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* Divisione IV Clinica Medica,
Dipartimento di Medicina Sperimentale e Patologia,
Università di Roma "La Sapienza", Dipartimento di Medicina Interna, Università di Roma "Tor Vergata," Rome, Italy
1Correspondence: Divisione IV Clinica Medica, Dipartimento di Medicina Sperimentale e Patologia, Università di Roma "La Sapienza." Policlinico Umberto I, Rome 00185, Italy. E-mail: francesco.violi{at}uniroma1.it
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: oxidative stress • polyphenols • platelet recruitment
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Experimental studies in animals demonstrated that red wine and grape juice reduce platelet activation in stenosed canine coronary arteries (2)
and platelet-dependent mural thrombosis (3)
. Even if ethanol inhibits platelet function, this effect is not achievable with moderate alcohol consumption (4)
; therefore, other mechanisms are likely to be implicated in the antithrombotic effect of moderate consumption of wine.
Much attention has recently focused on the possibility that the cardioprotective effect of wine is dependent on polyphenols, which represent its nonalcoholic component. This suggestion is supported by several studies demonstrating an inverse association between polyphenol intake and cardiovascular events (5
, 6)
. Inhibition of platelet aggregation seems to be one mechanism through which polyphenols could reduce cardiovascular disease (7)
, however some authors questioned the antiplatelet activity exerted in vivo by these red wine components (8)
. In fact, administration of a single polyphenol such as quercetin or epigenin to healthy volunteers did not affect platelet function (8)
. This finding, however, did not exclude an antiplatelet role of polyphenols, because separating major groups of polyphenolic compounds from purple grape juice showed that only a few fractions exhibited antiplatelet activity (9)
.
The investigation of polyphenol effect on platelet function may be relevant not only in the context of the relationship between wine and cardiovascular disease but also for explaining the putative cardioprotection exerted by polyphenol-rich food. Thus several, although not all, epidemiologic studies demonstrated an inverse relationship between polyphenol intake and cardiovascular disease (10
11
12
13
14
15)
A previous study demonstrated that polyphenols inhibit platelet function by enhancing redox status but the mechanism was not fully elucidated (9)
. The balance between oxidative stress and platelet production of NO has a key role in the process of platelet recruitment, which is an important phase of platelet activation at the site of vascular injury (16)
. Recruitment of additional platelets on atherosclerotic plaque likely represents a crucial phase of thrombus growth. We hypothesized that the antioxidant property of polyphenols could affect the redox status of platelets and eventually retard platelet recruitment. The aim of the present study was to investigate whether polyphenols were able to affect 1) platelet recruitment, 2) the production of platelet superoxide anion (O2–) and NO, and 3) specific pathways generating platelet oxidative stress.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Preparation of platelets
Platelets were obtained from healthy donors (age between 30 and 40 years) who had provided their informed consent. Blood was taken between 8:00 a.m. and 9:00 a.m. from subjects after a 12 h fast; sampling was performed by venipuncture of the antecubital vein with minimum stasis using a G-21 needle. The sample was immediately mixed with 0.13 mM of sodium citrate (ratio 9:1). Blood samples were processed within 2 h of sampling. To obtain platelet-rich plasma (PRP) the samples were centrifuged at 160 g for 15 min and PRP was washed twice using acid/citrate/dextrose (a-chlorohydrin) (1:7 v/v) to avoid platelet activation. Plasma was discarded and the resulting platelets were resuspended in Ca2+-free Tyrode’s buffer containing 10 mM HEPES (pH 6.5).
The mean sample vol for all experiments was 300 µl and agonist and or antagonist vol was in the range of 1–10 µl, which did not affect platelet count. Platelet counts and complete blood cell counts were determined using a routine hematology cell counter. Red cell contamination of PRP was lower than 1% (RBC/platelets), while leukocyte contamination was lower than 0.2% (WBC/platelets).
