Objective(s): Radiotherapy is an effective treatment modality against cancer. Despite recent technical progresses in radiation delivery precision, toxicity to healthy tissues remains the main limiting factor. RasGAP is a regulator of the Ras and Rho pathway; it has either a pro- or anti-apoptotic activity depending on the level of caspase expressed in the cell. The RasGAP derived peptide: TAT-RasGAP317 - 326 is the minimal sequence known to sensitize cancer cells, but not healthy cells, to genotoxin-induced apoptosis. In this study the TAT-RasGAP317 - 326 radio-sensitizing effect was tested in vitro and in vivo.Materials/Methods: Two weeks clonogenic forming assays with 5 human cancer cells (PANC-1, HCT116, U87, U251 and HeLa) and a non tumorigenic cell line (HaCaT) were performed. Cells were exposed to 0, 1, 2 and 4 Gy with or without 20 mMTAT-RasGAP317 - 326. Twenty mMTAT peptide was also used as control. TAT-RasGAP317 - 326 effect was also tested in tumor xenograft mouse models. Mice bearing HCT116 tumors (WT or p53 mutant) received 1.65 mg/kg TAT-RasGAP317 - 326 i.p. injected and were locally irradiated for 10 days with 3 Gy. Tumor volume was then followed during a minimum of 20 days. Control mice were treated with a single modality, either with TAT-RasGAP317 - 326 or with radiotherapy.Results: At all the tested radiation doses TAT-RasGAP317 - 326 showed a significant supra additive radio-sensitizing effect on all the tested tumor cell lines. Furthermore, it showed no sensitizing effect on the non tumorigenic cell line. In vivo, TAT-RasGAP317 - 326 also showed a significantly radio-sensitizing effect as shown by a significant higher reduction in tumor volume as much as by a significant tumor growth delay.Conclusions: Taken together our data suggest that TAT-RasGAP317 - 326 has a radio-sensitizing effect on in vivo and in vitro tumors without any effect on healthy tissues. Therefore TAT-RasGAP317 - 326 should be considered as a novel and attractive sensitizer compound allowing an improvement of the therapeutic interval.

Inflammatory diseases: is ubiquitinated NEMO at the hub?

Many patients with Crohn's disease carry mutations in NOD2, a molecule that can both activate and attenuate the pro-inflammatory effects of NF-kappa B. Recent studies implicate NOD2-induced ubiquitination of the NF-kappa B regulator NEMO as a potential means of manipulating the NF-kappa B signal.

Retinoid X receptor and peroxisome proliferator-activated receptor activate an estrogen responsive gene independent of the estrogen receptor.

Estrogen receptors regulate transcription of genes essential for sexual development and reproductive function. Since the retinoid X receptor (RXR) is able to modulate estrogen responsive genes and both 9-cis RA and fatty acids influenced development of estrogen responsive tumors, we hypothesized that estrogen responsive genes might be modulated by RXR and the fatty acid receptor (peroxisome proliferator-activated receptor, PPAR). To test this hypothesis, transfection assays in CV-1 cells were performed with an estrogen response element (ERE) coupled to a luciferase reporter construct. Addition of expression vectors for RXR and PPAR resulted in an 11-fold increase in luciferase activity in the presence of 9-cis RA. Furthermore, mobility shift assays demonstrated binding of RXR and PPAR to the vitellogenin A2-ERE and an ERE in the oxytocin promoter. Methylation interference assays demonstrated that specific guanine residues required for RXR/PPAR binding to the ERE were similar to residues required for ER binding. Moreover, RXR domain-deleted constructs in transfection assays showed that activation required RXR since an RXR delta AF-2 mutant completely abrogated reporter activity. Oligoprecipitation binding studies with biotinylated ERE and (35)S-labeled in vitro translated RXR constructs confirmed binding of delta AF-2 RXR mutant to the ERE in the presence of baculovirus-expressed PPAR. Finally, in situ hybridization confirmed RXR and PPAR mRNA expression in estrogen responsive tissues. Collectively, these data suggest that RXR and PPAR are present in reproductive tissues, are capable of activating estrogen responsive genes and suggest that the mechanism of activation may involve direct binding of the receptors to estrogen response elements.

Del "know how" al poder: la metodologia d'avaluació d'intangibles empresarials en competència i la seva aplicació en un entorn oligopolístic

Sometimes, behind the entrepreneurial profit the degree of monopoly is concealed. What it occurs with the models of investments evaluation when the skill of the employer consists on having capacity to capture the regulator?

