p38 mitogen-activated protein kinase activation during platelet storage: Consequences for platelet recovery and hemostatic function in vivo

Platelets undergo several modifications during storage that reduce their posttransfusion survival and functionality. One important feature of these changes, which are known as platelet storage lesion, is the shedding of the surface glycoproteins GPIb-α and GPV. We recently demonstrated that tumor necrosis factor-α converting enzyme (TACE/ADAM17) mediates mitochondrial injury-induced shedding of adhesion receptors and that TACE activity correlates with reduced posttransfusion survival of these cells. We now confirm that TACE mediates receptor shedding and clearance of platelets stored for 16 hours at 37°C or 22°C. We further demonstrate that both storage and mitochondrial injury lead to the phosphorylation of p38 mitogen-activated kinase (MAPK) in platelets and that TACE-mediated receptor shedding from mouse and human platelets requires p38 MAP kinase signaling. Protein kinase C, extracellular regulated-signal kinase MAPK, and caspases were not involved in TACE activation. Both inhibition of p38 MAPK and inactivation of TACE during platelet storage led to a markedly improved posttransfusion recovery and hemostatic function of platelets in mice. p38 MAPK inhibitors had only minor effects on the aggregation of fresh platelets under static or flow conditions in vitro. In summary, our data suggest that inhibition of p38 MAPK or TACE during storage may significantly improve the quality of stored platelets

The Lack of ADAM17 Activity during Embryonic Development Causes Hemorrhage and Impairs Vessel Formation

Background: ADAM17/TACE activity is important during embryonic development. We wished to investigate possible roles of this metalloprotease, focusing on vascular development. Methodology/Principal Findings: Mice mutant in the enzymatic activity of ADAM17 were examined at various stages of embryonic development for vascular pattern and integrity using markers for vessel wall cells. We observed hemorrhage and edema starting at embryonic day E14.5 and becoming more severe as development proceeded; prior to embryonic day E14.5, embryos appeared normal. Staining for PECAM-1/CD31 revealed abnormalities in the patterns of branching of the embryonic vasculature at E14.5. Conclusions/Significance: These abnormalities preceded association of pericytes or monocyte/macrophage cells with the affected vessels and, therefore, presumably arise from defects in endothelial function consequent upon failure of ADAM17 to cleave one or more substrates involved in vascular development, such as Notch, Delta, VEGFR2 or JAM-A. Our study demonstrates a role for ADAM17 in modulating embryonic vessel development and function

Expanded repertoire of RASGRP2 variants responsible for platelet dysfunction and severe bleeding.

Heritable platelet function disorders (PFDs) are genetically heterogeneous and poorly characterized. Pathogenic variants in RASGRP2, which encodes calcium and diacylglycerol-regulated guanine exchange factor I (CalDAG-GEFI), have been reported previously in 3 pedigrees with bleeding and reduced platelet aggregation responses. To better define the phenotype associated with pathogenic RASGRP2 variants, we compared high-throughput sequencing and phenotype data from 2042 cases in pedigrees with unexplained bleeding or platelet disorders to data from 5422 controls. Eleven cases harbored 11 different, previously unreported RASGRP2 variants that were biallelic and likely pathogenic. The variants included 5 high-impact variants predicted to prevent CalDAG-GEFI expression and 6 missense variants affecting the CalDAG-GEFI CDC25 domain, which mediates Rap1 activation during platelet inside-out αIIbβ3 signaling. Cases with biallelic RASGRP2 variants had abnormal mucocutaneous, surgical, and dental bleeding from childhood, requiring ≥1 blood or platelet transfusion in 78% of cases. Platelets displayed reduced aggregation in response to adenosine 5'-diphosphate and epinephrine, but variable aggregation defects with other agonists. There were no other consistent clinical or laboratory features. These data enable definition of human CalDAG-GEFI deficiency as a nonsyndromic, recessive PFD associated with a moderate or severe bleeding phenotype and complex defects in platelet aggregation

TACE/ADAM 17 et disponibilité biologique du TNF[Tumor Necrosis Factor]-alpha (rôle dans le développement de l'athérosclérose)

