Tyrosine Phosphorylation of the Fc Receptor -Chain in Collagen-stimulated Platelets

Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, United Kingdom.
Journal of Biological Chemistry (Impact Factor: 4.57). 08/1996; 271(30):18095-9. DOI: 10.1074/jbc.271.30.18095
Source: PubMed


Stimulation of platelets by the extracellular matrix protein collagen leads to activation of a tyrosine kinase-dependent mechanism resulting in secretion and aggregation. Tyrosine phosphorylation of the tyrosine kinase Syk and phospholipase Cgamma2 are early events in collagen-induced activation. We recently proposed that collagen-signaling in platelets involves a receptor or a receptor-associated protein containing an immunoreceptor tyrosine-based activation motif (ITAM) enabling interaction with Syk. In this report we show that collagen stimulation of platelets causes rapid tyrosine phosphorylation of the ITAM containing Fc receptor gamma-chain and that this is precipitated by the tandem Src homology 2 (SH2) domains of Syk expressed as a fusion protein. In addition we demonstrate an association between the Fc receptor gamma-chain with endogenous Syk in collagen-stimulated platelets. The Fc receptor gamma-chain undergoes tyrosine phosphorylation in platelets stimulated by a collagen-related peptide which does not bind the integrin alpha2beta1 and by the lectin wheat germ agglutinin. In contrast, cross-linking of the platelet low affinity receptor for immune complexes, FcgammaRIIA, or stimulation by thrombin does not induce phosphorylation of the Fc receptor gamma-chain. The present results provide a molecular basis for collagen activation of platelets which is independent of the integrin alpha2beta1 and involves phosphorylation of the Fc receptor gamma-chain, its association with Syk and subsequent phosphorylation of phospholipase Cgamma2. Collagen is the first example of a nonimmune receptor stimulus to signal through a pathway closely related to signaling by immune receptors.

