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

Synthese des proteoglycannes par les cellules musculaires lisses d'aorte de porc en culture : etude en fonction du cycle cellulaire, effet de l'insuline et de facteur(s) secrete(s) par les cellules endotheliales

SIGLECNRS T Bordereau / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Differential Involvement of ERK2 and p38 in Platelet Adhesion to Collagen

International audienceWe investigated the role of two MAP kinases, ERK2 and p38, in platelet adhesion and spreading over colla-gen matrix in static and blood flow conditions. P38 was involved in collagen-induced platelet adhesion and spreading in static adhesion conditions, whereas ERK2 was not. In blood flow conditions, with shear rates of 300 or 1500 s ؊1 , ERK2 and p38 displayed differential involvement in platelet adhesion, depending on the presence or absence of the von Willebrand factor (vWF). Low colla-gen coverage densities (0.04 g/cm 2) did not support vWF binding. During perfusions over this surface, plate-let adhesion was not affected by the inhibition of ERK2 phosphorylation by PD 98059. However, abolishing p38 activation by SB 203580 treatment reduced platelet adhesion by 67 ؎ 9% at 300 s ؊1 and 56 ؎ 2% at 1500 s ؊1. In these conditions, the p38 activity required for platelet adhesion depends on the ␣2␤1 collagen receptor. At higher collagen coverage densities (0.8 g/cm 2) supporting vWF binding, the inhibition of ERK2 activity by PD 98059 decreased adhesion by 47 ؎ 6% at 300 s ؊1 and 72 ؎ 3% at 1500 s ؊1 , whereas p38 inhibition had only a small effect. The ERK2 activity required for platelet adhesion was dependent on the interaction of vWF with GPIb. In conclusion, ERK2 and p38 have complementary effects in the control of platelet adhesion to collagen in a shear stress-dependent manner

LIM kinase/cofilin dysregulation promotes macrothrombocytopenia in severe von Willebrand disease-type 2B

International audiencevon Willebrand disease type 2B (VWD-type 2B) is characterized by gain-of-function mutations of von Willebrand factor (vWF) that enhance its binding to platelet glycoprotein Ibα and alter the protein's multimeric structure. Patients with VWD-type 2B display variable extents of bleeding associated with macrothrombocytopenia and sometimes with thrombopathy. Here, we addressed the molecular mechanism underlying the severe macrothrombocytopenia both in a knockin murine model for VWD-type 2B by introducing the p.V1316M mutation in the murine Vwf gene and in a patient bearing this mutation. We provide evidence of a profound defect in megakaryocyte (MK) function since: (a) the extent of proplatelet formation was drastically decreased in 2B MKs, with thick proplatelet extensions and large swellings; and (b) 2B MKs presented actin disorganization that was controlled by upregulation of the RhoA/LIM kinase (LIMK)/cofilin pathway. In vitro and in vivo inhibition of the LIMK/cofilin signaling pathway rescued actin turnover and restored normal proplatelet formation, platelet count, and platelet size. These data indicate, to our knowledge for the first time, that the severe macrothrombocytopenia in VWD-type 2B p.V1316M is due to an MK dysfunction that originates from a constitutive activation of the RhoA/LIMK/cofilin pathway and actin disorganization. This suggests a potentially new function of vWF during platelet formation that involves regulation of actin dynamics

Apoptotic Platelet Events Are Not Observed in Severe von Willebrand Disease-Type 2B Mutation p.V1316M

<div><p>Thrombocytopenia and increased platelet clearance observed in von Willebrand disease-type 2B (VWD-2B) may be explained by platelet apoptosis triggered by the constitutive binding of VWF to its receptor, glycoprotein Ib (GPIb). Apoptosis was assessed in platelets from two patients with a severe VWD-2B mutation VWF/p.V1316M and from mice transiently expressing VWF/p.V1316M. We now report that the VWD-2B mutation VWF/p.V1316M which binds spontaneously to its receptor GPIbα does not induce apoptosis. In 2 unrelated patients (P1 and P2) exhibiting different VWF plasma levels (70% and 36%, respectively, compared with normal pooled human plasma given as 100%), inner transmembrane depolarization of mitochondria, characteristic of apoptotic events was undetectable in platelets, whether washed or in whole blood. No or a moderate phosphatidyl serine (PS) exposure as measured by annexin-V staining was observed for P1 and P2, respectively. Expression of pro-apoptotic proteins Bak and Bax, and caspase-3 activity were similar to control platelets. In the VWD-2B mouse model expressing high levels of mVWF/p.V1316M (423%), similar to what is found in inflammatory pathologies, no significant difference was observed between mice expressing mVWF/WT and mVWF/p.V1316M. These results strongly argue against apoptosis as a mechanism for the thrombocytopenia of severe VWD-2B exhibiting the VWF/p.V1316M mutation.</p></div

Expression of apoptotic proteins in patients with VWD-type 2B.

<p>Washed platelets (2.5 x10⁸ platelets/mL) from controls (C) or P1 and P2 were lysed and then equal numbers of platelets were loaded. Apoptotic proteins were assessed by immunoblotting with anti-Bak, anti-Bax, anti Bcl-xL and anti-14-3-3ζ antibodies. Data are expressed as the ratio of apoptotic protein expression versus 14-3-3ζ expression. Then, the ratio of an apoptotic protein for P1 or P2 was compared with the corresponding ratio for control (100%). Results are means ± Standard Error of the Mean (SEM) from three independent experiments. *p = 2.8.0x10^-2, **p = 2.3x10^-3, ***p = 2.9x10^-4.</p