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Biochemical, Molecular, and Genetic Mechanisms

Resveratrol, at Concentrations Attainable with Moderate Wine Consumption, Stimulates Human Platelet Nitric Oxide Production^1–3,

⁴ Department of Internal Medicine, Division of Internal and Cardiovascular Medicine, University of Perugia, 06100 Perugia, Italy; ⁵ Division of IV Clinical Medicine, Department of Pathology and Experimental Medicine, Umberto I Hospital, 00185 Rome, Italy; and ⁶ National Institute for Food and Nutrition Research, Università di Roma "La Sapienza", 00185 Rome, Italy

^* To whom correspondence should be addressed. E-mail: grespa{at}unipg.it.

	ABSTRACT

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
The mechanisms through which moderate wine consumption reduces ischemic cardiovascular events are not yet fully unraveled. Grape extracts or a mixture of the polyphenols contained in wine were previously shown to increase nitric oxide (NO); however, little information is available on the effect of resveratrol, one of the main polyphenols of wine, on platelet NO production. We assessed the effects of resveratrol, at the concentrations attainable after moderate wine intake, on platelet NO production and the mechanism of this activity. Twenty healthy volunteers were studied before and after 15 d of controlled white or red wine intake (300 mL/d). After wine intake, plasma resveratrol and the release of NO by stimulated platelets increased significantly. Resveratrol, at the concentrations detected in plasma after wine intake, was incubated in vitro with washed platelets and several variables related to NO production and to signal transduction were measured. Resveratrol in vitro enhanced significantly the production of NO by stimulated platelets, the activity of platelet NO synthase (NOS), phosphorylation of protein kinase B, an activator of the endothelial NOS (eNOS), and phosphorylation of vasodilator-activated protein (VASP), an expression of the biologic activity of NO in platelets. Simultaneously, we observed decreased phosphorylation of P38 mitogen-activated protein kinase (p38MAPK), a proinflammatory pathway in human platelets, a reduction of the activity of NADPH oxidase, a major source of reactive oxygen species (ROS) and of the generation of O₂^– radicals, as detected by cytochrome C reduction. In conclusion, resveratrol, at concentrations attainable after moderate wine intake, activates platelet eNOS and in this way blunts the proinflammatory pathway linked to p38MAPK, thus inhibiting ROS production and ultimately platelet function. This activity may contribute to the beneficial effects of moderate wine intake on ischemic cardiovascular disease.

	Introduction

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

Studies in humans, as well as experiments on isolated tissues or organs, demonstrated that beneficial cardiovascular effects can be achieved also with de-alcoholized wine, suggesting that wine has beneficial effects partly dependent on components other than alcohol (1,4,12,13). Grapes contain a variety of phenolic antioxidants, including quercetin, catechin, epicatechin, proanthocyanidins and resveratrol (5), mainly present in grape skin.

The levels of the phenolic compounds in wines are highly variable, due to differences in fruit sources as well as in processing, ranging from 200 mg of total phenols in a glass of red wine to 40 mg in a glass of white wine (14–18). The protective effects of polyphenols against oxidative stress in biologic systems are ascribed to their capacity to chelate metal catalysts (19), activate antioxidant enzymes (20), reduce -tocopherol radicals (21), and inhibit oxidases (22,23). Specific polyphenols interact with arachidonic acid (AA)⁷ metabolism and inhibit platelet thromboxane A2 production (24). Red wine polyphenols have also been shown to increase the expression of endothelial nitric oxide (NO) synthase (eNOS) and consequently the release of NO by human umbilical vein endothelial cells in vitro; however, these actions were observed only with relatively high concentrations of polyphenols (25,26). It has been proposed that resveratrol, a relevant wine constituent, is involved in vascular protection. Fresh grape skin contains 50–100 µg/g of resveratrol, whereas the concentration of resveratrol in wine ranges from 0.1 to 14 mg/L for red wine and from 0.04 to 3.5 mg/L for white wine (with the highest concentrations reported in wines from pinot noir grapes) (14,15).

