(FcR γ-chain), the non-receptor tyrosine kinase Syk and Bristol BS8 1TD, UK phospholipase Cγ2 (PLCγ2) (Blake et al., 1994; Daniel et al., 1994a; Fujii et al., 1994;Yanaga et al., 1995; 5 Corresponding author Asazuma et al., 1996;Gibbins et al., 1996). Syk assembles A.Poole, J.M.Gibbins and M.Turner contributed equally to this work into signalling complexes at the plasma membrane via interaction between its tandem Src homology 2 (SH2) Activation of mouse platelets by collagen is associated domains and a tyrosine-phosphorylated immunoreceptor with tyrosine phosphorylation of multiple proteins tyrosine-based activation motif (ITAM). We recently proincluding the Fc receptor γ-chain, the tyrosine kinase posed a model for collagen-induced signalling in human Syk and phospholipase Cγ2, suggesting that collagen platelets in which receptor clustering induced by collagen signals in a manner similar to that of immune receptors.leads to tyrosine phosphorylation of the FcR γ-chain, This hypothesis has been tested using platelets from possibly by a Src family kinase, allowing binding of Syk, mice lacking the Fc receptor γ-chain or Syk. Tyrosine which becomes tyrosine phosphorylated and activated phosphorylation of Syk and phospholipase Cγ2 by (Gibbins et al., 1996). This initiates a series of events collagen stimulation is absent in mice lacking the Fc which may involve other kinases and adapter proteins receptor γ-chain. Tyrosine phosphorylation of phospholeading to tyrosine phosphorylation and activation of lipase Cγ2 by collagen stimulation is also absent in mice PLCγ2. This model has been evaluated in the present platelets which lack Syk, although phosphorylation of study using platelets from genetically modified mice which the Fc receptor γ-chain is maintained. In contrast, lack the FcR γ-chain or Syk. tyrosine phosphorylation of platelet proteins by the G protein-coupled receptor agonist thrombin is maintained in mouse platelets deficient in Fc receptor Results γ-chain or Syk. The absence of Fc receptor γ-chain or Syk is accompanied by a loss of secretion and aggrega-Tyrosine phosphorylation in collagen-and tion responses in collagen-but not thrombin-stimulated thrombin-stimulated platelets platelets. These observations provide the first direct Collagen and thrombin stimulated distinct but overlapping evidence of an essential role for the immunoreceptor increases in whole cell tyrosine phosphorylation in platetyrosine-based activation motif (ITAM) in signalling lets (Figures 1 and 2A). Both agonists stimulated increases by a non-immune receptor stimulus.in tyrosine phosphorylation of proteins of~42, 70-75, Keywords: collagen/Fc receptor γ-chain/ITAM/platelets/ 100, 110 and 130 kDa in platelets from B6 mice; the 42 Syk and 110 kDa proteins were more heavily phosphorylated in thrombin-stimulated cells. The largest increase in tyrosine phosphorylation was in the 70-75 kDa proteins. Collagen but not thrombin also stimulated marked tyrosine phos-

• Platelet PDI regulates a IIb b 3 integrin activation without affecting platelet activation and inside-out integrin signaling.• Platelet PDI is essential for platelet accumulation but not for fibrin generation and 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 wildtype 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 a IIb b 3 integrin activation but not P-selectin exposure, Ca 21 mobilization, b 3 -talin1 interaction, or platelet spreading on immobilized fibrinogen. Inhibition of ERp57 further diminished a IIb b 3 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. (Blood. 2013;122(6):1052-1061 IntroductionPlatelets play a central role in hemostasis and atherothrombosis. Following vascular injury, platelets rapidly adhere to activated endothelial cells and/or subendothelial matrix proteins such as collagen and von Willebrand factor through receptor-ligand interactions.1 Subsequently, activated platelets expose P-selectin from a-granules to the plasma membrane and release other granular molecules such as adenosine diphosphate (ADP), which activates other platelets and facilitates a IIb b 3 integrin-mediated platelet accumulation at the site of vascular injury. Although it is not fully understood how integrin function is regulated, it has been postulated that thiol rearrangement in integrins could be one of the regulatory mechanisms. [2][3][4] Previous studies showed that a IIb b 3 integrin has an endogenous isomerase activity and exposes free sulfhydryl groups during platelet activation. [4][5][6] Consistently, reducing agents such as reduced glutathione and cysteine affect platelet aggregation. 2,7,8 Using a IIb b 3 integrin with mutations on Cys residues, Mor-Cohen et al 9 reported that different disulfide bonds in the b 3 subunit change the structure and function of a IIb b 3 integrin. Moreover, disruption of the disulfide bonds of Cys5-Cys435 or Cys663...

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.

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-b-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 lM (± 0.77) and 9.72 lM (± 1.38) 30 min after ingestion of 150-mg and 300-mg doses of quercetin-4¢-O-b-Dglucoside, 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-b-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 Cc2, 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-b-D-glucoside by supplementation, and implicates quercetin as a dietary inhibitor of platelet cell signaling and thrombus formation.

Stimulation of platelets by the extracellular matrix protein collagen leads to activation of a tyrosine kinasedependent mechanism resulting in secretion and aggregation. Tyrosine phosphorylation of the tyrosine kinase Syk and phospholipase C ␥ 2 are early events in collageninduced activation. We recently proposed that collagensignaling in platelets involves a receptor or a receptorassociated 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 ␥-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 ␥-chain with endogenous Syk in collagen-stimulated platelets. The Fc receptor ␥-chain undergoes tyrosine phosphorylation in platelets stimulated by a collagen-related peptide which does not bind the integrin ␣ 2 ␤ 1 and by the lectin wheat germ agglutinin. In contrast, cross-linking of the platelet low affinity receptor for immune complexes, Fc␥RIIA, or stimulation by thrombin does not induce phosphorylation of the Fc receptor ␥-chain. The present results provide a molecular basis for collagen activation of platelets which is independent of the integrin ␣ 2 ␤ 1 and involves phosphorylation of the Fc receptor ␥-chain, its association with Syk and subsequent phosphorylation of phospholipase C ␥ 2. Collagen is the first example of a nonimmune receptor stimulus to signal through a pathway closely related to signaling by immune receptors.The adhesive and stimulatory properties of the extracellular matrix protein collagen on platelets are vital for the maintenance of hemeostasis. Upon vascular damage, platelets adhere to subendothelial collagen which stimulates a tyrosine kinase dependent pathway leading to platelet degranulation, aggregation and development of a hemeostatic plug. The mechanism of collagen stimulation of platelets is poorly understood, and the distinction between adhesion and stimulation ill defined. Several platelet glycoproteins have been implicated as potential collagen receptors, including the integrin ␣ 2 ␤ 1 (1), glycoprotein IV (GPIIIb, CD36) (2), glycoprotein VI (3), and uncharacterized 65-kDa (4) and 85-90-kDa glycoproteins (5). Patients whose platelets express abnormally low numbers of these proteins, or who possess autoantibodies to them, have limited bleeding defects (3, 5-9).Collagen stimulation of platelets activates tyrosine kinasedependent mechanisms which involve tyrosine phosphorylation of Syk and phospholipase C ␥ 2 (PLC ␥ 2) 1 (10 -12). Syk is a nonreceptor tyrosine kinase which is assembled into signaling complexes via interaction between its tandem Src homology 2 (SH2) domains and a tyrosine phosphorylated activation motif found in receptors of the immune system or their associated chains. The motif, termed the immunoreceptor tyrosine-based activation motif (ITAM), has the ...