Enantio-pyochelin: a novel siderophore produced by Pseudomonas fluorescens CHA0

RESUME Pour favoriser sa croissance en condition limitante de fer, le pathogène opportunistePseudomonas aeruginosa PAO1 sécrète un sidérophore nommé pyochéline. Celui-ci estproduit par un mécanisme de "thiotemplate", à partir de l'acide salicylique et de deuxmolécules de cystéine, et existe sous forme d'une paire de diastéréoisomèresinterconvertibles: pyochéline I (4'R, 2?R, 4?R) et pyochéline II (4'R, 2?S, 4?R). Deprécédentes études ont montré que la pyochéline induit l'expression de ses propres gènes debiosynthèse via le régulateur transcriptionnel PchR qui appartient à la famille AraC/XylS. Lapyochéline est donc non seulement un sidérophore mais également une molécule signale.Nous avons découvert que Pseudomonas fluorescens CHA0 sécrète une pyochélinestéréochimiquement distincte de celle produite par P. aeruginosa. Ce nouveau sidérophorefavorise la croissance de P. fluorescens en condition limitante en fer et induit l'expression deses propres gènes de biosynthèse. Cependant, cette molécule n'est pas reconnue commesidérophore ou molécule signale par P. aeruginosa. Réciproquement, la pyochéline estincapable de stimuler la croissance et la signalisation chez P. fluorescens. La structure dusiderophore de P. fluorescens CHA0 a été déterminée comme étant un antipode optique de lapyochéline et nommé énantio-pyochéline.La stéréospécificité de l'induction des gènes de biosynthèse de la pyochéline/énantiopyochélineest basée sur la stéréospécificité des protéines PchR de P. aeruginosa et P.fluorescens envers leur sidérophores-ligands respectifs. PchR est fonctionnel chez l'espècehétérologue, mais uniquement en présence de son propre ligand. Les récepteurs spécifiquesdes sidérophores pyochéline/enantio-pyochéline ne sont pas indispensables à la signalisationmais sont essentiels à l'incorporation du fer et à la croissance en carence de fer. Laconstruction de protéines hybrides et tronquées a révélé que le domaine N-terminal de PchRest l'élément déterminant pour la spécificité de la protéine vis-à-vis de son ligand. SUMMARY : The siderophore pyochelin is produced by the opportunistic pathogen Pseudomonas aeruginosa PAO1 and promotes growth under iron limitation. Pyochelin is made by a thiotemplate mechanism from salicylate and two molecules of cysteine and exists as a pair of interconvertible diastereoisomers: pyochelin I (4'R, 2"R, 4"R) and pyochelin II (4'R, 2"S, 4"R). Pyochelin induces the expression of its biosynthesis and uptake genes via the transcriptional AraC/Xy1S family regulator PchR in a process termed pyochelin signaling. Pseudomonas fluorescens CHAO was found to make a stereochemically distinct pyochelin to P. aeruginosa. This siderophore promoted the growth of P. fluorescens under iron limitation and induced the expression of its biosynthesis genes but was not recognised as a siderophore or signaling molecule by P. aeruginosa. Reciprocally, pyochelin was unable to promote growth or signaling in P. fluorescens. The structure of the P. fluorescens CHAO siderophore was determined and found to be enantio-pyochelin, the optical antipode of pyochelin. Stereospecificity in induction of pyochelin/enantio-pyochelin biosynthesis genes was found to be due to stereospecificity of the homologous PchR proteins of P. aeruginosa and P. fluorescens towards their respective siderophore ligands. PchR was able to function in the heterologous species, but only if supplied with its native ligand. The pyochelin/enantiopyochelin receptors were not essential for signaling although both receptors are essential for iron uptake and growth under iron limitation. Construction of hybrid and truncated PchR proteins revealed that the N-terminal domain of PchR is responsible for siderophore recognition/stereospecificity.

Genetic vulnerability factor for schizophrenia: association with glutamate cysteine ligase modifier gene and abnormal enzyme activity

Background: We previously reported in schizophrenia patients a decreased level of glutathione ([GSH]), the principal non-protein antioxidant and redox regulator, both in cerebrospinal-fluid and prefrontal cortex. To identify possible genetic causation, we studied genes involved in GSH metabolism. Methods: Genotyping: mass spectrometry analysis of polymerase chain reaction (PCR) amplified DNA fragments purified from peripheral blood. Gene expression: real-time PCR of total RNA isolated from fibroblast cultures derived from skin of patients (DSM-IV) and healthy controls (DIGS). Results: Case-control association study of single nucleotide polymorphisms (SNP) from the GSH key synthesizing enzyme glutamate-cysteine-ligase (GCL) modifier subunit (GCLM) was performed in two populations: Swiss (patients/controls: 40/31) and Danish (349/348). We found a strong association of SNP rs2301022 in GCLM gene (Danish: c2=3.2; P=0.001 after correction for multiple testing). Evidence for GCLM as a risk factor was confirmed in linkage study of NIMH families. Moreover, we observed a decrease in GCLM mRNA levels in patient fibroblasts, consistently with the association study. Interestingly, Dalton and collaborators reported in GCLM knock-out mice an increased feedback inhibition of GCL activity, resulting in 60% decrease of brain [GSH], a situation analogous to patients. These mice also exhibited an increased sensitivity to oxidative stress. Similarly, under oxidative stress conditions, GCL enzymatic activity was also decreased in patient fibroblasts. Conclusions: These results at the genetic and functional levels, combined with observations that GSH deficient models reveal morphological, electrophysiological, and behavioral anomalies analogous to those observed in patients, suggest that GCLM allelic variant is a vulnerability factor for schizophrenia.

