There is growing evidence that platelets play an important role in atherogenesis, plaque formation and plaque rupture. In clinical practice, inhibition of platelet activity with aspirin significantly reduces the odds of serious atherothrombotic vascular events and death in patients at high risk.1,2 However, in many patients aspirin is not effective. Among the many possible causes of aspirin (ASA) treatment failures, ASA resistance (ASA-R) emerges as a major therapeutic challenge. Clinical studies showed that there is a significant correlation between ASA resistance (diagnosed as insufficient inhibition of platelet aggregation by ASA) and myocardial infarction (MI), or cerebrovascular accident in patients with stable cardiovascular disease.3,4 Still, the etiology of ASA resistance is poorly understood. Because ASA nonresponder status may contribute to the failure of ASA therapy in the secondary prevention of cardiovascular and cerebrovascular incidents in as many as 30% to 40 % of patients, it is important to identify alternative pharmacological treatments that effectively inhibit aggregation of ASA-R platelets.1,2,5
Epidemiological studies suggest that Mediterranean diets are associated with a reduced risk of cardiovascular disease.6,7 It has been proposed that resveratrol is one of the most important dietary constituents involved in vasculoprotection. Resveratrol is one of a group of compounds called phytoalexins that are produced in plants during times of environmental stress such as microbial (fungal) infection or UV irradiation.8 Resveratrol has been identified in >70 species of plants, including grapevines (Vitis vinifera), mulberries, eucalyptus, and peanuts. Fresh grape skin contains ≈50 to 100 μg/g of resveratrol, whereas red wine concentrations range from ≈0.1 to 14 mg/L (highest concentrations reported in wines prepared from pinot noir grapes).9,10 The relatively high concentrations of resveratrol (and likely other polyphenols) in red wine is thought to be the explanation for the so-called “French paradox,”11,12 the unexpectedly low cardiovascular morbidity in Mediterranean populations. In line with this notion, epidemiological studies have linked moderate intake of resveratrol-containing red wine with a significant decrease in the risk of coronary artery disease.
Resveratrol and other natural polyphenol compounds are thought to have diverse antiatherogenic activities,13-17 such as the inhibition of low-density lipoprotein oxidation, regulation of vascular smooth muscle proliferation, and modulation of NO production. Importantly, previous studies suggested that resveratrol may suppress platelet aggregation.18-22 One can speculate that this action may be responsible, at least in part, for the protection against recurrent MI.
On the basis of the aforementioned studies we hypothesized that resveratrol is able to inhibit aggregation of platelets in high-risk, ASA-R cardiovascular patients. To test the hypothesis platelet-rich plasma (PRP) was isolated from ASA-sensitive (ASA-S) and ASA-R patients (ASA resistance was defined as higher-than-expected aggregation to collagen and epinephrine after oral treatment with 100 mg/day ASA) and aggregation to adenosine diphosphate (ADP), collagen, and epinephrine was measured in the absence and presence of resveratrol.
MATERIALS AND METHODS
Patients hospitalized after MI or with stable or unstable angina pectoris, peripheral artery disease, and/or scheduled for cardiac catheterization as potential candidates for percutaneous coronary intervention were eligible for the study if they were undergoing treatment with ASA (≥100 mg/d). We did not include patients with acute MI according to the American Heart Association/American College of Cardiology criteria, patients with HIV, patients with chronic oral anticoagulation, and patients with contraindication to ASA. All patients gave written informed consent, and our institutional ethics committee approved the study.
Study Protocol and Platelet Function Assays
After inclusion in the study, blood was drawn for platelet function assays with tubes that contained 3.8% sodium citrate. Platelet function was evaluated ex vivo by optical aggregometry (TX-4 aggregometer, CARAT Ltd, Budapest, Hungary) with 2 concentrations of ADP (5 and 10 μmol/L), collagen (2 μg/mL), and epinephrine (10 μmol/L) in the absence and presence of resveratrol (10−5 mol/L) according to the modified protocols of Ungvari et al,23 with PRP adjusted to 275 to 325 × 103 platelets/μL. The coefficient of variation of this optical aggregometry assay is <6% after stimulation with ADP. ASA resistance was defined as higher-than-expected aggregation to collagen and epinephrine (≥40%) after oral treatment with 100 mg/d ASA. All drugs and chemicals were purchased from Sigma Chemical Company (St. Louis, MO).