PFA-100k closure time
In all PFA-100k determinations, we used collagen/ADP cartridges. Briefly, blood samples were incubated for 30 min at 37°C with diphenyleneiodonium chloride (DPI) (10 µM), apocynin (100 µM), morpholinosidonimine (SIN1) (300 µM), vitamin C (50 µM), nitro-L-arginine methyl ester (L-NAME) (300 µM), sodium nitroprusside (SNP) (5 µM), the cGMP inhibitor LY83583 (5 µM), catechin (6–100 µM), quercetin (1.25–20 µM), or mix (catechin 6–100 µM + quercetin 1.25–20 µM) before activation. Solvents were used as controls. The samples were then subjected to the determination of closure time in collagen/ADP (CTCADP) cartridge (17)
.
Platelet recruitment
Platelet recruitment was performed according to the method described by Freedman and colleagues (16)
. Samples were incubated (30 min, 37°C) with DPI (10 µM), apocynin (100 µM), SIN1 (300 µM), vitamin C (50 µM), L-NAME (300 µM), SNP (5 µM), the cGMP inhibitor LY83583 (5 µM), catechin (6–100 µM), quercetin (1.25–20 µM), or mix (catechin 6–100 µM + quercetin 1.25–20 µM) before activation with collagen (7 µg/ml). Solvents were used as controls.
In some experiments catechin and quercetin were removed from the supernatant after a 30 min incubation to evaluate the recovery of platelet reactivity. Collagen-induced platelet aggregation was measured for 10 min. Then an equal portion of untreated platelets was added to each tube, which increased the density of the solution and hence caused a reduction in light transmission. Aggregation of the newly added platelet portion in the presence of an existing aggregate was then measured for 5 min and expressed as percentage of the aggregation that had been reached initially.
Aggregation was measured according to Born’s method (18)
and calculated as light transmission difference (LT%) between PRP and platelet poor plasma (PPP) as described (19)
.
Platelet primary aggregation
Platelet primary aggregation was measured according to Born’s method (18)
and calculated as light transmission difference (LT%) between PRP and platelet poor plasma (PPP) as described previously (19)
.
Samples were incubated (30 min, 37°C) with catechin (6–100 µM), quercetin (1.25–20 µM), or mix (catechin 6–100 µM + quercetin 1.25–20 µM) before activation with collagen (7 µg/ml). Solvents were used as controls. In some experiments catechin and quercetin were removed from the supernatant after a 30 min incubation to evaluate the recovery of platelet reactivity.
Superoxide anion evaluation
Superoxide anion (O2–) production was measured by lucigenin (5 µM) probe in a chemoluminescence system as previously reported (20)
. Samples were incubated for 30 min, at 37°C with catechin (6–100 µM), quercetin (1.25–20 µM), or mix (catechin 6–100 µM + quercetin 1.25–20 µM) before activation with collagen 7 µg/ml. Solvents were used as controls. In some experiments catechin and quercetin were removed from the supernatant after 30 min incubation to evaluate the recovery of platelet reactivity. O2– production was expressed as Stimulation Index (SI=mean concentration of stimulated platelet luminescence/average concentration of luminescence in unstimulated platelets).
NO measurement
A colorimetric assay kit was used to determine NO metabolites nitrite and nitrate in the supernatant of platelets (3x108/ml) activated at 37°C for 10 min. Samples were incubated for 30 min at 37°C with catechin (6–100 µM), quercetin (1.25–20 µM), or mix (catechin 6–100 µM + quercetin 1.25–20 µM) before activation with collagen (7 µg/ml). Solvents were used as controls. In some experiments catechin and quercetin were removed from the supernatant after 30 min incubation to evaluate the recovery of platelet reactivity.
Platelet NADPH oxidase activity
Measurement of platelets NADPH oxidase activity was performed in platelet homogenates as described previously (20)
. Briefly, washed platelets were suspended in a homogenic buffer containing: 50 mM Tris/HCl (ph 7.4), 1.0 mM EDTA, 2.0 mM leupeptin and 2.0 mM pepsatin A, then homogenized. Platelet homogenates were incubated for 10 min at 37°C° with 25 µM NADPH and added with or without catechin (25 µM), quercetin (5 µM), or catechin (25 µM) + quercetin (5 µM). The assay solution contained 400 µl Tyrode buffer and 0.5 µM lucigenin. The reaction was started by adding 100 µl of platelet homogenates to the assay solution in the presence or absence of arachidonic acid (AA) 0.5 mM.