In particular, concerning the role of resveratrol in potentiating NO production, most of the previous in vitro studies used concentrations of this flavonoid (from 1 to 1000 µmol/L) (24,27,28) much higher than those attainable in plasma after moderate wine consumption (29).

The effects of polyphenols on platelet function have been investigated by several authors and, among other activities, an increase of NO production associated with a decrease in the formation of reactive oxygen species (ROS) has been described, either when using extracts from grape components or a mixture of polyphenols mimicking the flavonoids detected in plasma after wine intake (23,29,30), but little data on the effects of resveratrol at in vivo attainable concentrations on platelet function are available.

In a previous study testing the hypothesis that moderate wine consumption inhibits oxidative stress in healthy subjects, we found that plasma total resveratrol (free resveratrol plus the glucuronated and sulfated metabolites, but not the hydroxylated and methylated derivatives), measured with an HPLC-electrochemical method (31), is 1.33 ± 0.3 µmol/L after white wine intake and 1.72 ± 0.1 µmol/L after red wine intake, with a mean increase of plasma resveratrol induced by wine intake of 0.7 ± 0.4 µmol/L (29).

In the present study, we assessed the effects of resveratrol, at concentrations attainable by moderate intake of red or white wine, on platelet NO production and studied the mechanism of this activity.

	Materials and Methods

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
Materials

Collagen from equine tendon was obtained from Mascia Brunelli. The nonenzymatic Nitric Oxide Assay kit was purchased from OXIS International. NOSdetect Assay kit, for the measurement of NO synthase (NOS) activity, was purchased from Stratagene Alexis Biochemicals. (³H)L-Arginine was purchased from Amersham-Pharmacia Biotech, and AA, L-N^G-monomethyl-L-arginine, resveratrol, and S-nitroso-N-acetyl-DL-penicillamine (SNAP) were purchased from Sigma-Aldrich.

Anti-p38 mitogen-activated protein kinase (p38MAPK) and anti-AKT (protein kinase B) antibodies were purchased from Santa Cruz Biotechnology and anti-vasodilator-stimulated phosphoprotein (VASP) IgG1 mouse monoclonal antibody was purchased from Becton Dickinson.

Ex vivo study

Citrated (1/10 v:v trisodium citrate 3.8%) blood samples were taken at baseline and after 15 d of controlled white wine or red wine intake (300 mL/d) in the morning, under fasting conditions, from 20 healthy human volunteers with no evidence of cardiovascular disease or risk factors for atherosclerosis and who had not taken antioxidants, vitamins, or antiplatelet drugs for at least 1 mo, participating in a previously reported study (29). All gave informed consent to participate in the study, which was approved by the Ethical Committee of the University of Rome "La Sapienza."

Briefly, after a run-in period of 1 wk, during which the subjects refrained from consuming wine or alcohol and nonsteroidal antiinflammatory drugs, they were randomly allocated to consume a total of 300 mL/d of red (total polyphenolic concentration, 1.8 g/L) (n = 10, 4 males and 6 females, mean age, 45 ± 6 y) or white (total polyphenolic concentration, 0.25 g/L) (n = 10, 5 males and 5 females, mean age, 42 ± 5 y) wine during dinner. At baseline and after 15 d of follow-up, a blood sample was taken from each subject in the morning after a 12-h fast [for more details, see (29)].

Platelet-rich plasma (PRP) was prepared by centrifugation at 160 x g for 10 min at room temperature and 250-µL aliquots (200,000 platelets/µL) were stimulated with AA 0.5 mmol/L (5 min, 37°C) in an aggregometer cuvette under continuous stirring. After stimulation, samples were centrifuged (10,000 x g; 5 min, room temperature) and the cell-free supernatant was stored at –80°C for later measurement of nitrite plus nitrate. The concentration of AA used was selected on the basis of previous reports (30–32) and on the basis of the consideration that the presence of plasmatic proteins, especially albumin, acting as carriers for AA, mediate its slow release in proximity of cell membranes, preserving cell integrity (33).

