A global proteomics approach identifies novel phosphorylated signaling proteins in GPVI-activated platelets: involvement of G6f, a novel platelet Grb2-binding membrane adapter.

Collagen-related peptide (CRP) stimulates powerful activation of platelets through the glycoprotein VI (GPVI)-FcR gamma-chain complex. We have combined proteomics and traditional biochemistry approaches to study the proteome of CRP-activated platelets, focusing in detail on tyrosine phosphorylation. In two separate approaches, phosphotyrosine immunoprecipitations followed by 1-D-PAGE, and 2-DE, were used for protein separation. Proteins were identified by MS. By following these approaches, 96 proteins were found to undergo PTM in response to CRP in human platelets, including 11 novel platelet proteins such as Dok-1, SPIN90, osteoclast stimulating factor 1, and beta-Pix. Interestingly, the type I transmembrane protein G6f was found to be specifically phosphorylated on Tyr-281 in response to platelet activation by CRP, providing a docking site for the adapter Grb2. G6f tyrosine phoshporylation was also found to take place in response to collagen, although not in response to the G protein-coupled receptor agonists, thrombin and ADP. Further, we also demonstrate for the first time that Grb2 and its homolog Gads are tyrosine-phosphorylated in CRP-stimulated platelets. This study provides new insights into the mechanism of platelet activation through the GPVI collagen receptor, helping to build the basis for the development of new drug targets for thrombotic disease.

Platelet protease-activated receptor 1 and membrane expression of P-selectin in pulmonary arterial hypertension

Introduction: Pulmonary arterial hypertension (PAH) is frequently associated with thrombotic events, particularly involving the pulmonary microcirculation at sites of vascular injury. We therefore decided to analyse protease-activated receptor 1 (PAR1), a key element in the activation of human platelets by thrombin, in PAH patients in stable clinical condition. Methods: Using flow cytometry, we analyzed platelet PAR1 density, PAR1-mediated exposure of P-selectin and the formation of platelet-leukocyte aggregates in 30 PAH patients aged 11 to 78 years (median 50.5 years). The control group consisted of 25 healthy subjects with the same age range as patients. Results: In patients, total platelet PAR1 density and uncleaved PAR1 density correlated negatively with platelet count (r(2) = 0.33 and r(2) = 0.34 respectively, p < 0.0015). In patients with a low platelet count (<150 x 10(9) platelets/L), both densities were increased relative to controls (82% and 33% respectively, p < 0.05). Thrombin peptide-induced platelet exposure of P-selectin was directly related to total and uncleaved PAR1 density (respectively, r(2) = 0.33 and r(2) = 0.29, p < 0.0025) and increased in subjects with low platelet count (46% versus those with normal platelet count, p < 0.05). Patients with low platelet count had decreased in vitro thrombin-induced formation of platelet-leukocyte aggregates (57% decrease versus controls, p < 0.05). Conclusions: There seems to be a subpopulation of PAH patients with increased propensity to thrombotic events as suggested by increased platelet PAR1 expression and PAR-mediated surface exposure of P-selectin associated with decreased platelet count. (C) 2009 Elsevier Ltd. All rights reserved.

Studies on IL-18 in HIV infection : effects of the cytokine on intestinal integrity, and platelets as a new source of the cytokine

