Flavonoids inhibit the platelet TxA(2) signalling pathway and antagonize TxA(2) receptors (TP) in platelets and smooth muscle cells

What is already known about this subject center dot Flavonoids are largely recognized as potential inhibitors of platelet function, through nonspecific mechanisms such as antioxidant activity and/or inhibition of several enzymes and signalling proteins. center dot In addition, we, and few others, have shown that certain antiaggregant flavonoids may behave as specific TXA2 receptor (TP) ligands in platelets. center dot Whether flavonoids interact with TP isoforms in other cell types is not known, and direct evidence that flavonoid-TP interaction inhibits signalling downstream TP has not been shown. What this study adds center dot This study first demonstrates that certain flavonoids behave as ligands for both TP isoforms, not only in platelets, but also in human myometrium and in TP-transfected HEK 293T cells. center dot Differences in the effect of certain flavonoids in platelet signalling, induced by either U46619 or thrombin, suggest that abrogation of downstream TP signalling is related to their specific blockage of the TP, rather than to a nonspecific effect on tyrosine kinases or other signalling proteins. Flavonoids may affect platelet function by several mechanisms, including antagonism of TxA(2) receptors (TP). These TP are present in many tissues and modulate different signalling cascades. We explored whether flavonoids affect platelet TP signalling, and if they bind to TP expressed in other cell types. Platelets were treated with flavonoids, or other selected inhibitors, and then stimulated with U46619. Similar assays were performed in aspirinized platelets activated with thrombin. Effects on calcium release were analysed by fluorometry and changes in whole protein tyrosine phosphorylation and activation of ERK 1/2 by Western blot analysis. The binding of flavonoids to TP in platelets, human myometrium and TP alpha- and TP beta-transfected HEK 293T cells was explored using binding assays and the TP antagonist H-3-SQ29548. Apigenin, genistein, luteolin and quercetin impaired U46619-induced calcium mobilization in a concentration-dependent manner (IC50 10-30 mu M). These flavonoids caused a significant impairment of U46619-induced platelet tyrosine phosphorylation and of ERK 1/2 activation. By contrast, in aspirin-treated platelets all these flavonoids, except quercetin, displayed minor effects on thrombin-induced calcium mobilization, ERK 1/2 and total tyrosine phosphorylation. Finally, apigenin, genistein and luteolin inhibited by > 50% H-3-SQ29548 binding to different cell types. These data further suggest that flavonoids may inhibit platelet function by binding to TP and by subsequent abrogation of downstream signalling. Binding of these compounds to TP occurs in human myometrium and in TP-transfected HEK 293T cells and suggests that antagonism of TP might mediate the effects of flavonoids in different tissues.

Platelet PECAM-1 inhibits thrombus formation in vivo

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a cell surface glycoprotein receptor expressed on a range of blood cells, including platelets, and on vascular endothelial cells. PECAM-1 possesses adhesive and signaling properties, the latter being mediated by immunoreceptor tyrosine-based inhibitory motifs present on the cytoplasmic tail of the protein. Recent studies in vitro have demonstrated that PECAM-1 signaling inhibits the aggregation of platelets. In the present study we have used PECAM-1-deficient mice and radiation chimeras to investigate the function of this receptor in the regulation of thrombus formation. Using intravital microscopy and laser-induced injury to cremaster muscle arterioles, we show that thrombi formed in PECAM-1-deficient mice were larger, formed more rapidly than in control mice, and were more stable. Larger thrombi were also formed in control mice that received transplants of PECAM-1-deficient bone marrow, in comparison to mice that received control transplants. A ferric chloride model of thrombosis was used to investigate thrombus formation in carotid arteries. In PECAM-1-deficient mice the time to 75% vessel occlusion was significantly shorter than in control mice. These data provide evidence for the involvement of platelet PECAM-1 in the negative regulation of thrombus formation.

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) mu mol/l. Collagen-stimulated (0.5 mu g/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.