    Platelet-released nitrite plus nitrate. Nitrite plus nitrate was measured by the Griess reagent in the supernatant plasma of arachidonic-acid–stimulated PRP obtained by centrifugation (as described above), using a commercially available colorimetric kit (nonenzymatic nitric oxide assay kit, Oxis) after reduction of nitrate to nitrite with a redox reaction catalyzed by cadmium, as previously described (34).

Before the test, granulated cadmium was washed with water, then with 0.1 mol/L HCl and finally with 0.1 mol/L NH₄OH, pH 9.6. Samples (50 µL adjusted with water to 190 µL) were deproteinized by precipitation with ZnSO₄ [10 µL of 30% (wt:v) solution] for 15 min at room temperature, then centrifuged and the supernatants were transferred into tubes containing cadmium beads and incubated overnight at room temperature under continuous agitation. Before the assay, samples were recentrifuged to remove beads and supernatants were then added to micro-well plates; color reagent (sulphanilamide and N-[1-naphtyl] ethylenediamine dihydrochloride) was added and 540-nm absorbance was read in a microplate reader. A standard curve was built to determine nitrite concentration for each assay.

In vitro study

For the in vitro studies, preliminary experiments with increasing concentrations of resveratrol (up to 1 µmol/L) indicated that 0.5 µmol/L was the minimum concentration of resveratrol giving a potentiation of NO production (data not shown). Because this concentration is largely within the range of the increase in plasma from the ex vivo study, we decided to use 0.5 µmol/L as the maximum concentration for our in vitro studies.

    Platelet preparation. Venous blood was drawn in trisodium citrate [3.8%, 1/10 (v:v)] from fasting healthy volunteers who had not ingested any drugs affecting platelet function for at least 14 d.

Platelets prepared from single volunteers were used for individual experiments. Blood was centrifuged at 160 x g for 10 min at room temperature and the supernatant PRP was separated. Washed platelets were prepared by the method of Mustard, as previously described (35); we counted platelets (Genius, S.E.A.C) and adjusted then to 2 x 10¹¹/L using Tyrode's buffer without Ca²⁺ and Mg²⁺. For the preparation of gel-filtered platelets (GFP), PRP was passed over a Sepharose-2B column equilibrated with Tyrode's HEPES-buffered saline [140 mmol/L NaCl, 6 mmol/L HEPES, 2 mmol/L Na₂HPO₄, 2 mmol/L MgSO₄, 0.1% dextrose, pH 7.4, and 0.4% bovine serum albumin (BSA)], as described (36–38). Prior to gel filtration, PRP was incubated (1 h at room temperature) with 0.2 or 0.5 µmol/L resveratrol. After gel filtration, the cell suspension was adjusted to a final concentration of 1 x 10¹¹ platelets/L. Platelets were activated with collagen (1.5 mg/L) for 5 min if not otherwise stated.

In preliminary experiments, the effects of resveratrol on NO production by stimulated platelets were tested using either collagen or AA as a stimulus and a similar potentiating effect of resveratrol was observed with both agonists (data not shown), as also confirmed by the strong correlation (Pearson r = 0.833; P < 0.001) of nitrite plus nitrate produced from collagen- and AA-stimulated platelets in samples treated with resveratrol (Supplemental Figs. 1 and 2).

    Platelet NO production. A NO-selective microelectrode (Inter Medical), able to detect nanomolar amounts of gaseous NO, was adapted for use in a standard platelet aggregometer (P.I.C.A., Chronolog) to monitor platelet NO production and aggregation simultaneously, as previously described (39). The NO monitoring device consists of a headstage amplifier with a built-in power supply and 2 electrodes. The working electrode (0.2-mm diameter, made from a Pt/Ir alloy) and the counter-electrode (carbon fiber) were inserted in an aggregometer microcuvette containing a stirring bar. The aggregometer, electrode, and headstage amplifier were housed in a Faraday cage to reduce electrical interference. The electrode current was continuously recorded with a Dual Channel Chart Recorder (Chronolog) and production of NO was quantified by integrating the area under the curve. All experiments were conducted at 37°C. SNAP was used to derive a standard curve that was linear from 10 to 350 pA over the range of 10–500 nmol/L SNAP concentration; r was typically 0.97. Platelet aggregation and NO production were studied using 1.5 mg/L collagen as a stimulus. Alternatively, nitrite plus nitrate in GFP supernatants were measured by the Griess reagent, as above described.