L'interleukine IL-18 (IL-18), un membre de la famille de l’IL-1, est une cytokine pro-inflammatoire multifonctionnelle. Elle est produite par les monocytes, les macrophages, les cellules dendritiques, les cellules épithéliales, les kératinocytes et le cortex surrénal dans le corps humain. Cette cytokine est d'abord produite comme une protéine précurseure inactive, qui est par la suite clivée en une forme mature par la caspase-1 activée. La caspase, en elle-même, existe comme précurseur inactif dans les cellules humaines et requiert l'assemblage d'inflammasomes pour son activation. L'IL-18 pour joue un rôle clé dans la médiation des conditions inflammatoires. Notre laboratoire et d'autres ont montré que l'infection par le VIH est accompagnée d'une augmentation des taux circulants d'IL-18 avec une diminution des niveaux de son antagoniste, l'interleukine-18 binding protein (IL-18BP). Dans cette thèse, nous démontrons pour que l'IL-18 est également produite et sécrétée par les plaquettes humaines lors de leur activation. Les plaquettes contiennent des composants de l'inflammasome. Ils assemblent et activent la caspase-1, qui ensuite traite le précurseur de l'IL-18 dans sa forme mature au cours du processus d'activation des plaquettes. La cytokine est synthétisée de novo lors de l'activation des plaquettes. Contrairement à l'IL-18, les plaquettes expriment constitutivement l’IL-18BP, et la libèrent de manière constitutive, ainsi que lors de l'activation. L'IL-18 et l'IL-18BP sont colocalisés avec CD63, un marqueur pour les granules α des plaquettes. L'IL-18 libéré des plaquettes constitue la source principale de cette cytokine dans la circulation humaine chez les individus sains. Nous avons identifié des concentrations faibles de cette cytokine dans les lysats de plaquettes chez les individus infectés par le VIH par rapport à ceux en santé. D'autre part, les concentrations ont été augmentées dans le sérum et le plasma pauvre en plaquettes chez les individus infectés. Des résultats similaires ont été obtenus avec l'IL-18BP dans les lysats de plaquettes d'individus sains et infectés par le VIH. Cependant, des quantités plus faibles de cet antagoniste ont été trouvées dans le sérum et le plasma pauvre en plaquettes d'individus infectés par le VIH par rapport à ceux en santé. Nos résultats ont des implications importantes pour les maladies inflammatoires chroniques dans laquelle une activité accrue de l'IL-18 joue un rôle pathogène. Le VIH est également accompagné par une inflammation intestinale et une diminution de l'intégrité intestinale, mesurée par la réparation de la muqueuse, la régénération et la perméabilité. Cependant, on en sait peu sur la relation entre le niveau élevé de l'IL-18 associé à l'infection au VIH et la perméabilité intestinale: ceci n'a jamais été étudié. Dans cette thèse, nous démontrons le rôle du virus et sa protéine Tat à augmenter la production d'IL-18 chez deux lignées de cellules épithéliales intestinales (HT29 et Caco2) ainsi qu'une diminution de l'IL-18BP. L'IL-18 induit une hyperperméabilité de la barrière épithéliale en perturbant à la fois les jonctions serrées et adhérentes, et ce, en modulant l'expression et la distribution de l'occludine, de claudine-2 et de la bêta-caténine. Une désorganisation de l'actine F a également été observée dans les cellules lors de l'incubation avec l'IL-18. Les mêmes observations ont été faites avec la protéine Tat du VIH-1. Après une incubation prolongée, l'IL-18 a causé la mort des cellules intestinales et induit l'apoptose par l'activation de la caspase-1 et la caspase-3. Fait intéressant, les taux plasmatiques de lipopolysaccharides chez trois catégories différentes de patients au VIH (ART-naïf, ART-traitée et contrôleurs élite) sont en corrélation avec les niveaux plasmatiques de l'IL-18. Enfin, nous avons étudié la voie de signalisation à travers laquelle l'IL-18 induit une perméabilité intestinale accrue. En bref, nos études identifient les plaquettes comme une source importante d'IL-18, et leur activation lors d'une infection à VIH contribue à des concentrations accrues de cette cytokine. Le virus entraine également l'augmentation de la production de cytokines par les cellules épithéliales intestinales. L'activité biologique accrue de ces cytokines contribue à la pathogenèse du sida en augmentant la perméabilité intestinale et en causant la mort des cellules intestinales. L'IL-18 pourrait servir de cible moléculaire pour retarder la progression du sida et réduire l'inflammation chronique dans un stade précoce d'une infection à VIH.

Platelets release novel thiol isomerase enzymes which are recruited to the cell surface following activation