For all statistical analyses, we used the GraphPad Prism software package, version 3.02. Discrete variables are reported as counts (percentages) and continuous variables as mean ± SD. We tested differences between groups with the χ2 test for discrete variables and with 1-way ANOVA followed by a Scheffé test for continuous variables.
The present study included 50 high-risk cardiac patients. Table 1 shows the baseline demographic and clinical characteristics of the study cohort.
ASA-R was defined as higher- than-expected aggregation to collagen and epinephrine (≥40%). Using this criteria 38% of our patients were diagnosed as ASA-R. ADP-induced platelet aggregation was only slightly elevated in ASA-R patients. Maximal aggregation to 5 μmol/L ADP was only slightly affected by resveratrol (Fig. 1A). Similar results were obtained using 10 μmol/L ADP (Fig. 1B). In contrast, maximal aggregation of ASA-R platelets to collagen was significantly decreased by resveratrol, whereas resveratrol had only marginal effects on ASA-S platelets (Fig. 1C). Resveratrol also significantly attenuated maximal aggregation of ASA-R platelets to epinephrine (Fig. 1D). ASA-R patients had a significantly higher level of platelet inhibition than did ASA-S patients (Fig. 1D). There was a correlation between the magnitudes of collagen- and epinephrine-induced aggregation, whereas collagen-induced aggregation did not correlate with ADP-induced responses. By all platelet function tests, we did not find any significant correlation between age, plasma triglyceride, and cholesterol concentration or any other variables shown in Table 1 and platelet aggregability.
Our study investigated the antiplatelet effects of resveratrol in a cohort of 50 high-risk cardiac patients. The major finding of this study is that resveratrol can inhibit aggregation of platelets from high-risk cardiac patients (Fig. 1). The mechanisms by which resveratrol exerts its antiplatelet effects are likely multifaceted. Resveratrol was shown to inhibit cyclooxygenase-1 (COX-1),24 which likely contributes to its ASA-like effects. Structure-activity relationships on methoxy-resveratrol analogs showed that the m-hydroquinone moiety is essential for irreversible inactivation of COX-1.24 Indeed, resveratrol (similar to ASA) exerts a marked inhibitory effect on collagen- and epinephrine-induced aggregation of human platelets (Fig. 1). Although both ASA and resveratrol inhibit ADP-induced platelet responses, this inhibition is less than their effect on collagen-induced responses.25-27 The likely explanation for these phenomena is that collagen- and epinephrine-induced aggregation depends on the COX-1/thromboxane A2 synthase axis, whereas this pathway plays a lesser role in ADP-induced signaling. Other potential mechanisms of the antiplatelet action of resveratrol involves inhibition of signal transduction pathways including mitogen-activated protein kinases18 and polyphosphoinositide signaling.22
It is significant that resveratrol is able to effectively inhibit aggregation of platelets from patients with ASA resistance (Fig. 1C, D). Interestingly, this inhibition is greater compared to that observed with platelets from control patients. ASA resistance is significantly associated with an increased risk of death, MI, or cerebrovascular accident compared with ASA-S patients.1,2,5 Recent studies suggest ASA resistance may promote plaque-associated thrombus formation in patients with coronary artery and cerebrovascular disease. In ASA-R patients an increased platelet activity also results in the release of prothrombotic and proinflammatory microvesicles (“platelet microparticles”) and likely promotes the development of atherosclerosis. Our study suggests that ASA nonresponders may obtain significant benefits from an increased resveratrol intake with respect to cardiovascular events.
The pathological mechanisms underlying ASA resistance are likely multifactorial.28 Previous studies suggested that increased oxidative stress-related increased COX-independent isoprostane formation,29 cigarette smoking, increased levels of norepinephrine caused by extended periods of mental stress, decreased bioavailability of ASA, and/or increased platelet sensitivity to collagen may contribute to clinical ASA resistance. It was also proposed that ASA may not be cardioprotective in patients with hyperlipidemia30; however, our data did not reveal a correlation between plasma lipids and platelet aggregability (Table 1). A contributing factor may be age itself. Our data show that 26% of high-risk cardiac patients under age 60 (5/19) was resistant to ASA treatment. In contrast, among older adults (>60 years old) 45% of patients (14/31) exhibited ASA resistance.
