Jonathan M. Gibbins

The Fc receptor γ-chain and the tyrosine kinase Syk are essential for activation of mouse platelets by collagen

Activation of mouse platelets by collagen is associated with tyrosine phosphorylation of multiple proteins including the Fc receptor γ‐chain, the tyrosine kinase Syk and phospholipase Cγ2, suggesting that collagen signals in a manner similar to that of immune receptors. This hypothesis has been tested using platelets from mice lacking the Fc receptor γ‐chain or Syk. Tyrosine phosphorylation of Syk and phospholipase Cγ2 by collagen stimulation is absent in mice lacking the Fc receptor γ‐chain. Tyrosine phosphorylation of phospholipase Cγ2 by collagen stimulation is also absent in mice platelets which lack Syk, although phosphorylation of the Fc receptor γ‐chain is maintained. In contrast, tyrosine phosphorylation of platelet proteins by the G protein‐coupled receptor agonist thrombin is maintained in mouse platelets deficient in Fc receptor γ‐chain or Syk. The absence of Fc receptor γ‐chain or Syk is accompanied by a loss of secretion and aggregation responses in collagen‐ but not thrombin‐stimulated platelets. These observations provide the first direct evidence of an essential role for the immunoreceptor tyrosine‐based activation motif (ITAM) in signalling by a non‐immune receptor stimulus.

Glycoprotein VI is the collagen receptor in platelets which underlies tyrosine phosphorylation of the Fc receptor γ‐chain

We have recently shown that collagen activates platelets through a pathway dependent on the Fc receptor γ‐chain and the tyrosine kinase Syk. We report here that the Fc receptor γ‐chain and the candidate collagen receptor glycoprotein VI (GPVI) co‐associate. Furthermore, cross‐linking GPVI stimulates a similar pattern of tyrosine phosphorylation to that stimulated by collagen, including tyrosine phosphorylation of Fc receptor γ‐chain. These results support a model where GPVI couples collagen‐stimulation of platelets to phosphorylation of the Fc receptor γ‐chain leading to activation of Syk and phospholipase Cγ2.

Tyrosine phosphorylation of the Fc receptor gamma-chain in collagen-stimulated platelets

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 Cγ2 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 γ-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 γ-chain with endogenous Syk in collagen-stimulated platelets. The Fc receptor γ-chain undergoes tyrosine phosphorylation in platelets stimulated by a collagen-related peptide which does not bind the integrin α2β1 and by the lectin wheat germ agglutinin. In contrast, cross-linking of the platelet low affinity receptor for immune complexes, FcγRIIA, or stimulation by thrombin does not induce phosphorylation of the Fc receptor γ-chain. The present results provide a molecular basis for collagen activation of platelets which is independent of the integrin α2β1 and involves phosphorylation of the Fc receptor γ-chain, its association with Syk and subsequent phosphorylation of phospholipase Cγ2. Collagen is the first example of a nonimmune receptor stimulus to signal through a pathway closely related to signaling by immune receptors.

Platelet adhesion signalling and the regulation of thrombus formation

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 α2β1. 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 αIIbβ3, a platelet thrombus is formed. Although increasing evidence indicates that each of the adhesion receptors GPIb-V-IX and GPVI and integrins α2β1 and αIIbβ3 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.

Collagen receptor signalling in platelets: extending the role of the ITAM

Abstract Recent studies have shown that activation of platelets by collagen is mediated through the same pathway as that used by immune receptors, with pivotal roles for an immunoreceptor tyrosine-based activation motif (ITAM) sequence on the Fc receptor γ-chain and the tyrosine kinase Syk. Here, Steve Watson and Jonathan Gibbins discuss the implications for the physiological and pathological regulation of blood platelets.

Ingestion of quercetin inhibits platelet aggregation and essential components of the collagen-stimulated platelet activation pathway in humans

Summary.  Background: Quercetin, a flavonoid present in the human diet, which is found in high levels in onions, apples, tea and wine, has been shown previously to inhibit platelet aggregation and signaling in vitro. Consequently, it has been proposed that quercetin may contribute to the protective effects against cardiovascular disease of a diet rich in fruit and vegetables. Objectives: A pilot human dietary intervention study was designed to investigate the relationship between the ingestion of dietary quercetin and platelet function. Methods: Human subjects ingested either 150 mg or 300 mg quercetin‐4′‐O‐β‐D‐glucoside supplement to determine the systemic availability of quercetin. Platelets were isolated from subjects to analyse collagen‐stimulated cell signaling and aggregation. Results: Plasma quercetin concentrations peaked at 4.66 µm (± 0.77) and 9.72 µm (± 1.38) 30 min after ingestion of 150‐mg and 300‐mg doses of quercetin‐4′‐O‐β‐D‐glucoside, respectively, demonstrating that quercetin was bioavailable, with plasma concentrations attained in the range known to affect platelet function in vitro. Platelet aggregation was inhibited 30 and 120 min after ingestion of both doses of quercetin‐4′‐O‐β‐D‐glucoside. Correspondingly, collagen‐stimulated tyrosine phosphorylation of total platelet proteins was inhibited. This was accompanied by reduced tyrosine phosphorylation of the tyrosine kinase Syk and phospholipase Cγ2, components of the platelet glycoprotein VI collagen receptor signaling pathway. Conclusions: This study provides new evidence of the relatively high systemic availability of quercetin in the form of quercetin‐4′‐O‐β‐D‐glucoside by supplementation, and implicates quercetin as a dietary inhibitor of platelet cell signaling and thrombus formation.

