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

Abstract

Aims: 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.

Methods: 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 TPalpha- and TPbeta-transfected HEK 293T cells was explored using binding assays and the TP antagonist (3)H-SQ29548.

Results: Apigenin, genistein, luteolin and quercetin impaired U46619-induced calcium mobilization in a concentration-dependent manner (IC(50) 10-30 microm). 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% (3)H-SQ29548 binding to different cell types.

Conclusions: 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.

Figures

Figure 1

Inhibition of [Ca2+]i mobilization by apigenin. A representative example of interference of [Ca2+]i mobilization by some flavonoids. Oregon Green BAPTA-1 AM-loaded platelets were stimulated with 2 µm U46619 in the absence or in the presence of increasing concentrations of apigenin and [Ca2+]i mobilization was recorded vs. time as stated in Materials and methods. The arrow indicates the addition of agonist

Figure 2

Effect of flavonoids on U46619- and thrombin-induced [Ca2+]i mobilization. Oregon green BAPTA-1 AM-loaded platelets, or aspirin-treated platelets, were incubated with dimethylsulphoxide (DMSO), flavonoids (50 µm) or with the TP antagonist SQ29548 (1 µm) and stimulated with 2 µm U46619 or 0.5 U ml−1 thrombin. Data represent mean percentage of inhibition compared with mean peak calcium concentration with DMSO [n = 3; *indicates significant differences (P< 0.05) in [Ca2+]i mobilization upon activation with U46619 or thrombin]. Open bars represent U46619-activated platelets; black bars indicate thrombin-stimulation of aspirin-treated platelets. API, Apigenin; GEN, genistein; LUT, luteolin; QUE, quercetin; RUT, rutin; SQ, SQ29548

Figure 3

Effect of flavonoids on U46619-induced tyrosine phosphorylation of platelet proteins. Washed platelets, in the presence of ethylene glycol tetraacetic acid (1 mm), were incubated with vehicle (dimethylsulphoxide), flavonoids (100 µm), the TP antagonist SQ29548 (10 µm) or with the Src kinase inhibitor PP2 (10 µm) and stimulated with 5 µm U46619 for 2 min. Platelets were then lysed and phosphotyrosine-containing proteins were identified by Western blot using PY20 antibody. A representative blot is shown in the upper panel. In each case, tyrosine phosphorylation was quantified by densitometry using Quantity One software. The lower panel shows comparative results of tyrosine phosphorylation achieved with platelets stimulated with U46619 in the absence or presence of flavonoids, SQ29548 or PP2. Data are mean ± SD from four separate experiments. API, Apigenin; GEN, genistein; LUT, luteolin; QUE, quercetin; RUT, rutin; SQ, SQ29548. *P < 0.05 compared with phosphotyrosine content in U46619-stimulated platelets in the absence of inhibitors

Figure 4

Inhibition of U46619-induced phosphorylation of ERK 1/2 by flavonoids. Washed platelets, containing ethylene glycol tetraacetic acid (1 mm), were incubated with vehicle [dimethylsulphoxide (DMSO)], flavonoids (100 µm), SQ29548 (10 µm) or the MEK inhibitor U0126 (10 µm), before activation with 5 µm U46619. Platelet lysates were subjected to sodium dodecyl sulphate–polyacrylamide gel electrophoresis and Western blotting with a specific phospho-p44/42 ERK monoclonal antibody. Equal protein loading was verified using antibody against βI tubulin. The upper plot is a representative example from six separate experiments with distinct platelet samples. The lower panel compares the densitometric quantification, using Quantity One software, of U46619-promoted ERK phosphorylation achieved in presence or absence of flavonoids, or U0126 (mean ± SD, n = 6). API, Apigenin; GEN, genistein; LUT, luteolin; QUE, quercetin; RUT, rutin; SQ, SQ29548. *P < 0.05 compared with ERK phosphorylation detected in stimulated platelets in the absence of inhibitors (+DMSO)

Figure 5

Effect of flavonoids on thrombin-induced protein tyrosine phosphorylation in aspirinized platelets. Washed platelets treated with 1 mm aspirin were incubated with flavonoids (100 µm), SQ29548 (10 µm) or with the Src kinase inhibitor PP2 (10 µm) and then stimulated for 2 min with thrombin (0.5 U ml−1). Phosphotyrosine proteins were identified by Western blot as described in Figure 3 and quantified by densitromety using Quantity One software. The upper plot is a representative blot and the lower panel shows comparative results of the densitometric analysis (mean ± SD, n = 4). API, Apigenin; GEN, genistein; LUT, luteolin; QUE, quercetin; RUT, rutin; SQ, SQ29548. *P < 0.05 compared with platelet phosphotyrosine content detected upon thrombin activation in the absence of inhibitors (+dimethylsulphoxide)

Figure 6

Effect of flavonoids on thrombin-induced phosphorylation of ERK 1/2 in aspirinized platelets. In vitro aspirinized platelets were incubated with flavonoids (100 µm), SQ29548 (10 µm) or the MEK inhibitor U0126 (10 µm) and then stimulated for 2 min with thrombin (0.5 U ml−1). Phospho-p44/42 ERK in platelet lysates were identified by Western blot as described in Figure 4. The upper plot is a representative example of phosho-ERK detection and the lower panel shows comparative results of the densitometric quantification performed with Quantity One software (mean ± SD, n = 4). API, Apigenin; GEN, genistein; LUT, luteolin; QUE, quercetin; RUT, rutin; SQ, SQ29548. *P < 0.05 compared with ERK phosphorylation detected in the absence of inhibitors (+dimethylsulphoxide)

Figure 7

Effect of flavonoids on 3H-SQ29548 binding to (A) platelets, (B) myometrium, (C) TPα-transfected HEK 293T cells and (D) TPβ-transfected HEK 293T cells. Washed cells or tissue were incubated with 5 nm3H-SQ29548 in the absence or in the presence of U46619 (5 µm), I-BOP (5 µm) or flavonoids (100 µm) as competitors. Nonspecific binding was determined as the residual binding of 3H-SQ29548 in the presence of 10 µm SQ29548. Data represent percentage of 3H-SQ29548-specific binding, considering 100% of binding as that achieved in the absence of competitor [dimethylsulphoxide (DMSO)]. API, Apigenin; GEN, genistein; LUT, luteolin; QUE, quercetin; RUT, rutin. Data are mean ± SD from three different experiments. *P < 0.05 compared with binding in samples without competitor (+DMSO)