The chemiluminescent signal was calculated as counts per minute (cpm) for an average of 10 min and corrected by protein concentration (cpm/mg). Values were expressed as relative chemiluminescence units (20)
.
In vitro phosphorylation of platelet proteins
The platelet suspensions (2x109/ml) were incubated for 1 h at 37°C with 32Pi (2 µCi/ml of cell suspension), separated from plasma proteins and from excess of 32Pi by centrifugation and suspended in Tyrode’s buffer containing 0.2% BSA, 5 µM glucose and 10 mM HEPES, pH 7.35. The final concentration of platelets was adjusted at 2 x 108 cells/ml.
32P-labeled platelets were incubated 30 min at 37°C with or without catechin (25 µM), quercetin (5 µM), or catechin (25 µM) + quercetin (5 µM), then stimulated with collagen (7 µg/ml). The reaction was stopped by addition of an equal vol of twice concentrate Laemli’ buffer, followed by incubation at 95C° for 5 min.
Protein samples were analyzed by 12% sodium dodecyl sulfate-polycrylamide gel electrophoresis (SDS-PAGE); for Western blot, proteins were electrotransferred to nitro-cellulose membranes.
The rate of protein kinase C (PKC) phosphorylation (expressed as phosphorylation of 47-Kda PKC specific substrate) was analyzed by autoradiography. The developed spots were calculated by densitometric analysis on an NIHimage 1.62f analyzer. The amount of phosphorylation was determined by dividing the areas of the phosphorylated spots of stimulated platelets by the area of unstimulated platelets. The value was expressed as decrement percentage of phosphorylation (%) (20)
.
Flow cytometry analysis of PAC 1
PAC 1 is an antibody (Abs) that recognizes an epitope on the glycoprotein (GP) IIb/IIIa of activated platelets at/or near the platelet fibrinogen receptor.
PAC 1 binding on platelets membrane was analyzed using the specific FITC-labeled monoclonal antibodies anti PAC1(Mab). All assays included samples to which an irrelevant isotype-matched Ab (FITC-labeled IgM) was added.
Platelet suspension (200 µl, 2x108/ml) was incubated 30 min at 37°C with or without L-NAME(300 µM), catechin (6–100 µM), quercetin (1.25–20 µM), or catechin (6–100 µM) + quercetin (1.25–20 µM) and stimulated with collagen (7 µg/ml). In some experiments catechin and quercetin were removed from the supernatant after a 30 min incubation to evaluate the recovery of platelet reactivity. Platelets were fixed with (2%) paraphormaldeide (0.1% BSA) for 60 min at room temperature; the suspension was treated with Mab (20 µl) for 60 min at 4C°. The unbound Mab was removed by centrifugation at 300 g for 3 min (twice) after the addition of PBS (0.1% BSA). Fluorescence intensity was analyzed on an Epics XL-MCL Cytometer (Coulter Electronics, FL, USA) equipped with an argon laser at 488 nM. For every histogram, 50,000 platelets were counted to evaluate the percentage of positive platelets. Ab reactivity is reported as mean fluorescence intensity.
Statistical analysis
Data are reported as mean ± SD. The comparison between variables was analyzed by Student’s t test for unpaired data.
When comparing >2 groups, 2-way ANOVA test followed by Kruskall-Wallis as a post hoc test was used. Correlation analysis was performed by Pearson’s correlation coefficient. Significance was accepted at a P < 0.05 concentration
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Both assays were sensitive to oxidative stress: thus, platelet recruitment was directly correlated(r=0.51 P=0.02) and closure time by PFA100 inversely correlated(r=–0.52 P=0.02) with platelet O2– formation (Fig. 2
A, B).