Future innovations in anti-platelet therapies

Platelets have long been recognized to be of central importance in haemostasis, but their participation in pathological conditions such as thrombosis, atherosclerosis and inflammation is now also well established. The platelet has therefore become a key target in therapies to combat cardiovascular disease. Anti‐platelet therapies are used widely, but current approaches lack efficacy in a proportion of patients, and are associated with side effects including problem bleeding. In the last decade, substantial progress has been made in understanding the regulation of platelet function, including the characterization of new ligands, platelet‐specific receptors and cell signalling pathways. It is anticipated this progress will impact positively on the future innovations towards more effective and safer anti‐platelet agents. In this review, the mechanisms of platelet regulation and current anti‐platelet therapies are introduced, and strong, and some more speculative, potential candidate target molecules for future anti‐platelet drug development are discussed.

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

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 γ‐chain, Syk, LAT and phospholipase Cγ2. Inhibition of phosphorylation of the Fc receptor γ‐chain suggests that quercetin inhibits early signaling events following stimulation of platelets with collagen. The activity of the kinases that phosphorylate the Fc receptor γ‐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 components of the GPVI signaling pathway, and may begin to explain the proposed health benefits of high quercetin intake.

Ingestion of onion soup high in quercetin inhibits platelet aggregation and essential components of the collagen-stimulated platelet activation pathway in man: a pilot study

Epidemiological data suggest that those who consume a diet rich in quercetin-containing foods may have a reduced risk of CVD. Furthermore, in vitro and ex vivo studies have observed the inhibition of collagen-induced platelet activation by quercetin. The aim of the present study was to investigate the possible inhibitory effects of quercetin ingestion from a dietary source on collagen-stimulated platelet aggregation and signalling. A double-blind randomised cross-over pilot study was undertaken. Subjects ingested a soup containing either a high or a low amount of quercetin. Plasma quercetin concentrations and platelet aggregation and signalling were assessed after soup ingestion. The high-quercetin soup contained 69 mg total quercetin compared with the low-quercetin soup containing 5 mg total quercetin. Plasma quercetin concentrations were significantly higher after high-quercetin soup ingestion than after low-quercetin soup ingestion and peaked at 2.59 (sem 0.42) mumol/l. Collagen-stimulated (0.5 mug/ml) platelet aggregation was inhibited after ingestion of the high-quercetin soup in a time-dependent manner. Collagen-stimulated tyrosine phosphorylation of a key component of the collagen-signalling pathway via glycoprotein VI, Syk, was significantly inhibited by ingestion of the high-quercetin soup. The inhibition of Syk tyrosine phosphorylation was correlated with the area under the curve for the high-quercetin plasma profile. In conclusion, the ingestion of quercetin from a dietary source of onion soup could inhibit some aspects of collagen-stimulated platelet aggregation and signalling ex vivo. This further substantiates the epidemiological data suggesting that those who preferentially consume high amounts of quercetin-containing foods have a reduced risk of thrombosis and potential CVD risk.

Platelet protein disulfide isomerase is required for thrombus formation but not for hemostasis in mice.

Protein disulfide isomerase (PDI) derived from intravascular cells is required for thrombus formation. However, it remains unclear whether platelet PDI contributes to the process. Using platelet-specific PDI-deficient mice, we demonstrate that PDI-null platelets have defects in aggregation and adenosine triphosphate secretion induced by thrombin, collagen, and adenosine diphosphate. Such defects were rescued by wild-type but not mutant PDI, indicating that the isomerase activity of platelet surface PDI is critical for the regulatory effect. PDI-deficient platelets expressed increased levels of intracellular ER protein 57 (ERp57) and ERp72. Platelet PDI regulated αIIbβ3 integrin activation but not P-selectin exposure, Ca(2+) mobilization, β3-talin1 interaction, or platelet spreading on immobilized fibrinogen. Inhibition of ERp57 further diminished αIIbβ3 integrin activation and aggregation of activated PDI-deficient platelets, suggesting distinct roles of PDI and ERp57 in platelet functions. We found that platelet PDI is important for thrombus formation on collagen-coated surfaces under shear. Intravital microscopy demonstrates that platelet PDI is important for platelet accumulation but not initial adhesion and fibrin generation following laser-induced arteriolar injury. Tail bleeding time in platelet-specific PDI-deficient mice were not significantly increased. Our results provide important evidence that platelet PDI is essential for thrombus formation but not for hemostasis in mice.