PtdIns(5)P activates the host cell PI3-kinase/Akt pathway during Shigella flexneri infection

The virulence factor IpgD, delivered into nonphagocytic cells by the type III secretion system of the pathogen Shigella flexneri, is a phosphoinositide 4-phosphatase generating phosphatidylinositol 5 monophosphate (PtdIns(5) P). We show that PtdIns(5) P is rapidly produced and concentrated at the entry foci of the bacteria, where it colocalises with phosphorylated Akt during the first steps of infection. Moreover, S. flexneri-induced phosphorylation of host cell Akt and its targets specifically requires IpgD. Ectopic expression of IpgD in various cell types, but not of its inactive mutant, or addition of short-chain penetrating PtdIns(5) P is sufficient to induce Akt phosphorylation. Conversely, sequestration of PtdIns(5) P or reduction of its level strongly decreases Akt phosphorylation in infected cells or in IpgD-expressing cells. Accordingly, IpgD and PtdIns(5) P production specifically activates a class IA PI 3-kinase via a mechanism involving tyrosine phosphorylations. Thus, S. flexneri parasitism is shedding light onto a new mechanism of PI 3-kinase/Akt activation via PtdIns(5) P production that plays an important role in host cell responses such as survival.

Isolation and partial characterisation of acid phosphatase isozymes from dormant oilseed of Corylus avellana L.

The acid phosphatase (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.2) complement from dormant hazel (Corylus avellana L.) seeds was found to exhibit significant electrophoretic heterogeneity partially attributable to the presence of distinct molecular forms. In axiferous tissue, total acid phosphatase activity increased in a biphasic fashion during chilling, a treatment necessary to alleviate seed dormancy. Three acid phosphatase isozymes were isolated from cotyledons of dormant hazel seeds by successive ammonium sulphate precipitation, size-exclusion, Concanavalin A affinity, cation- and anion-exchange chromatographies resulting in 75-, 389- and 191-fold purification (APase1, APase2, APase3, respectively). The three glycosylated isoforms were isolated to catalytic homogeneity as determined by electrophoretic, kinetic and heat-inactivation studies. The native acid phosphatase complement of hazel seeds had an apparent Mr of 81.5±3.5 kDa as estimated by size-exclusion chromatography, while the determined pI values were 5.1 (APase1), 6.9 (APase2) and 7.3 (APase3). The optimum pH for p-nitrophenyl phosphate hydrolysis was pH 3 (APase1), pH 5.6 (APase2) and pH 6 (APase3). The hazel isozymes hydrolysed a variety of phosphorylated substrates in a non-specific manner, exhibiting low Km and the highest specificity constant (Vmax/Km) for pyrophosphate. They were not primary phytases since they could not initiate phytic acid hydrolysis, while APase2 and APase3 had significant phospho-tyrosine phosphatase activity. Inorganic phosphate was a competitive inhibitor, while activity was significantly impaired in the presence of vanadate and fluoride.

Platelet 12-lipoxygenase activation via glycoprotein VI: involvement of multiple signaling pathways in agonist control of H(P)ETE synthesis

Lipoxygenases (LOX) contribute to vascular disease and inflammation through generation of bioactive lipids, including 12-hydro(pero xyeicosatetraenoic acid (12-H(P)ETE). The physiological mechanisms that acutely control LOX product generation in mammalian cells are uncharacterized. Human platelets that contain a 12-LOX isoform (p12-LOX) were used to define pathways that activate H(P)ETE synthesis in the vasculature. Collagen and collagen-related peptide (CRP) (1 to 10 g/mL) acutely induced platelet 12-H(P)ETE synthesis. This implicated the collagen receptor glycoprotein VI (GPVI), which signals via the immunoreceptor-based activatory motif (ITAM)- containing FcR chain. Conversely, thrombin only activated at high concentrations ( 0.2 U/mL), whereas U46619 and ADP alone were ineffective. Collagen or CRP-stimulated 12-H(P)ETE generation was inhibited by staurosporine, PP2, wortmannin, BAPTA/AM, EGTA, and L-655238, implicating src-tyrosine kinases, PI3-kinase, Ca2 mobilization, and p12-LOX translocation. In contrast, protein kinase C (PKC) inhibition potentiated 12-H(P)ETE generation. Finally, activation of the immunoreceptor tyrosine-based inhibitory motif (ITIM)– containing platelet endothelial cell adhesion molecule (PECAM-1) inhibited p12-LOX product generation. This study characterizes a receptor-dependent pathway for 12-H(P)ETE synthesis via the collagen receptor GPVI, which is negatively regulated by PECAM-1 and PKC, and demonstrates a novel link between immune receptor signaling and lipid mediator generation in the vasculature. (Circ Res. 2004;94:1598-1605.)

