G_13 is an essential mediator of platelet activation in hemostasis and thrombosis

Platelet activation at sites of vascular injury is essential for primary hemostasis, but also underlies arterial thrombosis leading to myocardial infarction or stroke. Platelet activators such as adenosine diphosphate, thrombin or thromboxane A_2 (TXA_2) activate receptors that are coupled to heterotrimeric G proteins. Activation of platelets through these receptors involves signaling through G_q, G_i and G_z (refs. 4, 5, 6). However, the role and relative importance of G12 and G13, which are activated by various platelet stimuli, are unclear. Here we show that lack of Galpha_13, but not Galpha_12, severely reduced the potency of thrombin, TXA2 and collagen to induce platelet shape changes and aggregation in vitro. These defects were accompanied by reduced activation of RhoA and inability to form stable platelet thrombi under high shear stress ex vivo. Galpha_13 deficiency in platelets resulted in a severe defect in primary hemostasis and complete protection against arterial thrombosis in vivo. We conclude that G_13-mediated signaling processes are required for normal hemostasis and thrombosis and may serve as a new target for antiplatelet drugs

Class I PI 3-kinases signaling in platelet activation and thrombosis: PDK1/Akt/GSK3 axis and impact of PTEN and SHIP1.

International audienceClass I phosphoinositide 3-kinases (PI3K) have been extensively studied in different models these last years and several isoforms are now promising drug targets to treat cancer and immune diseases. Blood platelets are non-nucleated cells critical for hemostasis and strongly involved in arterial thrombosis, a leading cause of death worldwide. Besides their role in hemostasis and thrombosis, platelets provide an interesting model to characterize the implication of the different isoforms of PI3K in signaling. They are specialized for regulated adhesion, particularly under high shear stress conditions found in arteries and use highly regulated signaling mechanisms to form and stabilize a thrombus. In this review we will highlight the role of class I PI3K in these processes and the pertinence of targeting them in the context of antithrombotic strategies but also the potential consequences on the bleeding risk of inhibiting the PI3K signaling in cancer therapy. The implication of upstream regulators of the most important isoforms of PI3K in platelets and their downstream effectors such as protein kinase B (PKB or Akt) and its target glycogen synthase kinase 3 (GSK3) will be discussed as well as the impact of PTEN and SHIP phosphatases as modulators of this pathway

Des ITIM du lymphocyte aux integrines de la plaquette: SHIP, une proteine a la croisee des chemins?

SCOPUS: ar.jinfo:eu-repo/semantics/publishe

An uncommon cause of allergic fungal sinusitis: Rhizopus oryzae.

International audienceWe report what we believe is the first case of allergic fungal rhinosinusitis (AFRS) caused by the fungus Rhizopus oryzae. Our patient was a 32-year-old woman who presented with unilateral nasal polyps and chronic nasal dysfunction. Computed tomography of the sinuses detected left-sided pansinusitis and bone erosion. T2-weighted magnetic resonance imaging demonstrated a signal void that suggested the presence of a fungal infection. The patient underwent unilateral ethmoidectomy. Histologic examination of the diseased tissue identified allergic mucin with 70% eosinophils and no fungal hyphae. Mycologic culture detected R oryzae. After a short period of improvement, the patient experienced a recurrence, which was confirmed by radiology. A second surgery was performed, and the same fungal hyphae were found in the mucus and on culture, which led us to suspect AFRS. Since no IgE test for R oryzae was available, we developed a specific immunologic assay that confirmed the presence of specific IgG, which identified a high degree of immunologic reaction against our homemade R oryzae antigens. With a long course of systemic antifungal treatment, the patient's symptoms resolved and no recurrence was noted at 5 years of follow-up

A novel mass assay to quantify the bioactive lipid PtdIns3P in various biological samples.

International audiencePtdIns3P is recognized as an important player in the control of the endocytotic pathway and in autophagy. Recent data also suggest that PtdIns3P contributes to molecular mechanisms taking place at the plasma membrane and at the midbody during cytokinesis. This lipid is present in low amounts in mammalian cells and remains difficult to quantify either by traditional techniques based on radiolabelling followed by HPLC to separate the different phosphatidylinositol monophosphates, or by high-sensitive liquid chromatography coupled to MS, which is still under development. In the present study, we describe a mass assay to quantify this lipid from various biological samples using the recombinant PtdIns3P 5-kinase, PIKfyve. Using this assay, we show an increase in the mass level of PtdIns3P in mouse and human platelets following stimulation, loss of this lipid in Vps34-deficient yeasts and its relative enrichment in early endosomes isolated from BHK cells