The thiol isomerase enzymes protein disulphide isomerase (PDI) and endoplasmic reticulum protein 5 (ERp5) are released by resting and activated platelets. These re-associate with the cell surface where they modulate a range of platelet responses including adhesion, secretion and aggregation. Recent studies suggest the existence of yet uncharacterised platelet thiol isomerase proteins. This study aimed to identify which other thiol isomerase enzymes are present in human platelets. Through the use of immunoblotting, flow cytometry, cell-surface biotinylation and gene array analysis, we report the presence of five additional thiol isomerases in human and mouse platelets and megakaryocytes, namely; ERp57, ERp72, ERp44, ERp29 and TMX3. ERp72, ERp57, ERp44 and ERp29 are released by platelets and relocate to the cell surface following platelet activation. The transmembrane thiol isomerase TMX3 was also detected on the platelet surface but does not increase following activation. Extracellular PDI is also implicated in the regulation of coagulation by the modulation of tissue factor activity. ERp57 was identified within platelet-derived microparticle fractions, suggesting that ERp57 may also be involved in the regulation of coagulation as well as platelet function. These data collectively implicate the expanding family of platelet-surface thiol isomerases in the regulation of haemostasis.

Proteinase-activated receptors, targets for kallikrein signaling

Serine proteinases like thrombin can signal to cells by the cleavage/activation of proteinase-activated receptors (PARs). Although thrombin is a recognized physiological activator of PAR(1) and PAR(4), the endogenous enzymes responsible for activating PAR(2) in settings other than the gastrointestinal system, where trypsin can activate PAR(2), are unknown. We tested the hypothesis that the human tissue kallikrein (hK) family of proteinases regulates PAR signaling by using the following: 1) a high pressure liquid chromatography (HPLC)-mass spectral analysis of the cleavage products yielded upon incubation of hK5, -6, and -14 with synthetic PAR N-terminal peptide sequences representing the cleavage/activation motifs of PAR(1), PAR(2), and PAR(4); 2) PAR-dependent calcium signaling responses in cells expressing PAR(1), PAR(2), and PAR(4) and in human platelets; 3) a vascular ring vasorelaxation assay; and 4) a PAR(4)-dependent rat and human platelet aggregation assay. We found that hK5, -6, and -14 all yielded PAR peptide cleavage sequences consistent with either receptor activation or inactivation/disarming. Furthermore, hK14 was able to activate PAR(1), PAR(2), and PAR(4) and to disarm/inhibit PAR(1). Although hK5 and -6 were also able to activate PAR(2), they failed to cause PAR(4)-dependent aggregation of rat and human platelets, although hK14 did. Furthermore, the relative potencies and maximum effects of hK14 and -6 to activate PAR(2)-mediated calcium signaling differed. Our data indicate that in physiological settings, hKs may represent important endogenous regulators of the PARs and that different hKs can have differential actions on PAR(1), PAR(2), and PAR(4).

Anti-inflammatory effect of parenteral fish oil lipid emulsion on human activated mononuclear leukocytes

Background & aim: To compare the effect of fish oil-based (FO) lipid emulsions (LE) for parenteral administration with standard LE and a new FO containing LE composed of four different oils on the antigen presentation and inflammatory variables. Methods: Phytohemagglutinin (PHA) activated human mononuclear leukocytes were cultured with different LE - Control: without LE; SO: soybean oil; SO/FO: soybean and FO (4:1); MCT/SO: medium chain triglycerides and SO (1:1); MCT/SO/FO: MCT/SO and FO (4:1) and SMOF: a new LE containing FO. Cytokine production was evaluated by ELISA, the expression of antigen-presenting and co-stimulatory surface molecules were analyzed by flow cytometry and lymphocyte proliferation was assessed by H(3)-Thymidine incorporation, after tetanus toxoid-induced activation. Results: All LE decreased the HLA-DR and increased CD28 and CD152 expression on monocytes/macrophages and lymphocytes surface (p < 0.05). SO/FO and MCT/SO/FO decreased lymphocyte proliferation (p<0.05). All LE decreased IL-2 product ion, but this effect was enhanced with MCT/SO/FO and SMOF (p < 0.05). MCT/SOTO decreased IL-6 and increased IL-10, whereas SO had the opposite effect (p < 0.05). Conclusion: FO LE inhibited lymphocyte proliferation and had an anti-inflammatory effect. These effects seem to be enhanced when FO is mixed with MCT/SO. SMOF had a neutral impact on lymphocyte proliferation and IL-6 and IL-10 production.