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The rate of platelet recruitment was inversely related to platelet NO (r=–0.54 P=0.001) (Fig. 2C
); conversely closure time by PFA100 was directly related to platelet NO(r=0.45 P=0.04) (Fig. 2D
). An inverse correlation between platelet NO and O2– production was observed (n=20, r=–0.9 P<0.01).
Incubation of platelets with two inhibitors of NADPH oxidase, namely DPI and apocynin, significantly reduced platelet recruitment (–83% and –90%, respectively) and prolonged closure time measured by PFA100(+58% and+30%, respectively) (Fig. 3
A, B). Similar findings were obtained in samples incubated with two NO donors, SIN1 and SNP (–63% and –65%, respectively, for platelet recruitment; +90% and+87%, respectively, for closure time measured by PFA100), and the antioxidant ascorbic acid (vitamin C) (–85% for platelet recruitment and +58% for closure time measured by PFA100), (Fig. 3A, B
). Inhibition of platelet recruitment and prolongation of closure time by PFA100 induced by SNP was counteracted by platelet incubation with LY83583, an inhibitor of cGMP (+80% and –39%, respectively) (Fig. 3A, B
). Finally, incubation of platelet with the NO synthase inhibitor L-NAME significantly enhanced platelet recruitment and reduced the closure time by PFA100 (+10% and –29%, respectively) (Fig. 3A, B
). Figure 3C
showed a representative tracing of recruitment in platelets treated or not with L-NAME, apocynin, DPI, or superoxide dismutase.
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Effect of polyphenols on platelet function
Incubation of platelets with quercetin or catechin alone did not influence platelet recruitment compared to control (Fig. 4
A). Conversely in samples added with both quercetin and catechin platelet recruitment was significantly inhibited depending on the concentration used (Fig. 4A
). Removing catechin and quercetin from the sample after the incubation period did not reduce their effect on platelet recruitment (not shown). Similar findings were obtained with PFA100; closure time was unaffected in samples incubated with quercetin or catechin alone (Fig. 4B
); conversely, closure time was significantly prolonged in samples with added quercetin and catechin in a dose-dependent manner (Fig. 4B
).
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Platelet primary aggregation induced by collagen (7 µg/ml) was unaffected by treatment with single polyphenol; a combination of as high as 10 µM quercetin and 50 µM catechin resulted in weak but significant (–21%, P<0.004) reduction of platelet primary aggregation.
To see whether platelet NO and polyphenols inhibited platelet function via modulating GP IIb/IIIa, collagen-induced platelet GP IIb/IIIa, conformational change was measured by PAC 1 in samples added with and without L-NAME or polyphenols. Incubation of platelets with L-NAME resulted in a significant increase of GP IIb/IIIa expression (Fig. 4D
).
Single polyphenol did not influence PAC1 binding; conversely, platelet PAC1 binding was significantly inhibited in platelets added with both quercetin and catechin depending on the concentration used (Fig. 4C
). Removing catechin and quercetin form the sample after the incubation period did not reduce their effect on platelet PAC1 binding.
Relationship between inhibition of platelet function by polyphenols and NO/O2– production.
First we analyzed whether polyphenols influenced platelet NO production in response to collagen. Single polyphenol did not significantly influence platelet NO (Fig. 5
A); conversely platelet NO production was significantly increased in platelets added with both quercetin and catechin depending on the concentration used.
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We also examined whether the two polyphenols influenced intraplatelet oxidative stress by measuring platelet O2– formation elicited by collagen. While quercetin and catechin alone did not affect platelet O2–, incubation of platelets with both quercetin and catechin resulted in a significant inhibition of platelet O2– production depending on the concentration used (Fig. 5B
). Removing catechin and quercetin form the sample after the incubation period did not reduce their effect on platelet production of both NO and O2–.
An inverse correlation between the reduction of O2– and the increase of NO was observed in poliphenol-treated platelets (r=–0.6, P=0.02)
To investigate the role of NO on the inhibition of platelet recruitment elicited by polyphenols, the experiment was repeated in the presence or absence of L-NAME. The study showed that L-NAME significantly counteracted the inhibition of platelet recruitment when polyphenols were used at low concentration; at high polyphenol concentration the inhibition of platelet recruitment persisted despite L-NAME treatment (Fig. 5C, D
).