To assess platelet NOS activity, we used a commercially available kit (NOSdetect assay kit) that measures NOS activity by monitoring the enzymatic conversion of (³H)L-arginine to (³H)L-citrulline. Briefly, the platelet suspension was incubated with 37 MBq/L of (³H)L-arginine for 30 min at 37°C, then platelets were diluted with HEPES Tyrode's to a final concentration of 2 x 10¹¹/L; prior to trigger the reaction using 1.5 mg/L collagen as a stimulus, Ca²⁺ concentration was restored by adding CaCl₂ 2mmol/L. After stimulation for 5 min, samples were centrifuged, the pellet was extracted with the homogenization buffer provided with the kit, and the supernatant was added to an ion exchange resin, also provided with the kit. (³H)L-citrulline does not bind to the resin; thus, the effluent was recovered and added to scintillation tubes and counted with a β-counter. A standard curve using increasing (³H)L-arginine concentrations was built for each assay.

For the calculation of platelet NO production, as measured using any of the above-described methods, levels observed in the same samples after treatment with L-N^G-monomethyl-L-arginine were subtracted to give the net stimulus- and/or resveratrol-induced NO production.

    Platelet p38MAP kinase, AKT, and VASP activities. p38MAP kinase, AKT, and VASP phosphorylation in resting or collagen- (6 mg/L) stimulated washed platelets was analyzed by flow cytometry using specific antibodies (p38MAPK IgG goat polyclonal, anti AKT IgG goat polyclonal, and anti VASP IgG1 mouse monoclonal). In all assays, an irrelevant isotype-matched antibody was used as a negative control.

Antibodies (1 mg/L) were added to 200 µL of platelet suspension (2 x 10¹¹/L), previously fixed with (2%) paraformaldehyde (0.1% BSA) for 60 min at room temperature and then permeabilized with digitonin (100 µmol/L) for 30 min at room temperature (40). Unbound antibody was removed by adding 0.1% BSA PBS and subsequent centrifugation at 3000 x g for 3 min (twice). Fluorescence intensity was analyzed on an Epics XL-MCL cytometer (Coulter Electronics) equipped with an argon laser at 488 nmol/L. For every histogram, 50,000 platelets were counted to determine the proportion of positive platelets. We reported antibody reactivity in arbitrary units obtained by multiplying the number of positive events resulting from platelet stimulation by the mean values of the fluorescence observed when the specific antibody was used and by correcting for the values obtained in unstimulated samples treated with the same antibody.

    Platelet NADPH oxidase activity. Measurement of platelet NADPH oxidase activity was carried out in platelet homogenates according to Seno et al. (41). Washed platelets were suspended in homogenate buffer containing: 50 mmol/L Tris/HCl (pH 7.4), 1.0 mmol/L EDTA, 2.0 mmol/L leupeptin, and 2.0 mmol/L pepstatin A and then homogenized. Platelet homogenates were incubated for 10 min at 37°C with 100 µmol/L NADPH in the presence of resveratrol (0.1 and 0.5 µmol/L) or its vehicle. The assay solution contained 400 µL Tyrode's buffer and 0.25 mmol/L lucigenin. After preincubation at 37°C for 3 min, we started the reaction by adding 100 µL of platelet homogenates together with AA 0.5 mmol/L or its vehicle. The chemiluminescent signal was expressed as relative chemiluminescence units for a mean of 10 min and corrected for protein concentration (relative chemiluminescence units/mg) (34,41). Protein concentrations were determined by the method of Lowry.

    O₂^– generation evaluated by cytochrome c reduction. Generation of superoxide anion radicals (O₂^–) in resting platelets and in platelets stimulated with collagen (6 mg/L), after preincubation with resveratrol (0.1 and 0.5 µmol/L) or its vehicle, was measured by cytochrome c reduction, as described earlier (42,43). After a 10-min activation, platelets were sedimented by centrifugation at 2000 x g for 5 min, the supernatants were transferred into spectrophotometer cuvettes, and the reduction of cytochrome c was measured at 550 nm. To calculate the molar concentration of O₂^–, an extinction coefficient for cytochrome c of 18,700 (mol/L)^–1 cm^–1 was used (42,43).

Statistical analysis

For the ex vivo studies, the comparisons among groups before and after red or white wine intake were conducted using 1-way and repeated-measures ANOVA or, in case of nonhomogeneous variances (assessed with the Levene's test), with nonparametric tests (Wilcoxon's and Kolmogorov-Smirnov (z) tests; StatViewII software, Abacus Concepts) [for more details, see ref. 29)].

For the in vitro studies, statistical analyses were carried by the ANOVA followed by the Bonferroni post hoc test for selected pairs (GraphPad Prism 4.01 for Windows, GraphPad Software), as indicated. A P-value of < 0.05 was considered significant. Data are expressed as means ± SEM.

	Results

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
Ex vivo study

    Platelet nitrite plus nitrate production. After 15 d of controlled wine intake by healthy volunteers, plasma resveratrol concentrations increased from 0.72 ± 0.3 to 1.33 ± 0.3 µmol/L for white wine and from 0.71 ± 0.02 to 1.72 ± 0.1 µmol/L for red wine. The increased plasma resveratrol concentration, assessed by HPLC after the hydrolysis of the samples with β-glucuronidase and sulfatase, was 1.0 ± 0.3 µmol/L after red wine and 0.6 ± 0.3 µmol/L after white wine, with a mean increase of 0.7 ± 0.4 µmol/L (29).

Nitrite plus nitrate concentrations in the supernatant of AA (0.5 mmol/L)-stimulated PRP increased significantly after 15 d of controlled red [from 13.9 ± 3.4 µmol/L to 24.1 ± 3.6 µmol/L (P = 0.037)] or white wine [from 10.6 ± 2.4 µmol/L to 19.8 ± 4.8 µmol/L (P = 0.01)] intake. The changes in the nitrite plus nitrate released by activated platelets after red or white wine intake did not differ.

In vitro studies

    Platelet NO production. GFP stimulated with collagen (1.5 mg/L) released 25.8 ± 12.0 pmol/10⁸ platelets of NO (Fig. 1A). Preincubation with resveratrol (0.2–0.5 µmol/L) produced an increase of NO production that was significant at the concentration of 0.5 µmol/L (Fig. 1A). At the same time, platelet aggregation decreased and this was significant at 0.5 µmol/L resveratrol (P = 0.046) (Fig. 1B).

View larger version (21K): [in this window] [in a new window]   FIGURE 1  Effect of resveratrol on collagen (Coll)-induced NO production and platelet aggregation. (A) NO release from collagen-stimulated (1.5 mg/L) gel-filtered platelets measured with a NO-sensitive electrode. (B) Maximal amplitude of aggregation of collagen-stimulated washed platelets. (C) Nitrite plus nitrate release in the supernatant of collagen-stimulated platelets. (D) NOS activity of collagen-stimulated washed platelets. Values are means ± SEM, n = 11. #Different from collagen, P < 0.05.

Platelet NOS activity upon stimulation with collagen was 16.9 ± 8.7 pmol (³H)L-citrulline/10⁸ platelets. In resveratrol-treated platelets, (³H)L-citrulline production increased significantly at 0.5 µmol/L (Fig. 1D).

    Collagen-induced AKT phosphorylation. Stimulation with collagen induced the phosphorylation of platelet AKT compared with unstimulated platelets (P < 0.005) (Fig. 2A); preincubation with resveratrol (0.1–0.5 µmol/L) dose-dependently enhanced AKT phosphorylation induced by collagen, with a significant increase when the concentration was at least 0.1 µmol/L (Fig. 2A).