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    • "Once vascular injury has occurred, platelets are principally activated by locally exposed collagen in the vascular wall and locally generated thrombin, initiating hemostasis[1]. The binding of collagen to GPVI on platelets results in receptor clustering and thereby stimulates the tyrosine phosphorylation of specific tyrosine residues within an associated trans-membrane protein, the Fc receptor γ-chain (FcRγ-chain)[2,3]. This leads to the recruitment of signaling proteins such as the Src kinase, the tyrosine kinase Syk, PLCγ2, phosphoinositide 3-kinase (PI3K) and MAPKS[3,4], resulting in the inside-out activation of the integrin αIIbβ3 and the release of the secondary mediators, such as ADP and thromboxane A2 (TxA2), culminating in platelet aggregation mediated by fibrinogen binding to αIIbβ3 and thrombus formation. "
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    ABSTRACT: Platelets play a pivotal role in atherothrombosis and the antiplatelet agents have been proved to be useful in preventing onset of acute clinical events including myocardial infarction and stroke. Increasing number of natural compounds has been identified to be potential antiplatelet agents. Here we report the antiplatelet effect of glaucocalyxin A (GLA), an ent-diterpenoid that we isolated and purified from the aerial parts of Rabdosia japonica (Burm. f.) var. glaucocalyx (Maxim.) Hara, and investigate the molecular mechanisms by which GLA inhibits platelet activation and thrombus formation. The effect of GLA on platelet activation was measured using platelets freshly isolated from peripheral blood of healthy donors. Results showed that pretreatment of human platelets with lower concentrations of GLA (0.01μg/ml, 0.1μg/ml) significantly inhibited platelet aggregation induced by collagen (P<0.001) and CRP (P<0.01), a synthetic GPVI ligand, but not by ADP and U46619. Accordingly, GLA inhibited collagen-stimulated tyrosine phosphorylation of Syk, LAT, and phospholipase Cγ2, the signaling events in collagen receptor GPⅥ pathway. GLA also inhibited platelet p-selectin secretion and integrin activation by convulxin, a GPVI selective ligand. Additionally, GLA was found to inhibit low-dose thrombin-induced platelet activation. Using a flow chamber device, GLA was found to attenuate platelet adhesion on collagen surfaces in high shear condition. In vivo studies showed that GLA administration increased the time for complete occlusion upon vascular injury in mice, but did not extend tail-bleeding time when mice were administered with relatively lower doses of GLA. Therefore, the present results provide the molecular basis for the inhibition effect of GLA on platelet activation and its in vivo effect on thrombus formation, suggesting that GLA could potentially be developed as an antiplatelet and antithrombotic agent.
    Full-text · Article · Dec 2013 · PLoS ONE
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    • "GPVI complexes with FcR γ-chain dimers and is the major signalling receptor for collagen on the surface of platelets [1] [2]. Interaction of the receptor with sub-endothelial collagen and subsequent clustering results in phosphorylation of the immuno-receptor tyrosine-based activation motif (ITAM) by the Src family kinases Fyn and Lyn [3] [4]. Subsequently, spleen tyrosine kinase (Syk) is recruited and is activated by autophosphorylation [5]. "
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    ABSTRACT: Introduction: Platelet Glycoprotein (GP)VI is a member of the immunoglobulin superfamily expressed only on platelets, and is the major signalling receptor for collagen. Histone deacetylase inhibitors (HDACi) are anti-cancer agents used for the treatment of haematological malignancies, and we examined the effects of administration of HDACi to mice on platelet function including responses to agonists including collagen related peptide (CRP). Materials and methods: C57BL/6 mice were injected with two structurally different HDACi, panobinostat and romidepsin, for three days and platelet receptor levels and responses to agonists were assessed by flow cytometry and western blot. Results: Platelets from mice treated with either HDACi were impaired in their ability to respond to CRP, but not thrombin or adenosine diphosphate (ADP). HDACi treatment increased acetylation of megakaryocytic GPVI, resulting in loss of intact (~60-65-kDa) GPVI and formation of ~10-kDa remnant GPVI. Circulating platelets had reduced surface and total expression of GPVI. Platelets from mice treated with HDACi had impaired GPVI signalling following treatment with CRP, resulting in inhibition of Syk phosphorylation and activation, and the final common pathways of platelet activation. Conclusions: Administration of HDACi in vivo may ablate platelet responses to agonists and platelet function.
    Full-text · Article · May 2013 · Thrombosis Research
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    • "An ITAM has two YxxLs separated by 6–12 amino acids. Clustering of ITAM receptors such as the collagen receptor GPVI leads to Src family kinase-dependent phosphorylation of the two conserved tyrosines and subsequent binding and activation of Syk tyrosine kinase [1]. This initiates a signalling cascade that culminates in the activation of PLCγ2, which in turn mediates cellular activation through production of the secondary messengers inositol-1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DAG). "
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    ABSTRACT: Collagen activates mammalian platelets through a complex of the immunoglobulin (Ig) receptor GPVI and the Fc receptor γ-chain, which has an immunoreceptor tyrosine-based activation motif (ITAM). Cross-linking of GPVI mediates activation through the sequential activation of Src and Syk family kinases and activation of PLCγ2. Nucleated thrombocytes in fish are activated by collagen but lack an ortholog of GPVI. In this study we show that collagen activates trout thrombocytes in whole blood and under flow conditions through a Src kinase driven pathway. We identify the Ig receptor G6f-like as a collagen receptor and demonstrate in a cell line assay that it signals through its cytoplasmic ITAM. Using a morpholino for in vivo knock-down of G6f-like levels in zebrafish, we observed a marked delay or absence of occlusion of the venous and arterial systems in response to laser injury. Thus, G6f-like is a physiologically relevant collagen receptor in fish thrombocytes which signals through the same ITAM-based signalling pathway as mammalian GPVI, providing a novel example of convergent evolution.
    Full-text · Article · Dec 2012 · PLoS ONE
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