Fyn and Lyn phosphorylate the Fc receptor γ chain downstream of glycoprotein VI in murine platelets, and Lyn regulates a novel feedback pathway

Quek, Lynn; Pasquet, Jean‐Max; Hers, Ingeborg; Cornall, Richard J.; Knight, Graham; Barnes, Michael J.; Hibbs, Margaret L.; Dunn, Ashley R.; Lowell, Clifford A.; Watson, Steve P.

doi:10.1182/blood.v96.13.4246.h8004246_4246_4253

Cited by 22 publications

(28 citation statements)

References 25 publications

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“…Quercetin was found to inhibit tyrosine phosphorylation of the FcR g-chain and therefore appears to have an inhibitory effect at the top of the collagen-stimulated activation pathway. Tyrosine phosphorylation of the FcR g-chain is thought to involve the activity of two Src-family kinases, namely Fyn and Lyn [26,27]. We have also shown that quercetin is able to inhibit the kinase activity of Fyn and Lyn in a concentration-dependent manner, both via GPVI (in whole cells) and directly.…”

Section: Discussionmentioning

confidence: 76%

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Quercetin inhibits collagen-stimulated platelet activation through inhibition of multiple components of the glycoprotein VI signaling pathway

Hubbard

Stevens

Cicmil

et al. 2003

Journal of Thrombosis and Haemostasis

115

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Summary. Background: The regulation of platelet function by pharmacological agents that modulate platelet signaling has proven a successful approach to the prevention of thrombosis. A variety of molecules present in the diet have been shown to inhibit platelet activation, including the antioxidant quercetin. Objectives: In this report we investigate the molecular mechanisms through which quercetin inhibits collagen-stimulated platelet aggregation. Methods: The effect of quercetin on platelet aggregation, intracellular calcium release, whole cell tyrosine phosphorylation and intracellular signaling events including tyrosine phosphorylation and kinase activity of proteins involved in the collagen-stimulated glycoprotein (GP) signaling pathway were investigated. Results: We report that quercetin inhibits collagen-stimulated whole cell protein tyrosine phosphorylation and intracellular mobilization of calcium, in a concentration-dependent manner. Quercetin was also found to inhibit various events in signaling generated by the collagen receptor GPVI. This includes collagen-stimulated tyrosine phosphorylation of the Fc receptor g-chain, Syk, LATand phospholipase Cg2. Inhibition of phosphorylation of the Fc receptor g-chain suggests that quercetin inhibits earlysignaling events following stimulation of platelets with collagen. The activity of the kinases that phosphorylate the Fc receptor g-chain, Fyn and Lyn, as well as the tyrosine kinase Syk and phosphoinositide 3-kinase was also inhibited by quercetin in a concentration-dependent manner, both in whole cells and in isolation. Conclusions: The present results provide a molecular basis for the inhibition by quercetin of collagen-stimulated platelet activation, through inhibition of multiple componentsof the GPVI signaling pathway, and may begin to explain the proposed health benefits of high quercetin intake.

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Section: Discussionmentioning

confidence: 76%

“…The tyrosine phosphorylation of the FcR g-chain following collagen binding to GPVI is mediated by the Src-family kinases Fyn and Lyn [26,27]. This leads to the recruitment and activa- tion of the cytosolic tyrosine kinase Syk.…”

Section: The Kinase Activities Of the Src-family Kinases Fyn And Lyn mentioning

confidence: 99%

Quercetin inhibits collagen-stimulated platelet activation through inhibition of multiple components of the glycoprotein VI signaling pathway

Hubbard

Stevens

Cicmil

et al. 2003

Journal of Thrombosis and Haemostasis

115

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“…Importantly, the level of c-Cbl expression was similar in platelets de®cient in the two tyrosine kinases. These results could re¯ect an indirect role of Fyn and Lyn, which mediate tyrosine phosphorylation of the FcR g-chain [4], but could also re¯ect a direct contribution to c-Cbl tyrosine phosphorylation.…”