We have used citrate as an anticoagulant, based on protocols used in previous studies.3,31-33 Although lowering calcium levels may affect, at least in theory, platelet responses from certain species (eg, aggregation of rabbit platelets are more calcium-dependent), citrate-treated human platelets aggregate well in response to ADP, collagen, and epinephrine. Because in our study PRP preparation was performed identically in all cases, the effects of resveratrol could be compared between ASA-S and ASA-R groups.
We have not demonstrated the actual bioavailability of resveratrol in this study. Because the amounts of resveratrol used for research are higher than that found in wine further studies are definitely needed when both ASA and resveratrol are given orally to patients.
Human studies and animal experiments suggest that resveratrol is readily absorbed from the gastrointestinal tract. However, the oral bioavailability of free resveratrol seems to be small because of rapid and extensive metabolism and the consequent formation of various metabolites as resveratrol glucuronides and resveratrol sulfates. Although resveratrol conjugates are likely to exhibit biological activities similar to resveratrol, further bioequivalence studies are needed. It is also important to note that food that is rich in resveratrol (eg, grapes, wine, berries, peanuts, peanut products) also contain different polyphenol compounds (including trans-astringin, trans-piceid, trans-resveratrol, ε-viniferin, trans-δ-viniferin ),34,35 which are likely to exert similar biological effects. Recent studies estimate that people eating a Mediterranean diet consume ≈6 mg/d stilbenes from red wine alone.34,35 Thus, one can assume that in humans postprandial plasma levels of resveratrol, resveratrol metabolites, and other polyphenols overlap with the antiaggregatory concentrations of these compounds. Our findings encourage the search for structurally related compounds with good bioavailability for clinical use in high-risk cardiac patients with ASA resistance. In addition to resveratrol, trans-piceid and δ-viniferin also contribute to a significant proportion of stilbenes in wine dietary intake, but their effects on ASA-R platelets have not yet been investigated. Other polyphenols that may exert biological effects similar to resveratrol include various anthocyanins and flavonols (eg, quercetin, myricetin, kaempferol).36 Dietary counseling to promote the consumption of polyphenol-containing foods could be a part of cardiac rehabilitation programs37 initiated to achieve coronary risk reduction in these patients.
Collectively, dietary intake of resveratrol and related polyphenols is likely to exert significant cardioprotective effects in part by inhibiting platelet aggregation. We propose that high-risk cardiovascular ASA-R patients will especially benefit from resveratrol consumption.
We thank the 2 anonymous reviewers for their helpful suggestions on our article.
1. Antithrombotic-Trialists'-Collaboration. Collaborative meta-analysis of randomised trials of antiplatelet therapy for prevention
of death, myocardial infarction, and stroke in high risk patients. BMJ
2. Altman R, Luciardi HL, Muntaner J, et al. The antithrombotic profile of aspirin. Aspirin resistance, or simply failure? Thromb J
3. Gum PA, Kottke-Marchant K, Welsh PA, et al. A prospective, blinded determination of the natural history of aspirin resistance among stable patients with cardiovascular disease. J Am Coll Cardiol
4. Gum PA, Kottke-Marchant K, Poggio ED, et al. Profile and prevalence of aspirin resistance in patients with cardiovascular disease. Am J Cardiol
5. Grundmann K, Jaschonek K, Kleine B, et al. Aspirin non-responder status in patients with recurrent cerebral ischemic attacks. J Neurol
6. Keys A, Menotti A, Karvonen MJ, et al. The diet and 15-year death rate in the seven countries study. Am J Epidemiol
7. de Lorgeril M, Salen P, Martin JL, et al. Mediterranean diet, traditional risk factors, and the rate of cardiovascular complications after myocardial infarction: final report of the Lyon Diet Heart Study. Circulation
8. Versari A, Parpinello GP, Tornielli GB, et al. Stilbene compounds and stilbene synthase expression during ripening, wilting, and UV treatment in grape cv. Corvina. J Agric Food Chem
9. Mark L, Nikfardjam MS, Avar P, et al. A validated HPLC method for the quantitative analysis of trans-resveratrol and trans-piceid in hungarian wines. J Chromatogr Sci