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) 1b-V-IX receptor complex, GPV1 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 alphaIIbbeta(3), a platelet thrombus is formed. Although increasing evidence indicates that each of the adhesion receptors GPIb-V-IX and GPV1 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.

The role of polyphenolic compounds in the diet as inhibitors of platelet function

Platelets play a substantial role in cardiovascular disease, and for many years there has been a search for dietary components that are able to inhibit platelet function and therefore decrease the risk of cardiovascular disease. Platelets can be inhibited by alcohol, dietary fats and some antioxidants, including a group of compounds, the polyphenols, found in fruits and vegetables. A number of these compounds have been shown to inhibit platelet function both in vitro and in vivo. In the present study the effects of the hydroxycinnamates and the flavonoid quercetin on platelet activation and cell signalling in vitro were investigated. The hydroxycinnamates inhibited platelet function, although not at levels that can be achieved in human plasma by dietary intervention. However, quercetin inhibited platelet aggregation at levels lower than those previously reported. Quercetin was also found to inhibit intracellular Ca mobilisation and whole-cell tyrosine protein phosphorylation in platelets, which are both processes essential for platelet activation. The effect of polyphenols on platelet aggregation in vivo was also investigated. Twenty subjects followed a low-polyphenol diet for 3 d before and also during supplementation. All subjects were supplemented with a polyphenol-rich meal every lunchtime for 5 d. Platelet aggregation and plasma flavonols were measured at baseline and after 5 d of dietary supplementation. Total plasma flavonoids increased significantly after the dietary intervention period (P = 0.001). However, no significant changes in ex vivo platelet aggregation were observed. Further investigation of the effects of individual polyphenolic compounds on platelet function, both in vitro and in vivo, is required in order to elucidate their role in the relationship between diet and the risk of cardiovascular disease.

Water-soluble tripeptide A beta(9-11) forms amyloid-like fibrils and exhibits neurotoxicity

A water-soluble, hydrophilic tripeptide GYE, having sequence identity with the N-terminal segment of amyloid peptides A,beta(9-11), upon self-association exhibits amyloid-like fibrils and significant neurotoxicity towards the Neuro2A cell line. However, the tripeptides GFE and GWE, in which the centrally located tyrosine residue has been replaced by phenylalanine or tryptophan, fail to show amyloidogenic behavior and exhibit little or no neurotoxicity.

A new motif in the formation of peptide nanotubes: the crystallographic signature

Terminally protected acyclic tripeptides Boc-Tyr(1)-Val(2)-Tyr(3)-OMe 1 and Boc-Tyr(1)-lle(2)-Tyr(3)-OMe 2 self-assemble into nanotubes in crystals through various noncovalent interactions with an average internal diameter of 5 Angstrom (0.5 nm), and the tubular ensemble is developed through the hydrogen-bonded side chains of tyrosine residues. The inside of the hollow nanotubular structures is hydrophilic; however, no solvent molecules have been crystallographically detected.

Flavonoids: antioxidants or signalling molecules?

Many studies are accumulating that report the neuroprotective, cardioprotective, and chemopreventive actions of dietary flavonoids. While there has been a major focus on the antioxidant properties, there is an emerging view that flavonoids, and their in vivo metabolites, do not act as conventional hydrogen-donating antioxidants but may exert modulatory actions in cells through actions at protein kinase and lipid kinase signalling pathways. Flavonoids, and more recently their metabolites, have been reported to act at phosphoinositide 3-kinase (PI 3-kinase), Akt/protein kinase B (Akt/PKB), tyrosine kinases, protein kinase C (PKC), and mitogen activated protein kinase (MAP kinase) signalling cascades. Inhibitory or stimulatory actions at these pathways are likely to affect cellular function profoundly by altering the phosphorylation state of target molecules and by modulating gene expression. A clear understanding of the mechanisms of action of flavonoids, either as antioxidants or modulators of cell signalling, and the influence of their metabolism on these properties are key to the evaluation of these potent biomolecules as anticancer agents, cardioprotectants, and inhibitors of neurodegeneration (C) 2004 Elsevier Inc. All rights reserved.