Inhibition of human platelet aggregation by eosinophils

AIMS: The relationship between the activity of eosinophils and platelets has been observed in recent decades by many scientists. These observations include increased numbers of eosinophils associated with platelet disorders, including changes in the coagulation cascade and platelet aggregation. Based on these observations, the interaction between eosinophils and platelets in platelet aggregation was analyze. MAIN METHODS: Human platelets were incubated with eosinophil cytosolic fraction, promyelocytic human HL-60 clone 15 cell lineage, and eosinophil cationic protein (ECP). Platelet rich plasma (PRP) aggregation was induced by adenosine diphosphate, platelet activating factor, arachidonic acid, and collagen, and washed platelets (WP) were activated by thrombin. KEY FINDINGS: Aggregation induced by all agonists was dose dependently inhibited by eosinophil cytosolic fraction. This inhibition was only partially reversed by previous incubation of the eosinophils with l-Nitro-Arginine-Methyl-Ester (l-NAME). Previous incubation with indomethacin did not prevent the cytosolic fraction induced inhibition. The separation of eosinophil cytosolic fraction by gel filtration on Sephadex G-75 showed that the inhibitory activity was concentrated in the lower molecular weight fraction. HL-60 clone 15 cells differentiated into eosinophils for 5 and 7 day were able to inhibit platelet aggregation. The ECP protein inhibited the platelet aggregation on PRP and WP. This inhibition was more evident in WP, and the citotoxicity MTT assay proved the viability of tested platelets, showing that the observed inhibition by the ECP protein does not occur simply by cell death. SIGNIFICANCE: Our results indicate that eosinophils play a fundamental role in platelet aggregation inhibition

Converting enzyme-independent release of tumor necrosis factor α and IL-1β from a stimulated human monocytic cell line in the presence of activated neutrophils or purified proteinase 3

Two important cytokines mediating inflammation are tumor necrosis factor α (TNFα) and IL-1β, both of which require conversion to soluble forms by converting enzymes. The importance of TNFα-converting enzyme and IL-1β-converting enzyme in the production of circulating TNFα and IL-1β in response to systemic challenges has been demonstrated by the use of specific converting enzyme inhibitors. Many inflammatory responses, however, are not systemic but instead are localized. In these situations release and/or activation of cytokines may be different from that seen in response to a systemic stimulus, particularly because associations of various cell populations in these foci allows for the exposure of procytokines to the proteolytic enzymes produced by activated neutrophils, neutrophil elastase (NE), proteinase 3 (PR3), and cathepsin G (Cat G). To investigate the possibility of alternative processing of TNFα and/or IL-1β by neutrophil-derived proteinases, immunoreactive TNFα and IL-1β release from lipopolysaccharide-stimulated THP-1 cells was measured in the presence of activated human neutrophils. Under these conditions, TNFα and IL-1β release was augmented 2- to 5-fold. In the presence of a specific inhibitor of NE and PR3, enhanced release of both cytokines was largely abolished; however, in the presence of a NE and Cat G selective inhibitor, secretory leucocyte proteinase inhibitor, reduction of the enhanced release was minimal. This finding suggested that the augmented release was attributable to PR3 but not NE nor Cat G. Use of purified enzymes confirmed this conclusion. These results indicate that there may be alternative pathways for the production of these two proinflammatory cytokines, particularly in the context of local inflammatory processes.

Staphylococcus Aureus Enterotoxins A And B Inhibit Human And Mice Eosinophil Chemotaxis And Adhesion In Vitro.