Insight in protein S deficiency from mouse models

Protein S (ProS) is an important negative regulator of blood coagulation. Its physiological importance is evident in purpura fulminans and other life-threatening thrombotic disorders typical of ProS deficient patients. Our previous characterization of ProS deficiency in mouse models has shown similarities with the human phenotypes: heterozygous ProS-deficient mice (Pros+/-) had increased thrombotic risk whereas homozygous deficiency in ProS (Pros-/-) was incompatible with life (Blood 2009; 114:2307-2314). In tissues, ProS exerts cellular functions by binding to and activating tyrosine kinase receptors of the Tyro3 family (TAM) on the cell surface.To extend the analysis of coagulation defects beyond the Pros-/- phenotype and add new insights into the sites of synthesis ProS and its action, we generated mice with inactivated ProS in hepatocytes (Proslox/loxAlbCre+) as well as in endothelial and hematopoietic cells (Proslox/loxTie2Cre+). Both models resulted in significant reduction of circulating ProS levels and in a remarkable increased thrombotic risk in vivo. In a model of tissue factor (TF)-induced venous thromboembolism (VTE), only 17% of Proslox/loxAlbCre+ mice (n=12) and only 13% of Proslox/loxTie2Cre+ mice (n=14) survived, compared with 86% of Proslox/lox mice (n=14; P<0.001).To mimic a severe acquired ProS deficiency, ProS gene was inactivated at the adult stage using the polyI:C-inducible Mx1-Cre system (Proslox/loxMx1Cre+). Ten days after polyI:C treatment, Proslox/loxMx1Cre+ mice developed disseminated intravascular coagulation with extensive lung and liver thrombosis.It is worth noting that no skin lesions compatible with purpura fulminans were observed in any of the above-described models of partial ProS deficiency. In order to shed light on the pathogenesis of purpura fulminans, we exposed the different ProS-deficient mice to warfarin (0.2 mg/day). We observed that Pros+/-, Proslox/loxAlbCre+ and Proslox/loxTie2Cre+ mice developed retiform purpura (characterized by erythematous and necrotic lesions of the genital region and extremities) and died after 3 to 5 days after the first warfarin administration.In human, ProS is also synthesized by megakaryocytes and hence stored at high concentrations in circulating platelets (pProS). The role of pProS has been investigated by generating megakaryocyte ProS-deficient model using the PF4 promoter as Cre driver (Proslox/loxPf4Cre+). In the TF-induced VTE model, Proslox/loxPf4Cre+ (n=15) mice showed a significant increased risk of thrombosis compared to Proslox/lox controls (n=14; survival rate 47% and 86%, respectively; P<0.05). Furthermore, preliminary results suggest survival to be associated with higher circulating ProS levels. In order to evaluate the potential role of pProS in thrombus formation, we investigated the thrombotic response to intravenous injection of collagen-epinephrine in vivo and platelet function in vitro. Both in vivo and in vitro experiments showed similar results between Proslox/loxPf4Cre+ and Proslox/lox, indicating that platelet reactivity was not influenced by the absence of pProS. These data suggest that pProS is delivered at the site of thrombosis to inhibit thrombin generation.We further investigated the ability of ProS to function as a ligand of TAM receptors, by using homozygous and heterozygous deficient mice for both the TAM ligands ProS and Gas6. Gas6-/-Pros-/- mice died in utero and showed comparable dramatic bleeding and thrombotic phenotype as described for Pros-/- embryos.In conclusion, like complete ProS deficiency, double deficiency in ProS and Gas6 was lethal, whereas partial ProS deficiency was not. Mice partially deficient in ProS displayed a prothrombotic phenotype, including those with only deficiency in pProS. Purpura fulminans did not occur spontaneously in mice with partial Pros deficiency but developed upon warfarin administration.Thus, the use of different mice models of ProS deficiency can be instrumental in the study of its highly variable thrombotic phenotype and in the investigation of additional roles of ProS in inflammation and autoimmunity through TAM signaling.