A mutation of the human EPHB2 gene leads to a major platelet functional defect.

The ephrin transmembrane receptor family of tyrosine kinases is involved in platelet function. We report the first EPHB2 variant affecting platelets in 2 siblings (P1 and P2) from a consanguineous family with recurrent bleeding and normal platelet counts. Whole-exome sequencing identified a c.2233C>T variant (missense p.R745C) of the EPHB2 gene. P1 and P2 were homozygous for this variant, while their asymptomatic parents were heterozygous. The p.R745C variant within the tyrosine kinase domain was associated with defects in platelet aggregation, αIIbβ3 activation, and granule secretion induced by G-protein-coupled receptor (GPCR) agonists and convulxin, as well as in thrombus formation on collagen under flow. In contrast, clot retraction, flow-dependent platelet adhesion, and spreading on fibrinogen were only mildly affected, indicating limited effects on αIIbβ3 outside-in signaling. Most importantly, Lyn, Syk, and FcRγ phosphorylation, the initial steps in glycoprotein VI (GPVI) platelet signaling were drastically impaired in the absence of platelet-platelet contact, indicating a positive role for EPHB2 in GPVI activation. Likewise platelet activation by PAR4-AP showed defective Src activation, as opposed to normal protein kinase C activity and Ca2+ mobilization. Overexpression of wild-type and R745C EPHB2 variant in RBL-2H3 (rat basophilic leukemia) cells stably expressing human GPVI confirmed that EPHB2 R745C mutation impaired EPHB2 autophosphorylation but had no effect on ephrin ligand-induced EPHB2 clustering, suggesting it did not interfere with EPHB2-ephrin-mediated cell-to-cell contact. In conclusion, this novel inherited platelet disorder affecting EPHB2 demonstrates this tyrosine kinase receptor plays an important role in platelet function through crosstalk with GPVI and GPCR signaling

In Vivo Analysis of the Role of O-Glycosylations of Von Willebrand Factor

The objective of this project was to study the function of O-glycosylations in von Willebrand factor (VWF) life cycle. In total, 14 different murine Vwf cDNAs mutated on one or several O-glycosylations sites were generated: 9 individual mutants, 2 doublets, 2 clusters and 1 mutant with all 9 murine glycosylation sites mutated (Del-O-Gly). We expressed each mutated cDNA in VWF deficient-mice by hydrodynamic injection. An immunosorbent assay with Peanut Agglutinin (PNA) was used to verify the O-glycosylation status. Wild-type (WT) VWF expressed by hepatocytes after hydrodynamic injection was able to bind PNA with slightly higher affinity than endothelial-derived VWF. In contrast, the Del-O-Gly VWF mutant did not bind PNA, demonstrating removal of O-linked glycans. All mutants displayed a normal multimeric pattern. Two mutants, Del-O-Gly and T1255A/T1256A, led to expression levels 50% lower than those induced by WT VWF and their half-life in vivo was significantly reduced. When testing the capacity of each mutant to correct the bleeding time of VWF-deficient mice, we found that S1486A, T1255A, T1256A and the doublet T1255A/T1256A were unable to do so. In conclusion we have shown that O-glycosylations are dispensable for normal VWF multimerization and biosynthesis. It also appears that some O-glycosylation sites, particularly the T1255 and T1256 residues, are involved in the maintenance of VWF plasma levels and are essential for normal haemostasis. As for the S1486 residue, it seems to be important for platelet binding as demonstrated in vitro using perfusion experiments

Rôle complémentaire des MAP Kinases ERK2, p38 et JNK1 dans l'activation plaquettaire

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Activation et régulation de la MAP kinase ERK2 dans les plaquettes humaines

Les MAP (Mitogen-Activated Protein) kinases jouent un rôle essentiel dans l'homéostasie cellulaire mais peu de littérature existe dans les plaquettes sanguines, responsables de la formation du thrombus, lors de lésion vasculaire. Nous avons alors étudié l'activation de la MAP Kinases ERK2 (Extracellular-Regulated protein Kinase) induite par la thrombine et mis en évidence les protéines de la cascade d'activation impliquée. Par ailleurs, nous avons étudié les relations entre ERK2 et l'intégrine aIIbb3 engagée lors de l'agrégation plaquettaire. Nos travaux montrent une régulation négative de l'activation de ERK2 par une ser/thr phosphatase dépendante de l'engagement de l'intégrine, dans le cytosol des plaquettes stimulées par la thrombine. Enfin, l'étude de l'activation de ERK2 par de faibles concentrations de collagène, a mis en évidence la coopération des récepteurs de l'ADP et du TXA2, couplés aux protéines G hétérotrimériques.The MAP kinases are essential in cellular functions but little is known about it in blood platelets which are responsible of thrombus formation in the case of vascular injury. So, we decided to study the activation of MAP kinase ERK2 induced by thrombin and the involvement of the different proteins of this pathway. Moreover, we studied the relationship between ERK2 and the 1IIbb3 integrin, responsible of the platelet aggregation. Our results shown a negative regulation of the ERK2 activation by a ser/thr phosphatase dependently of the integrin engagement. Finaly, activation of ERK2 by low collagen concentrations involves the coactivation of both protein G coupled receptors of the ADP an TXA2.PARIS5-BU Saints-Pères (751062109) / SudocSudocFranceF

Implication des MAP Kinases ERK2 et p38 dans l'activation plaquettaire

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Les MAP kinases JNKs dans l'hémostase et la thrombose

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

ACTIVATION ET REGULATION DES MAP KINASES EXTRACELLULAR SIGNAL-REGULATED KINASE 2 ET C-JUN NH2-TERMINAL KINASE 1 DANS LES PLAQUETTES HUMAINES

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF