Chocolate: Modern Science Investigates an Ancient Medicine

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1 Chocolate: Modern Science Investigates an Ancient Medicine Cocoa and Wine Polyphenols Modulate Platelet Activation and Function 1 Dietrich Rein,* Teresa G. Paglieroni, Debra A. Pearson,* Ted Wun, ** Harold H. Schmitz, Robert Gosselin and Carl L. Keen* 2 *Department of Nutrition, University of California, Davis, Davis, CA 95616; Sacramento Medical Foundation, Center for Blood Research, Sacramento, CA; **Division of Hematology and Oncology and Department of Pathology, University of California Davis Medical Center, Sacramento, CA and Mars Incorporated, Hackettstown, NJ ABSTRACT There is speculation that dietary polyphenols can provide cardioprotective effects due to direct antioxidant or antithrombotic mechanisms. We report in vitro and postingestion ex vivo effects of cocoa procyanidins, a procyanidin-rich cocoa beverage and dealcoholized red wine (DRW) on human platelet activation. In a series of in vitro studies, cocoa procyanidin trimers, pentamers or DRW (3 and 10 mol/l) were incubated with citrated peripheral whole blood in the presence and absence of platelet agonists. Platelet activation was detected using fluorescent-labeled monoclonal antibodies recognizing the fibrinogen binding conformation of GPIIb-IIIa (referred to herein as PAC-1 binding) and the activation-dependent platelet epitope CD62P (P-selectin). The percentage of CD42a-positive platelets coexpressing PAC-1 binding and/or CD62P was determined by multiparameter flow cytometry. Procyanidin trimers, pentamers and DRW added to whole blood in vitro increased PAC-1 binding and P-selectin expression. In contrast, procyanidin trimers, pentamers and DRW inhibited the platelet activation in response to epinephrine. The effects on platelet activation of cocoa beverage and DRW consumption were also studied in healthy subjects. Citrated blood was obtained before and 2 and 6 h after the ingestion of a cocoa beverage, a caffeine-containing beverage, DRW or water. Platelet activation was measured by flow cytometry. The consumption of DRW did not affect the expression of activation-dependent platelet antigens, either unstimulated or after ex vivo activation with epinephrine. However, the consumption of DRW increased PAC-1 binding in response to 100 mol/l ADP ex vivo. Cocoa consumption reduced platelet response to agonists ex vivo. The ingestion of water had no effect on platelet activation, whereas a caffeine-containing beverage augmented the response of platelets to epinephrine. In summary, select cocoa procyanidins and DRW added to whole blood in vitro increased expression of platelet activation markers in unstimulated platelets but suppressed the platelet activation response to epinephrine. In contrast, cocoa consumption suppressed unstimulated and stimulated platelet activation in whole blood. This suppressive effect observed on platelet reactivity may explain in part the reported cardioprotective effects of dietary polyphenols. J. Nutr. 130: 2120S 2126S, KEY WORDS: polyphenols red wine cocoa beverage platelet activation GPIIb-IIIa complex P-selectin thrombosis whole blood flow cytometry Regular consumption of polyphenol-rich foods is inversely associated with death from thrombotic and cardiovascular disease (CVD) 3 (Hertog et al and 1995, Keli et al. 1996, Yochum et al. 1999). The basis for this protective association is uncertain, but it has stimulated interest in the antioxidant 1 Published as part of a supplement to The Journal of Nutrition. Guest editors for the supplement publication were John W. Erdman, Jr., University of Illinois at Urbana-Champaign; Jo Wills, Mars, United Kingdom and D Ann Finley, University of California, Davis. 2 To whom reprint requests should be addressed. 3 Abbreviations used: CVD, cardiovascular disease; DRW, dealcoholized red wine; FITC, fluorescein isothiocyanate; PE, phycoerythrin; PerCP, peridinin chlorophyll protein. effects (Frankel et al. 1993) and health implications of food flavonoids (Kinsella et al. 1993, Kuhnau 1976). Red wine was the first polyphenol-rich food to be inversely associated with ischemic heart disease deaths in industrialized countries (St. Leger et al. 1979) and with deaths from coronary heart disease in segments of the French population (Renaud and de Lorgeril 1992). Cocoa is another polyphenol-rich food that has the potential to improve an individual s oxidant defense system (Rein et al. 2000a, Waterhouse et al. 1996). In addition, compounds in cocoa have other positive impacts on the immune system, such as the transcription of interleukin-2 by peripheral blood mononuclear cells (Mao et al. 1999, Sanbongi et al. 1997) /00 $ American Society for Nutritional Sciences. 2120S