View larger version (18K): [in this window] [in a new window]   FIGURE 2  Effect of resveratrol (Resv) on AKT (A), p38 MAPK (B), and VASP phosphorylation (C) in collagen (Coll)-stimulated platelets. Data are expressed as means ± SEM, n = 5. #Different from collagen alone, P < 0.001.

    VASP phosphorylation. VASP phosphorylation tended to increase upon collagen stimulation (Fig. 2C). When platelets were preincubated with resveratrol (0.1–0.5 µmol/L), VASP phosphorylation increased further, with a significant rise when the concentration was at least 0.1 µmol/L (Fig. 2C and Supplemental Fig. 3).

    Platelet NADPH oxidase activity and cytochrome c reduction. Platelet NADPH oxidase activity was enhanced by stimulation with AA (0.5 mmol/L) and an especially strong enhancement was observed when AA was added together with NADPH (100 µmol/L) compared with AA alone (Fig. 3A). Preincubation with resveratrol (0.1–0.5 µmol/L) decreased AA-mediated O₂- formation, with a significant reduction when the concentration was at least 0.1 µmol/L (Fig. 3A). Confirmatory experiments were conducted with a higher concentration of NADPH (25 mmol/L), giving comparable results (data not shown). Generation of O₂^– in control platelets was enhanced by stimulation with collagen (Fig. 3B) and preincubation with resveratrol (0.1–0.5 µmol/L) decreased collagen-mediated O₂^– production, with a significant reduction already at 0.1 µmol/L.

View larger version (15K): [in this window] [in a new window]   FIGURE 3  Effect of resveratrol (resv) on NADPH oxidase activity (A) and ROS production (B) by stimulated, washed human platelets. (A) NADPH oxidase activity of platelets stimulated with 0.5 mmol/L AA. (B) Cytochrome C-dependent ROS production by platelets stimulated with 6 mg/L collagen (Coll). Data are expressed as means ± SEM, n = 5. #Different from stimulus alone, P < 0.001.

	Discussion

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

Our results showed a marked increase of platelet-derived nitrite plus nitrate after wine consumption, which was similar for both red and white wine. In these subjects, the increase of platelet-released nitrite plus nitrate was paralleled by an increase in plasma resveratrol (29). We thus decided to test the hypothesis that resveratrol directly induces NO production from human blood platelets at concentrations attainable during moderate daily wine consumption.

Polyphenols, and in particular resveratrol, inhibit ROS production from stimulated platelets (27,29,34). Platelets, like endothelial cells, contain not only the L-arginine-NOS pathway but also a phagocyte-type NADPH oxidase that is a major source of ROS (41,42). Indeed, Pignatelli et al. (44) have recently demonstrated that human platelets express gp91^phox, the catalytic unit of NADPH oxidase, and that this enzyme is crucial for the production of ROS by platelets, because patients with hereditary deficiency of gp91^phox have an almost complete suppression of platelet superoxide anion production.

In our in vitro experiments, the stimulation of platelets with collagen induced the production of NO, both when NO was measured directly by an ultrasensitive electrochemical device and by the assay of the NO degradation products nitrite plus nitrate in the supernatant of stimulated platelets.

The activity of the platelet eNOS enzyme was also increased, upon collagen stimulation, as documented by an increased incorporation of (³H)L-citrulline in platelets preincubated with (³H)L-arginine.

Preincubation of blood platelets with low concentrations of resveratrol, i.e. concentrations corresponding to the increased plasma concentrations produced by moderate wine intake, significantly increased platelet NO production, both as detected directly by a NO-sensitive electrode and by the measurement of NO-degradation products in the supernatant of stimulated platelets. This increase was the consequence of the stimulation of platelet NOS activity, as documented by the increased formation of (³H)L-citrulline in platelets preincubated with (³H)L-arginine.