Staphylococcus aureus aggravates the allergic eosinophilic inflammation. We hypothesized that Staphylococcus aureus-derived enterotoxins directly affect eosinophil functions. Therefore, this study investigated the effects of Staphylococcal enterotoxins A and B (SEA and SEB) on human and mice eosinophil chemotaxis and adhesion in vitro, focusing on p38 MAPK phosphorylation and intracellular Ca(2+) mobilization. Eosinophil chemotaxis was evaluated using a microchemotaxis chamber, whereas adhesion was performed in VCAM-1 and ICAM-1-coated plates. Measurement of p38 MAPK phosphorylation and intracellular Ca(2+) levels were monitored by flow cytometry and fluorogenic calcium-binding dye, respectively. Prior incubation (30 to 240 min) of human blood eosinophils with SEA (0.5 to 3 ng/ml) significantly reduced eotaxin-, PAF- and RANTES-induced chemotaxis (P<0.05). Likewise, SEB (1 ng/ml, 30 min) significantly reduced eotaxin-induced human eosinophil chemotaxis (P<0.05). The reduction of eotaxin-induced human eosinophil chemotaxis by SEA and SEB was prevented by anti-MHC monoclonal antibody (1 μg/ml). In addition, SEA and SEB nearly suppressed the eotaxin-induced human eosinophil adhesion in ICAM-1- and VCAM-1-coated plates. SEA and SEB prevented the increases of p38 MAPK phosphorylation and Ca(2+) levels in eotaxin-activated human eosinophils. In separate protocols, we evaluated the effects of SEA on chemotaxis and adhesion of eosinophils obtained from mice bone marrow. SEA (10 ng/ml) significantly reduced the eotaxin-induced chemotaxis along with cell adhesion to both ICAM-1 and VCAM-1-coated plates (P<0.05). In conclusion, the inhibition by SEA and SEB of eosinophil functions (chemotaxis and adhesion) are associated with reductions of p38 MAPK phosphorylation and intracellular Ca(2+) mobilization.

MUC1 epithelial mucin (CD227) is expressed by activated dendritic cells

The MUC1 mucin (CD227) is a cell surface mucin originally thought to be restricted to epithelial tissues. We report that CD227 is expressed on human blood dendritic cells (DC) and monocyte-derived DC following in vitro activation. Freshly isolated murine splenic DC had very low levels of CD227; however, all DC expressed CD227 following in vitro culture. In the mouse spleen, CD227 was seen on clusters within the red pulp and surrounding the marginal zone in the white pulp. Additionally, we confirm CD227 expression by activated human T cells and show for the first time that the CD227 cytoplasmic domain is tyrosine-phosphorylated in activated T cells and DC and is associated with other phosphoproteins, indicating a role in signaling. The function of CD227 on DC and T cells requires further elucidation.

Formaldehyde-fixation of platelets for flow cytometric measurement of phosphatidylserine exposure is feasible.

Strong platelet activation results in a redistribution of negatively charged phospholipids from the cytosolic to the outer leaflet of the cellular membrane. Annexin V has a high affinity to negatively charged phospholipids and can be used to identify procoagulant platelets. Formaldehyde fixation can cause factitious Annexin V binding. Our aim was to evaluate a method for fixing platelets avoiding additional Annexin V binding. We induced expression of negatively charged phospholipids on the surface of a fraction of platelets by combined activation with convulxin and thrombin in the presence of Annexin V-fluorescein isothiocyanate and calcium. Aliquots of resting and activated platelets were fixed with a low concentration, calcium-free formaldehyde solution. Both native platelets and fixed platelets were analyzed by flow cytometry immediately and after a 24-h storage at 4°C. We observed that the percentage of Annexin V positive resting platelets ranged from 1.5 to 9.3% for the native samples and from 0.4 to 12.8% for the fixed samples (P=0.706, paired t-test). The amount of Annexin V positive convulxin/thrombin activated platelets varied from 12.9 to 35.4% without fixation and from 15.3 to 36.3% after formalin fixation (P=0.450). After a 24-h storage at 4°C, Annexin V positive platelets significantly increased both in the resting and in the convulxin/thrombin activated samples of native platelets (both P<0.001), while results for formalin fixed platelets did not differ from baseline values (P=0.318 for resting fixed platelets; P=0.673 for activated fixed platelets). We conclude that platelet fixation with a low concentration, calcium-free formaldehyde solution does not alter the proportion of Annexin V positive platelets. This method can be used to investigate properties of procoagulant platelets by multicolor flow-cytometric analysis requiring fixation steps.

Cross-presenting human gammadelta T cells induce robust CD8+ alphabeta T cell responses.