Loss of 3-chlorotyrosine by inflammatory oxidants: Implications for the use of 3-chlorotyrosine as a bio-marker in vivo

Activated neutrophils generate the potent oxidant hypochlorous acid (HOCl) from the enzyme myeloperoxidase (MPO). A proposed bio-marker for MPO-derived HOCl in vivo is 3-chlorotyrosine, elevated levels of which have been measured in several human inflammatory pathologies. However, it is unlikely that HOCl is produced as the sole oxidant at sites of chronic inflammation as other reactive species are also produced during the inflammatory response. The work presented shows that free and protein bound 3-chlorotyrosine is lost upon addition of the pro-inflammatory oxidants, HOCl, peroxynitrite, and acidified nitrite. Furthermore, incubation of 3-chlorotyrosine with activated RAW264.7 macrophages or neutrophil-like HL-60 cells resulted in significant loss of 3-chlorotyrosine. Therefore, at sites of chronic inflammation where there is concomitant ONOO- and HOCl formation, it is possible measurement of 3-chlorotyrosine may represent an underestimate of the true extent of tyrosine chlorination. This finding could account for some of the discrepancies reported between 3-chlorotyrosine levels in tissues in the literature. (c) 2008 Elsevier Inc. All rights reserved.

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

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-beta-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 mum (+/-0.77) and 9.72mum (+/-1.38) 30min after ingestion of 150-mg and 300-mg doses of quercefin-4'-O-beta-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-beta-D-glucoside. Correspondingly, collagen-stimulated tyrosine phosphorylation of total platelet proteins was inhibited. This was accorripanied by reduced tyrosine phosphorylation of the tyrosine kinase Syk and phospholipase Cgamma2, 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-beta-D-glucoside by supplementation, and implicates quercetin as a dietary inhibitor of platelet cell signaling and thrombus formation.

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

Background: The regulation of platelet function by pharmacological agents that modulate platelet signaling haspharmacolo 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 gamma-chain, Syk, LAT and phospholipase Cgamma2. Inhibition of phosphorylation of the Fc receptor gamma-chain suggests that quercetin inhibits early signaling events following stimulation of platelets with collagen. The activity of the kinases that phosphorylate the Fc receptor gamma-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.

Regulation of cell cycle and stress responses to hydrostatic pressure in fission yeast

We have investigated the cellular responses to hydrostatic pressure by using the fission yeast Schizosaccharomyces pombe as a model system. Exposure to sublethal levels of hydrostatic pressure resulted in G2 cell cycle delay. This delay resulted from Cdc2 tyrosine-15 (Y-15) phosphorylation, and it was abrogated by simultaneous disruption of the Cdc2 kinase regulators Cdc25 and Wee1. However, cell cycle delay was independent of the DNA damage, cytokinesis, and cell size checkpoints, suggesting a novel mechanism of Cdc2-Y15 phosphorylation in response to hydrostatic pressure. Spc1/Sty1 mitogen-activated protein (MAP) kinase, a conserved member of the eukaryotic stress-activated p38, mitogen-activated protein (MAP) kinase family, was rapidly activated after pressure stress, and it was required for cell cycle recovery under these conditions, in part through promoting polo kinase (Plo1) phosphorylation on serine 402. Moreover, the Spc1 MAP kinase pathway played a key role in maintaining cell viability under hydrostatic pressure stress through the bZip transcription factor, Atf1. Further analysis revealed that prestressing cells with heat increased barotolerance, suggesting adaptational cross-talk between these stress responses. These findings provide new insight into eukaryotic homeostasis after exposure to pressure stress.