Le développement de l athérosclérose est étroitement associé aux processus immunoinflammatoires. J ai étudié l implication des formes membranaire (tmTNF) et soluble (sTNF) du TNFa et de l enzyme de conversion du TNF (TACE/ADAM17) dans l athérosclérose.L utilisation de souris transgéniques soumises à une athérosclérose nutritionnelle, montre que la présence exclusive d un transgène non clivable du tmTNF n empêche pas la formation des lésions précoces mais en réduit significativement leur état inflammatoire caractérisé par une diminution des macrophages. En revanche, la déficience en TNF empêche totalement la formation des lésions (Canault et al. Atherosclerosis 2004). Nous avons émis l hypothèse que le tmTNF pouvait réduire la réactivité inflammatoire de la cellule endothéliale aortique. Les résultats ne montrent pas de modifications majeures du profil de sécrétion des cytokines par les cellules endothéliales exprimant exclusivement le tmTNF non clivable ce qui semble écarter la cellule endothéliale dans les phénomènes observés in vivo. (Canault et al. Thromb.Haemost. 2004). Ces résultats nous ont conduit, à étudier le rôle d ADAM17 dans l athérosclérose.Nous avons montré dans la souris apoE-/-, qu ADAM17 est exprimée dans les lésions athéromateuses aortiques et que cette expression suit la progression des lésions au cours du temps. Cette augmentation de TACE dans les lésions de l aorte rend compte d une capacité plus élevée de l aorte à libérer ex vivo les TNFR1 et TNFR2, substrats d ADAM17 et probablement aussi de l augmentation de la concentration plasmatique en ces récepteurs. Ces données suggèrent que dans la lésion athéroscléreuse, ADAM17 participe à la réaction inflammatoire et contribue à l élévation des taux circulants des récepteurs au TNF (Canault et al. Atherosclerosis, 2005 ). Dans les plaques humaines issues d endartériectomie carotidienne, ADAM17 est présente dans des zones acellulaires où sont présentes aussi des microparticules (MP). Nous avons montré que les MP de la plaque portaient la forme active d ADAM17 à leur surface et que ces MP étaient actives aussi bien in vitro sur un substrat synthétique mimétique de la zone de clivage du TNF que sur des cellules endothéliales en culture exprimant le TNF et le TNFR1. Ces données suggèrent fortement qu ADAM17 portée par les MP contribue à la régulation du processus inflammatoire lié à la formation des lésions d athérosclérose (Canault et al. soumis). En conclusion, mes résultats ont permis d avancer dans la connaissance du rôle des formes moléculaires du TNF dans l athérosclérose soulignant ainsi le rôle potentiel d ADAM17, protéase que nous mettons en évidence dans les lésions. Ces résultats ouvrent la voie à d autres recherches ayant pour objectifs d établir ADAM17 comme un acteur important de la régulation du potentiel pro-inflammatoire dans l athérosclérose.Abstract: The initiation of atherosclerosis is closely related to the immuno-inflammatory processes. TNFa (TNF) is produced as a transmembrane form (tmTNF) cleaved by TACE/ADAM17 into the soluble form (sTNF). During my Ph.D. thesis, I studied the implication of these molecular forms of TNF and of TACE/ADAM17 in atherosclerosis. To this end, we used three groups of mice: one expressing the wild type form of TNF (sTNF and tmTNF), one the uncleavable form of tmTNF, and one deficient in TNF, all studied in nutritional or genetic model of atherosclerosis. We showed that TNF deficiency significantly reduced the development of atherosclerotic lesions (Canault et al. Atherosclerosis 2004) and that tmTNF partly supported TNF effect depending on the vascular area considered. Then, we hypothesized that TNF phenotypes could influence the inflammatory reactivity of macrophages and aortic endothelial cells. Results showed that in macrophages, sTNF supports the expression of IL-6 and RANTES and tmTNF accounts for MCP-1 expression. TNF phenotype does not modify significantly the cytokine secretion profile of endothelial cells (Canault et al. Thromb. Haemost. 2004). These data underline the role of TNF phenotypes on macrophage inflammatory properties (manuscript in preparation). These results led us to study the role of TACE/ADAM17 in atherosclerosis. We showed that in apoE knock-out mice fed a high-fat diet, the expression of ADAM17 examined in aortic sinus and arch increased during atherosclerosis. This increase may account for the enhanced capability of aorta and macrophages to release ex vivo TNFR1 and TNFR2 that are also ADAM17 substrates and probably for the increased TNFR plasmatic concentrations during atherosclerosis (Canault et al. Atherosclerosis 2005). In human atherosclerotic plaques, ADAM17 is expressed on cellular but also acellular materials that we hypothesized to be microparticles (MP). We showed that plaque MPs carry active form of ADAM17 at their surface. These MPs are active in vitro on a synthetic substrate that mimics the cleavage zone of proTNF as well as on cultured endothelial cells expressing TNF and TNFR1 at their surface. These data strongly suggest that MPs participate to the regulation of TACE-dependent inflammatory processes being held in atherosclerotic lesion (Canault et al. submitted). To conclude, these results point out the role of the different forms of TNF on atherosclerosis, emphasizing ADAM17 as a key enzyme regulating inflammation linked to the equilibrium between these forms. This work opens new research areas aiming to determinate the role of ADAM17 in the regulation of inflammation in various pathologies in which inflammation is predominant (atherosclerosis, obesity).AIX-MARSEILLE2-BU Méd/Odontol. (130552103) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Etude des mécanismes de régulation du clivage du cd40 ligand plaquettaire (rôle de la phosphoinositide 3-kinase)

AIX-MARSEILLE2-BU Pharmacie (130552105) / SudocSudocFranceF

RasGRP2 Structure, Function and Genetic Variants in Platelet Pathophysiology

International audienceRasGRP2 is calcium and diacylglycerol-regulated guanine nucleotide exchange factor I that activates Rap1, which is an essential signaling-knot in "inside-out" αIIbβ3 integrin activation in platelets. Inherited platelet function disorder caused by variants of RASGRP2 represents a new congenital bleeding disorder referred to as platelet-type bleeding disorder-18 (BDPLT18). We review here the structure of RasGRP2 and its functions in the pathophysiology of platelets and of the other cellular types that express it. We will also examine the different pathogenic variants reported so far as well as strategies for the diagnosis and management of patients with BDPLT18

Letter by Laine et al Regarding Article, “Antithrombotic Agents: New Directions in Antithrombotic Therapy”

International audienc

Lipides peroxydés et réaction immuno-inflammatoire dans l’athérosclérose

Inflammation is now considered as a critical process that closely escorts lipid disturbances in the initiation and progression of atherosclerosis. Oxidative process, particularly oxidation of LDL, by creating neo-epitopes, is a key initiator of the inflammatory reaction as it triggers both innate and adaptive immunity. This further induces the production of pro-inflammatory cytokines and the dysregulation of endothelial and hemostatic functions leading to atherosclerotic plaque growth and rupture. The specific role of some cytokines and receptors in the dysregulation of the Th1/Th2 response is now emerging to better approach the complex mechanisms inducing disturbances of the immunoinflammatory process in atherosclerosis