Effect of polyphenols on platelet NADPH oxidase
As the above reported findings suggested that polyphenols could interfere with the production of 02–, we investigated whether quercetin and catechin, alone or in combination, affected the activity of platelet NADPH oxidase, which has a key role in the platelet production of 02– (21)
.
Compared to controls, incubation of platelets with quercetin or catechin alone did not influence platelet NADPH oxidase activity (Fig. 6
A); conversely in samples added with both quercetin and catechin platelet NADPH oxidase activity was significantly inhibited.
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As PKC has a prominent role on NADPH oxidase activation (22)
we investigated whether polyphenols affected PKC activity. Compared to control, incubation of platelets with quercetin or catechin alone did not influence platelet PKC activation (Fig. 6B
). In samples added with both quercetin and catechin, platelet PKC activation was almost completely suppressed (–94±6.6%, P<0.001 vs. collagen-stimulated platelet) (Fig. 6B
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Addition of polyphenols to platelets elicited profound functional changes consisting of marked reduction of platelet recruitment depending on the concentration of polyphenols used. However, a single polyphenol had weak inhibitory effect on platelet recruitment only when used at high concentration. Conversely, a combination of concentrations as low as 1 µM quercetin plus 6 µM catechin (Fig. 4A
) resulted in a significant inhibition of platelet recruitment. The concentration of polyphenols in human circulation is dependent on the source of polyphenols. In red wine, for instance, quercetin ranges from 5 to 104 µM and catechin from 172 to 645 µM (23)
. After wine intake the polyphenol concentration in human plasma is usually <1 µM, but may reach much higher values with other polyphenol-rich food; in the case of quercetin and catechin, the concentrations achievable in human blood may reach values close to 2 and 5 µM, respectively (24)
. That fact that we found a synergism between quercetin and catechin within a range of concentration potentially achievable in human circulation could be useful for explaining the putative beneficial effect of polyphenol-rich food against cardiovascular disease. These data support our hypothesis suggesting that the antiplatelet effect of polyphenol-rich food is likely to occur as a consequence of a synergism among the polyphenols (18)
.
NO and O2– produced by activated platelets have an opposite effect on platelet recruitment: thus, O2– production enhances and NO decreases platelet recruitment (16
,25)
. In accordance with these findings we observed that both methods exploring platelet activation were sensitive to O2–/NO production. In fact, incubation of platelets with an inhibitor of NADPH oxidase or with an inhibitor of NO synthase resulted in reduced and enhanced platelet recruitment, respectively. A previous study demonstrated that polyphenols inhibit platelet recruitment with a mechanism involving increased release of NO (9)
. Even whether polyphenols have been shown to enhance NO synthase (NOS) activation (26
, 27)
, this does not seem to occur in platelets where polyphenols did not affect NOS (9)
. Another attractive possibility is that the increase of platelet NO is a result of platelet O2– inhibition. Therefore, as O2– rapidly interacts with NO to form peroxynitrite, the inhibition of platelet O2– by polyphenols could enhance NO bioactivity and eventually retard platelet recruitment.
Several studies have shown that polyphenols possess an antioxidant effect including the inhibition of NADPH oxidase activation (28
29
30
31)
, which is the most important cellular producer of O2– (21)
. However, it has never been investigated whether polyphenols decrease the activation of platelet NADPH oxidase. The role of this enzymatic pathway in generating platelet O2– was recently supported by our group. We showed that in patients with hereditary deficiency of gp91phox, the central core of NADPH oxidase, the platelet production of O2– was almost completely absent (21)
. According to our previous study (20)
, incubation of platelet with NADPH enhanced platelet arachidonic acid induced O2– production. The addition of quercetin and catechin to platelet sample dose-dependently inhibited the formation of O2–, suggesting that polyphenols reduced the activation of this enzyme. To investigate the mechanism through which polyphenols inhibited the platelet NADPH oxidase, we tested in vitro whether they influenced the activation of PKC. A previous study demonstrated, in fact, that this enzyme activates NADPH oxidase via phosphorylation of p47 phox (32). Moreover, we have already shown that, in platelets, PKC activation has a pivotal role in inducing arachidonic acid-mediated NADPH oxidase activation (20)
. Therefore, we investigated whether polyphenols inhibited the activation of PKC and demonstrated that the combination of quercetin with catechin exerted an inhibitory effect on the activation of this enzyme.