Section: Phosphorylation Of C-cbl In Murine Platelets Activated By Gpmentioning

confidence: 94%

c-Cbl negatively regulates platelet activation by glycoprotein VI

Auger

Best

Snell

et al. 2003

Journal of Thrombosis and Haemostasis

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Summary. Background: The adapter protein c‐Cbl has emerged as having a potential role in negative regulation of immune receptor signaling. The major platelet‐signaling receptor for collagen, glycoprotein VI (GpVI), is associated with the Fc receptor (FcR) γ‐chain, and signals through a similar pathway to immune receptors. c‐Cbl is tyrosine‐phosphorylated in response to stimulation of GpVI, whereas phosphorylation of c‐Cbl in thrombin‐activated platelets is dependent on fibrinogen binding to the integrin GpIIb/IIIa. Objective: To investigate the role of c‐Cbl in platelet signaling. Methods: Murine platelets lacking functional c‐Cbl or Src family kinases were analyzed. Results: Phosphorylation of c‐Cbl through GpVI is reduced in murine platelets deficient in the Src‐family kinases Fyn and Lyn, demonstrating that they lie upstream of c‐Cbl phosphorylation. Phosphorylation of several proteins of the GpVI‐signaling pathway, including the FcR γ‐chain, Syk and phospholipase Cγ2 (PLCγ2), is increased in the absence of c‐Cbl. In line with this, aggregation is potentiated in response to the GpVI‐specific collagen‐related peptide (CRP) after a slight delay. A delay in potentiation is also seen in response to stimulation by thrombin. Conclusions: These observations demonstrate that c‐Cbl negatively regulates platelet responses to GpVI agonists and to thrombin, with the latter effect possibly being mediated downstream of GpIIb/IIIa. c‐Cbl may play a physiological role in helping to prevent unwanted platelet activation in vivo.

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“…The Src family kinases Fyn and Lyn play an early role in platelet activation by GPVI, upstream of the tyrosine kinase Syk and the effecter enzyme PLCγ2 (10)(11)(12). Roles for Src kinases in signaling downstream of other platelet receptors, including α2β1 (16) and GPIb-IX-V (26,32) have also recently been revealed.…”

Section: Src Kinase Inhibition Abolishes Platelet Aggregation and Parmentioning

confidence: 99%

“…The identification of the GPVI-Fc receptor (FcR) γ-chain complex as the principal receptor underlying activation by collagen (6)(7)(8)(9), and the elucidation of its Src kinase-dependent signaling cascade (10)(11)(12), provided the means to develop experimental tools to assess the two-site, twostep model. The availability of mouse platelets that are deficient in α2β1, GPVI, or FcR γ-chain was of particular importance in this regard, due to the small number of human patients that are deficient in these proteins.…”

mentioning

confidence: 99%

Adhesion of human and mouse platelets to collagen under shear: a unifying model

et al. 2005

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There is presently confusion as to the roles of alpha2beta1 and GPVI in supporting platelet adhesion and aggregate formation on collagen at intermediate/high shear. Recent studies have reported essential, partial, or dispensable roles for either receptor in supporting these events, and the possibility that there may be fundamental differences between their roles in human and mouse platelets has been proposed. Further, the recent recognition that Src family tyrosine kinases contribute to signaling by alpha2beta1 and other adhesive receptors, in addition to GPVI, has added to this debate. The present study compares the roles of alpha2beta1, GPVI, and Src-dependent kinases in supporting adhesion and aggregation in human and mouse platelets in whole blood using blocking antibodies, mutant mice, and a novel inhibitor of Src kinases, PD0173952, which is effective in plasma. The results demonstrate that the fundamental processes of adhesion and aggregate formation are conserved in mice and human platelets and that two mechanisms of stable adhesion and activation on collagen exist. These can be distinguished by the contributions of GPVI and alpha2beta1, with GPVI-mediated platelet activation either preceding or following integrin-mediated adhesion. The relative contribution of each pathway depends on environmental conditions and may also reflect platelet heterogeneity. These observations form the basis of a unifying two-state model of platelet adhesion and aggregate formation on collagen that is conserved between human and mouse platelets.

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Fyn and Lyn phosphorylate the Fc receptor γ chain downstream of glycoprotein VI in murine platelets, and Lyn regulates a novel feedback pathway

Cited by 22 publications

References 25 publications

Quercetin inhibits collagen-stimulated platelet activation through inhibition of multiple components of the glycoprotein VI signaling pathway

Quercetin inhibits collagen-stimulated platelet activation through inhibition of multiple components of the glycoprotein VI signaling pathway

c-Cbl negatively regulates platelet activation by glycoprotein VI

Adhesion of human and mouse platelets to collagen under shear: a unifying model

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