Gammadelta T cells are implicated in host defense against microbes and tumors but their mode of function remains largely unresolved. Here, we have investigated the ability of activated human Vgamma9Vdelta2(+) T cells (termed gammadelta T-APCs) to cross-present microbial and tumor antigens to CD8(+) alphabeta T cells. Although this process is thought to be mediated best by DCs, adoptive transfer of ex vivo antigen-loaded, human DCs during immunotherapy of cancer patients has shown limited success. We report that gammadelta T-APCs take up and process soluble proteins and induce proliferation, target cell killing and cytokine production responses in antigen-experienced and naïve CD8(+) alphabeta T cells. Induction of APC functions in Vgamma9Vdelta2(+) T cells was accompanied by the up-regulation of costimulatory and MHC class I molecules. In contrast, the functional predominance of the immunoproteasome was a characteristic of gammadelta T cells irrespective of their state of activation. Gammadelta T-APCs were more efficient in antigen cross-presentation than monocyte-derived DCs, which is in contrast to the strong induction of CD4(+) alphabeta T cell responses by both types of APCs. Our study reveals unexpected properties of human gammadelta T-APCs in the induction of CD8(+) alphabeta T effector cells, and justifies their further exploration in immunotherapy research.

PPAR beta/delta Agonists Modulate Platelet Function via a Mechanism Involving PPAR Receptors and Specific Association/Repression of PKC alpha-Brief Report

Objectives-Peroxisome proliferator-activated receptor beta/delta (PPAR beta/delta) is a nuclear receptor found in platelets. PPAR beta/delta agonists acutely inhibit platelet function within a few minutes of addition. As platelets are anucleated, the effects of PPAR beta/delta agonists on platelets must be nongenomic. Currently, the particular role of PPAR beta/delta receptors and their intracellular signaling pathways in platelets are not known. Methods and Results-We have used mice lacking PPAR beta/delta (PPAR beta/delta(-/-)) to show the effects of the PPAR beta/delta agonist GW501516 on platelet adhesion and cAMP levels are mediated specifically by PPAR beta/delta, however GW501516 had no PPAR beta/delta-specific effect on platelet aggregation. Studies in human platelets showed that PKC alpha, which can mediate platelet activation, was bound and repressed by PPAR beta/delta after platelets were treated with GW501516. Conclusions-These data provide evidence of a novel mechanism by which PPAR receptors influence platelet activity and thereby thrombotic risk. (Arterioscler Thromb Vasc Biol. 2009; 29: 1871-1873.)

CD40 signalling in platelet function

Le CD40 est un membre de la famille des récepteurs du facteur de nécrose tumorale ("Tumour necrosis factor", TNF), initialement identifié sur des cellules de carcinome de la vessie. L'interaction du CD40 avec son ligand (CD40L) est d'une importance cruciale pour le développement des cellules B et de la commutation d'isotype au cours de la réponse immunitaire acquise. L'expression du complexe CD40/CD40L était initialement cru d'être limiter aux cellules du système immunitaire, mais aujourd'hui il est bien connu que ce complexe est également exprimé sur les cellules du système circulatoire et vasculaire, et est impliqué dans diverses réactions inflammatoires; de sorte que le CD40L est maintenant considéré comme une molécule thrombo-inflammatoire prédictive des événements cardiovasculaires. Les plaquettes expriment constitutivement le CD40, alors que le CD40L n'est exprimé que suite à leur l'activation. Il est ensuite clivé en sa forme soluble (sCD40L) qui représente la majorité du sCD40L en circulation. Il fut démontré que le sCD40L influence l'activation plaquettaire mais son effet exact sur la fonction plaquettaire, ainsi que les mécanismes cellulaires et moléculaires sous-jacents à son action demeurent inconnus. Ainsi, ce projet a été entrepris dans le but d’adresser les objectifs spécifiques suivants: 1) évaluer les effets in vitro du sCD40L sur l'activation et l'agrégation plaquettaire; 2) identifier les récepteurs plaquettaires impliqués dans l’action du sCD40L; 3) élucider les voies signalétiques intracellulaires induits par le sCD40L; 4) évaluer les effets du sCD40L sur la formation de thrombus in vivo. Nous avons trouvé que le sCD40L augmente fortement l'activation et l'agrégation des plaquettes en réponse à de faibles concentrations d'agonistes. Les plaquettes humaines traitées avec une forme mutante du sCD40L qui n'interagit pas avec le CD40, et les plaquettes de souris déficientes en CD40 ne furent pas en mesure d'induire de telles réponses, indiquant que le récepteur principal du sCD40L au niveau des plaquettes est le CD40. En plus, nous avons identifié la présence de plusieurs membres de la famille du facteur associé du récepteur du TNF ("TNF receptor-associated factor", TRAF) dans les plaquettes et nous avons montré que seulement le TRAF2 s'associe avec le CD40 suite à la stimulation par le sCD40L. Nos résultats indiquent aussi que le sCD40L agisse sur les plaquettes au repos par l'entremise de deux voies signalétiques distinctes. La première voie implique l'activation de la petite GTPase Rac1 et de sa cible en aval, soit la protéine kinase p38 activée par le mitogène ("p38 mitogen-activated protein kinase", p38 MAPK ), menant au changement de forme plaquettaire et à la polymérisation de l'actine; alors que la deuxième voie implique l'activation de la cascade signalétique du NF-kB. Par ailleurs, à la suite d'une lésion artérielle induite par le chlorure de fer, le sCD40L exacerbe la formation de thrombus et l'infiltration leucocytaire au sein du thrombus dans les souris du type sauvage, mais pas chez les souris déficientes en CD40. En conclusion, ce projet a permis d'identifier pour la première fois deux voies signalétiques distinctes en aval du CD40 plaquettaire et a permis d'établir leur implication dans l'activation et l'agrégation plaquettaire en réponse au sCD40L. De manière plus importante, ce projet nous a permis d'établir un lien direct entre les niveaux élevés du sCD40L circulant et la formation de thrombus in vivo, tout en soulignant l'importance du CD40 dans ce processus. Par conséquent, l'axe CD40/CD40L joue un rôle important dans l'activation des plaquettes, les prédisposant à une thrombose accrue en réponse à une lésion vasculaire. Ces résultats peuvent expliquer en partie la corrélation entre les taux circulants élevés du sCD40L et l'incidence des maladies cardiovasculaires.