Taken together, these data show that polyphenols exert an antioxidant effect via inhibition of O2– generated by NADPH oxidase, and suggest that this effect could result in enhanced NO bioavailability. Consistent with this hypothesis, polyphenols on the one hand reduced O2– and, on the other, increased NO platelet formation, with a significant inverse correlation between these two variables.
These changes could help explain the antiplatelet effect of polyphenols, because O2–/NO platelet production is essential for platelet recruitment via modulation of platelet GP IIb/IIIa expression. Thus, Begonja et al. (33)
recently showed that NADPH oxidase inhibitors retard thrombus growth via down-regulation of platelet GP IIb/IIIa, suggesting that O2– overexpresses this membrane glycoprotein. Platelet NO has an opposite effect, as it down-regulates the platelet expression of GP IIb/IIIa (34
, 35)
. Accordingly, NO bioactivity is essential for fibrinogen binding platelets, as shown by demonstrating that low NO activity is associated with enhanced fibrinogen binding to GP IIb/IIIa (34
, 35)
. Based on these findings, we speculated that polyphenols could interfere with platelet recruitment via down-regulation of GP IIb/IIIa. To explore this issue, we investigated whether polyphenols influenced the PAC1 binding to platelets and demonstrated that it was significantly inhibited by the combination of quercetin with catechin, indicating that down-regulation of platelet IIb/IIIa had a pivotal role in the inhibition of platelet recruitment elicited by polyphenols.
Our study has some critical issues that deserve consideration. First, NO2/NO3 may also stem from NO interaction with O2– and potentially be affected by O2– decrease; however, NO2 and NO3 can also stem from NO interaction with other molecules and, in the case of O2– lowering, can be formed through alternative pathways (36)
. The inverse correlation between platelet NO2/NO3 and O2– favors this hypothesis and suggests that, in platelets, the increase of N02/NO3 could reflect an enhanced NO bioavailability.
Second, platelet NO seems be implicated in the retarded platelet recruitment elicited by polyphenols, because treatment of platelet with L-NAME reduced the antiplatelet effect of polyphenols. However, L-NAME partially counteracted the antiplatelet effect of polyphenols, suggesting that the increase of platelet NO was not fully responsible for the retarded recruitment elicited by polyphenols. Inhibition of O2– per se could offer an alternative explanation as it is able to overexpress platelet GP IIb/IIIa independent of NO scavenging (33)
. Further study is required to investigate the interplay between O2– and NO in the inhibition of platelet recruitment elicited by polyphenols.
Third, we cannot exclude that polyphenols inhibit platelet recruitment with mechanisms independent of antioxidant activity. For instance, PKC is able to activate platelet function via pleckstin-dependent cytoskeleton reorganization (37)
; therefore, it is possible that polyphenols also retard platelet recruitment by affecting this pathway. However, our experimental model was sensitive to intraplatelet redox status, so it is arguable that the effect of polyphenols on platelet NO and O2– production has a pivotal role in inhibiting platelet recruitment.
In conclusion, our study demonstrated that quercetin and catechin synergistically act in inhibiting platelet recruitment with a mechanism involving NO and O2– platelet production. Such an antiplatelet effect could further contribute to explain the mechanism through which polyphenols reduce atherosclerotic disease in animals and humans.
Received for publication November 7, 2005. Accepted for publication February 12, 2006.
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REFERENCES |
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IIbß3-integrin activation independent of the NO/cGMP pathway. Blood 15,2757-2760This article has been cited by other articles:
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