10. Pervaiz S. Resveratrol: from grapevines to mammalian biology. FASEB J
11. Kopp P. Resveratrol, a phytoestrogen found in red wine. A possible explanation for the conundrum of the “French paradox”? Eur J Endocrinol
12. Zern TL, Fernandez ML. Cardioprotective effects of dietary polyphenols. J Nutr
13. Wang Z, Zou J, Cao K, et al. Dealcoholized red wine containing known amounts of resveratrol suppresses atherosclerosis
in hypercholesterolemic rabbits without affecting plasma lipid levels. Int J Mol Med
14. Wang Z, Zou J, Huang Y, et al. Effect of resveratrol on platelet aggregation in vivo and in vitro. Chin Med J (Engl)
15. Zou J, Huang Y, Chen Q, et al. Effects of resveratrol on oxidative modification of human low density lipoprotein. Chin Med J (Engl)
16. Wang Z, Huang Y, Zou J, et al. Effects of red wine and wine polyphenol resveratrol on platelet aggregation in vivo and in vitro. Int J Mol Med
17. Zou J, Huang Y, Cao K, et al. Effect of resveratrol on intimal hyperplasia after endothelial denudation in an experimental rabbit model. Life Sci
18. Kirk RI, Deitch JA, Wu JM, et al. Resveratrol decreases early signaling events in washed platelets but has little effect on platalet in whole food. Blood Cells Mol Dis
19. Olas B, Wachowicz B. Resveratrol and vitamin C as antioxidants in blood platelets. Thromb Res
20. Olas B, Wachowicz B, Saluk-Juszczak J, et al. Effect of resveratrol, a natural polyphenolic compound, on platelet activation induced by endotoxin or thrombin. Thromb Res
21. Olas B, Wachowicz B. Resveratrol, a phenolic antioxidant with effects on blood platelet functions. Platelets
22. Olas B, Wachowicz B, Holmsen H, et al. Resveratrol inhibits polyphosphoinositide metabolism in activated platelets. Biochim Biophys Acta
23. Ungvari Z, Sarkadi-Nagy E, Bagi Z, et al. Simultaneously increased TxA2
activity in isolated arterioles and platelets of rats with hyperhomocysteinemia. Arterioscler Thromb Vasc Biol
24. Szewczuk LM, Forti L, Stivala LA, et al. Resveratrol is a peroxidase-mediated inactivator of COX-1 but not COX-2: a mechanistic approach to the design of COX-1 selective agents. J Biol Chem
25. Kuster LJ, Frolich JC. Platelet aggregation and thromboxane release induced by arachidonic acid, collagen, ADP and platelet-activating factor following low dose acetylsalicylic acid in man. Prostaglandins
26. Rinder CS, Student LA, Bonan JL, et al. Aspirin does not inhibit adenosine diphosphate-induced platelet alpha-granule release. Blood
27. Rupprecht HJ, Darius H, Borkowski U, et al. Comparison of antiplatelet effects of aspirin, ticlopidine, or their combination after stent implantation. Circulation
28. De Gaetano G, Cerletti C. Aspirin resistance: a revival of platelet aggregation tests? J Thromb Haemost
29. Csiszar A, Stef G, Pacher P, et al. Oxidative stress-induced isoprostane formation may contribute to aspirin resistance in platelets. Prostaglandins Leukocyte Essent Fatty Acids
30. Friend M, Vucenik I, Miller M. Research pointers: platelet responsiveness to aspirin in patients with hyperlipidaemia. BMJ
31. Borna C, Lazarowski E, van Heusden C, et al. Resistance to aspirin is increased by ST-elevation myocardial infarction and correlates with adenosine diphosphate levels. Thromb J
32. Zimmermann N, Kurt M, Wenk A, et al. Is cardiopulmonary bypass a reason for aspirin resistance after coronary artery bypass grafting? Eur J Cardiothorac Surg
33. Steinhubl SR, Varanasi JS, Goldberg L. Determination of the natural history of aspirin resistance among stable patients with cardiovascular disease. J Am Coll Cardiol
34. Vitrac X, Castagnino C, Waffo-Teguo P, et al. Polyphenols newly extracted in red wine from southwestern France by centrifugal partition chromatography. J Agric Food Chem
35. Vitrac X, Bornet A, Vanderlinde R, et al. Determination of stilbenes (delta-viniferin, trans-astringin, trans-piceid, cis- and trans-resveratrol, epsilon-viniferin) in Brazilian wines. J Agric Food Chem
36. Padilla E, Ruiz E, Redondo S, et al. Relationship between vasodilation capacity and phenolic content of Spanish wines. Eur J Pharmacol
37. Giannuzzi P, Saner H, Bjornstad H, et al. Secondary prevention
through cardiac rehabilitation: position paper of the Working Group on Cardiac Rehabilitation and Exercise Physiology of the European Society of Cardiology. Eur Heart J