Rôle de la PKC delta dans la fonction plaquettaire en réponse à la thrombine via le récepteur GPIb alpha

La famille des protéines kinases C (PKC) est essentielle pour la fonction plaquettaire en réponse à la thrombine qui signale et active les plaquettes via les proteases activated receptors (PAR-1 et PAR-4) et le GPIbα. Ces derniers constituent les récepteurs de moyenne/faible et de hautes affinités pour la thrombine, respectivement. L’isoforme PKCδ régule positivement ou négativement la fonction des plaquettes tout dépendamment de la nature du stimulus. Cependant, son importance dans la fonction plaquettaire en réponse à la thrombine en aval de la GPIbα reste inconnue. L’objectif principal de ce projet de doctorat était de déterminer l'implication de l'axe thrombine/GPIbα/PKCδ dans la fonction plaquettaire et d’évaluer le rôle de cet axe dans la régulation de la thrombose. Dans les plaquettes humaines, le prétraitement avec l'inhibiteur spécifique de la PKCδ δ(V1-1)TAT, a significativement potentialisé l'activation et l’agrégation des plaquettes en réponse à de faibles concentrations de α-thrombine, mais pas en réponse à la γ-thrombine ou aux agonistes des PARs. Ce phénomène de potentialisation a été associé à une sécrétion accrue de granules, de génération de thromboxane A2 (TXA2) et une phosphorylation de la PKCδ sur la Tyr311, qui ont toutes été prévenues par l’inhibition spécifique du GPIbα à l’aide d’un anticorps monoclonal bloquant. En outre, l'inhibition de la p38 MAPK, ERK1/2 et le TXA2 a inversé ce processus de potentialisation. Les plaquettes murines déficientes en PKCδ étaient aussi plus réactives à la thrombine et ont montré une augmentation significative de l'agrégation, alors qu’une étude menée in vivo chez la souris PKCδ- /- a montré, suite à une stimulation par α-thrombine, une réaction thrombotique accrue caractérisée par une diminution significative du temps de saignement ainsi qu’une formation de thrombo-embolies pulmonaires. En bloquant le GPIbα, ces effets ont été renversés. Cette étude ouvre de nouvelles perspectives quant au rôle de la PKCδ dans les plaquettes en aval de GPIbα, où elle régule négativement la fonction plaquettaire en réponse à la thrombine. Ainsi, l'axe thrombine/GPIbα/PKCδ dans les plaquettes pourrait représenter un régulateur critique de la fonction plaquettaire et l'hémostase, et le dysfonctionnement de cette voie pourrait conduire à des événements thrombotiques.