Tyrosine phosphorylation of the Fc receptor gamma-chain in collagen-stimulated platelets.
ABSTRACT 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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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.Thrombosis Research 05/2013; DOI:10.1016/j.thromres.2013.02.013 · 2.43 Impact Factor
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ABSTRACT: Molecular mechanisms underlying the links between dietary intake of flavonoids and reduced cardiovascular disease risk are only partially understood. Key events in the pathogenesis of cardiovascular disease, particularly thrombosis, are inhibited by these polyphenolic compounds via mechanisms such as inhibition of platelet activation and associated signal transduction, attenuation of generation of reactive oxygen species, enhancement of nitric oxide production and binding to thromboxane A(2) receptors. In vivo, effects of flavonoids are mediated by their metabolites, but the effects and modes of action of these compounds are not well-characterized. A good understanding of flavonoid structure-activity relationships with regard to platelet function is also lacking. Inhibitory potencies of structurally distinct flavonoids (quercetin, apigenin and catechin) and plasma metabolites (tamarixetin, quercetin-3'-sulphate and quercetin-3-glucuronide) for collagen-stimulated platelet aggregation and 5-hydroxytryptamine secretion were measured in human platelets. Tyrosine phosphorylation of total protein, Syk and PLCgamma2 (immunoprecipitation and Western blot analyses), and Fyn kinase activity were also measured in platelets. Internalization of flavonoids and metabolites in a megakaryocytic cell line (MEG-01 cells) was studied by fluorescence confocal microscopy. Key results: The inhibitory mechanisms of these compounds included blocking Fyn kinase activity and the tyrosine phosphorylation of Syk and PLCgamma2 following internalization. Principal functional groups attributed to potent inhibition were a planar, C-4 carbonyl substituted and C-3 hydroxylated C ring in addition to a B ring catechol moiety. The structure-activity relationship for flavonoids on platelet function presented here may be exploited to design selective inhibitors of cell signalling.British Journal of Pharmacology 02/2010; 159(6):1312-25. DOI:10.1111/j.1476-5381.2009.00632.x · 4.99 Impact Factor
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ABSTRACT: Platelets perform a central role in haemostasis and thrombosis. They adhere to subendothelial collagens exposed at sites of blood vessel injury via the glycoprotein (GP) Ib-V-IX receptor complex, GPVI and integrin alpha(2)beta(1). These receptors perform distinct functions in the regulation of cell signalling involving non-receptor tyrosine kinases (e.g. Src, Fyn, Lyn, Syk and Btk), adaptor proteins, phospholipase C and lipid kinases such as phosphoinositide 3-kinase. They are also coupled to an increase in cytosolic calcium levels and protein kinase C activation, leading to the secretion of paracrine/autocrine platelet factors and an increase in integrin receptor affinities. Through the binding of plasma fibrinogen and von Willebrand Factor to integrin alpha(IIb)beta(3), a platelet thrombus is formed. Although increasing evidence indicates that each of the adhesion receptors GPIb-V-IX and GPVI and integrins alpha(2)beta(1) and alpha(IIb)beta(3) contribute to the signalling that regulates this process, the individual roles of each are only beginning to be dissected. By contrast, adhesion receptor signalling through platelet endothelial cell adhesion molecule 1 (PECAM-1) is implicated in the inhibition of platelet function and thrombus formation in the healthy circulation. Recent studies indicate that understanding of platelet adhesion signalling mechanisms might enable the development of new strategies to treat and prevent thrombosis.Journal of Cell Science 08/2004; 117(Pt 16):3415-25. DOI:10.1242/jcs.01325 · 5.33 Impact Factor