Incubation with resveratrol also produced an increase of AKT phosphorylation in platelets, one of the effectors of NOS activation; in fact, agonist-dependent stimulation of PI3K and/or activation of calmodulin-dependent protein kinase kinase can activate AKT to phosphorylate eNOS, concomitantly to the increase in cytoplasmic calcium. The calcium-dependent activation of calmodulin will stimulate calmodulin-dependent protein kinase kinase and the recruitment of calmodulin and perhaps Hsp90 to eNOS, facilitating rapid eNOS activation and the burst-like release of NO (42–50). How phosphorylation of eNOS by AKT enhances NO release is not completely known, but it is likely to be related to changes in the sensitivity of the enzyme to calcium-activated calmodulin (46).

The increase of NOS activity and the consequent enhanced release of NO produced biologically relevant effects in platelets as shown by the enhanced PKG-dependent VASP phosphorylation induced by preincubation with resveratrol. p38 MAPK phosphorylation also decreased in resveratrol-preincubated platelets, which is likely to be an effect of the enhanced NO formation (43,49). When activated, p38 MAPK is able to induce a strong production of ROS (50). Moreover, p38MAPK phosphorylation induces the activation of platelet integrin _IIbβ₃; thus, the inhibition of p38MAPK phosphorylation is relevant for the suppression of platelet aggregation.

Together, these data suggest that resveratrol increases platelet NO production via PI3K-dependent AKT phosphorylation and the subsequent phosphorylation and activation of eNOS. The increased NO production in turn induces an increase of cGMP-dependent effects, such as VASP phosphorylation and inhibition of p38MAPK phosphorylation, with the consequent inhibition of platelet activation and a relevant reduction of ROS production (29,35,51). Recently, data showing resveratrol-dependent inhibition of p38MAPK in platelets have been reported (50).

Previous studies demonstrated that polyphenols inhibit NADPH oxidase activity by modulating the PKC pathway (25–29). We studied AA-induced NADPH oxidase activity and collagen-induced cytochrome c-dependent ROS production in platelets and found that resveratrol inhibits NADPH oxidase activity and ROS production at the same concentrations that induce NO production. Superoxide generated by NADPH-oxidase is one of the most effective agents to scavenge NO via the formation of peroxynitrite. Therefore, the enhanced release of NO from platelets upon treatment with resveratrol is probably the result of both an enhanced NO-synthase activity and a diminished NO inactivation.

These findings are in agreement with some of the inhibitory effects previously observed in platelets using polyphenols mixtures (13,24–26,39). Thus, the present study suggests that the wine polyphenol resveratrol alone, at the concentrations produced in plasma by moderate intake of wine, either red or white, exerts on human platelets a NO production-stimulating effect. This activity may be complemented by other activities of the polyphenols contained in wine, such as inhibition of LDL oxidation, increased HDL, the general improvement of the lipoprotein profile (51) and, at the cellular level, the downregulation of PKC-mediated NADPH oxidase activation, to generate the well-known protective effects of moderate wine consumption against cardiovascular ischemic events (6,18).

	FOOTNOTES

 
¹ Supported by a grant to P.G. from the Fondazione Cassa di Risparmio di Perugia (project no. 2007.0130.020).

² Author disclosures: P. Gresele, P. Pignatelli, G. Guglielmini, R. Carnevale, A. M. Mezzasoma, A. Ghiselli, S. Momi, and F. Violi, no conflicts of interest.

³ Supplemental Figures 1–3 are available with the online posting of this paper at jn.nutrition.org.

⁷ Abbreviations used: AA, arachidonic acid; AKT, protein kinase B; BSA, bovine serum albumin; eNOS, endothelial nitric oxide synthase; GFP, gel-filtered platelets; NOS, nitric oxide synthase; p38MAPK, p38 mitogen-activated protein kinase; PRP, platelet-rich plasma; ROS, reactive oxygen species; SNAP, S-nitroso-N-acetyl-DL-penicillamine; VASP, vasodilator-stimulated phosphoprotein.

Manuscript received 7 March 2008. Initial review completed 1 April 2008. Revision accepted 17 June 2008.

	LITERATURE CITED

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 

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