SHIP1 Controls Internal Platelet Contraction and α_IIbβ₃ Integrin Dynamics in Early Platelet Activation

The Src homology 2 domain-containing inositol 5-phosphatase 1 (SHIP1) is known to dephosphorylate PtdIns(3,4,5)P3 into PtdIns(3,4)P2 and to interact with several signaling proteins though its docking functions. It has been shown to negatively regulate platelet adhesion and spreading on a fibrinogen surface and to positively regulate thrombus growth. In the present study, we have investigated its role during the early phase of platelet activation. Using confocal-based morphometric analysis, we found that SHIP1 is involved in the regulation of cytoskeletal organization and internal contractile activity in thrombin-activated platelets. The absence of SHIP1 has no significant impact on thrombin-induced Akt or Erk1/2 activation, but it selectively affects the RhoA/Rho-kinase pathway and myosin IIA relocalization to the cytoskeleton. SHIP1 interacts with the spectrin-based membrane skeleton, and its absence induces a loss of sustained association of integrins to this network together with a decrease in αIIbβ3 integrin clustering following thrombin stimulation. This αIIbβ3 integrin dynamics requires the contractile cytoskeleton under the control of SHIP1. RhoA activation, internal platelet contraction, and membrane skeleton integrin association were insensitive to the inhibition of PtdIns(3,4,5)P3 synthesis or SHIP1 phosphatase activity, indicating a role for the docking properties of SHIP1 in these processes. Altogether, our data reveal a lipid-independent function for SHIP1 in the regulation of the contractile cytoskeleton and integrin dynamics in platelets

Platelets Are Critical Key Players in Sepsis

Host defense against infection is based on two crucial mechanisms: the inflammatory response and the activation of coagulation. Platelets are involved in both hemostasis and immune response. These mechanisms work together in a complex and synchronous manner making the contribution of platelets of major importance in sepsis. This is a summary of the pathophysiology of sepsis-induced thrombocytopenia, microvascular consequences, platelet-endothelial cells and platelet–pathogens interactions. The critical role of platelets during sepsis and the therapeutic implications are also reviewed

Chronic estradiol treatment reduces platelet responses and protects mice from thromboembolism through the hematopoietic estrogen receptor α.

International audienceAlthough estrogens are known to have a deleterious effect on the venous thrombosis risk and a preventive action on the development of arterial atheroma, their effect on platelet function in vivo remains unclear. Here, we demonstrate that a chronic high physiologic level of estradiol (E2) in mice leads to a marked decrease in platelet responsiveness ex vivo and in vivo compared with ovariectomized controls. E2 treatment led to increased bleeding time and a resistance to thromboembolism. Hematopoietic chimera mice harboring a selective deletion of estrogen receptors (ERs) α or β were used to demonstrate that the effects of E2 were exclusively because of hematopoietic ERα. Within ERα the activation function-1 domain was not required for resistance to thromboembolism, as was previously shown for atheroprotection. This domain is mandatory for E2-mediated reproductive function and suggests that this role is controlled independently. Differential proteomics indicated that E2 treatment modulated the expression of platelet proteins including β1 tubulin and a few other proteins that may impact platelet production and activation. Overall, these data demonstrate a previously unrecognized role for E2 in regulating the platelet proteome and platelet function, and point to new potential antithrombotic and vasculoprotective therapeutic strategies

Roles of the C-terminal tyrosine residues of LAT in GPVI-induced platelet activation; insights in the mechanism of PLC{gamma}2 activation.

Linker for activation of T cells (LAT) is an adaptor protein required for organisation of the signalling machinery downstream of the platelet collagen receptor, the glycoprotein VI (GPVI). Here, we investigated the effect of LAT mutations on specific signalling pathways and on platelet functions in response to GPVI triggering by convulxin (Cvx). Using mice containing tyrosine to phenylalanine mutations of the adaptor, we show the crucial role played by the tyrosine residues at position 175, 195 and 235 in the phosphorylation of LAT and in the whole pattern of protein tyrosine phosphorylation in response to Cvx. These three C-terminal tyrosine residues are important to recruit the tyrosine kinase Fyn which may be involved in LAT phosphorylation. Efficient phosphoinositide 3-kinase (PI3K) activation requires the three C-terminal tyrosine residues of LAT but not its tyrosine 136. Interestingly, single mutation of the tyrosine 136 results in the loss of phospholipase Cgamma2 (PLCgamma2) activation without affecting its PI3K-dependent membrane association, and is sufficient to impair platelet responses to Cvx. Thus, activation of PLCgamma2 via GPVI is dependent on two complementary events; its interaction with the tyrosine 136 of LAT and its membrane location which itself requires events mediated by the three C-terminal tyrosines of LAT