2 DIETARY POLYPHENOLS AND PLATELET FUNCTION 2121S Major polyphenol components of red wine and of cocoa, including the flavonoids, catechin and epicatechin, are absorbed and rapidly metabolized (Caccetta et al. 2000, Donovan et al. 1999, Richelle et al. 1999, Waterhouse et al. 1996). Physiological effects of wine polyphenols have been ascribed to their ability to inhibit LDL oxidation in both in vitro models (Kondo et al. 1996, Teissedre et al. 1996, Waterhouse et al. 1996) and after wine consumption (Aviram et al. 1997, Fuhrman et al. 1995, Nigdikar et al. 1998, Serafini et al. 1998). Certain cocoa and chocolates contain substantial amounts of flavan-3-ol procyanidin oligomers (Lazarus et al. 1999, Porter et al. 1991), and as such the consumption of this product can contribute substantially to the total dietary polyphenol intake, in a fashion similar to tea and red wine (Arts et al. 1999). Components in cocoa can inhibit the oxidation of LDL (Kondo et al. 1996), increase the plasma total antioxidant capacity (Rein et al. 2000a) and protect against the production of excessive peroxynitrite, a potent mediator of inflammation (Arteel and Sies 1999). Although there is considerable evidence in support of the concept that CVD is due in part to excessive oxidative damage, the role of oxidative damage as a primary cause of CVD has been questioned by some investigators (Parthasarathy et al. 1999), as has the hypothesis that antioxidants in the diet can reduce CVD onset and progression (Puddey and Croft 1999). Thus, it is important to consider the possibility that dietary plant polyphenols may have cardioprotective effects other than, or in addition to, that offered by their well known potential to act as antioxidants. In this regard, it has been suggested that some polyphenolics may affect thrombosis and CVD by interfering with platelet activation and function (Keevil et al. 2000, Maalej et al. 1997). Ruff (1999) has argued that only 50% of the antithrombotic effect of alcoholic beverages, including red wine, can be attributed to their HDL-raising effect and that the other 50% is related to decreased platelet activity. Blood platelets play a major role in CVD and thrombosis (Osterud 1997, White 1994). The GPIIb-IIIa complex mediates platelet aggregation by most physiological agonists (Coller et al. 1996) and thus is a target for therapeutic antagonists (Scarborough et al. 1999, The EPIC Investigators 1994). Preventive antithrombotic treatments include platelet inhibitors such as aspirin (Hennekens 1997) and perhaps antioxidants (Hennekens 1994). Polyphenolic components in plant foods, particularly red wine and cocoa, may also protect against CVD by inhibiting platelet activation and aggregation (Dobrydneva et al. 1999, Keevil et al. 2000, Maalej et al. 1997, Osman et al. 1998, Pace-Asciak et al. 1995). We present the results from a series of studies on the effects of cocoa procyanidins and dealcoholized red wine (DRW) on platelet function. In the first set of experiments, we tested whether cocoa procyanidins or DRW incubated with whole blood affected the unstimulated or agoniststimulated expression of PAC-1 binding and granular membrane activation-dependent platelet antigen P-selectin expression on the platelet surface. In the second set of experiments, we investigated whether the consumption of a cocoa beverage or DRW affected platelet activation in healthy humans. A preliminary report on the effects of cocoa consumption on in vivo platelet function has been previously published (Rein et al. 2000b). MATERIALS AND METHODS Subjects. Ten subjects (five men and five women, y old) participated in the in vitro study. Four groups, with 10 subjects each, participated in the consumption study (DRW group, five men and five women, y old; water group, four men and six women, y old; cocoa beverage group, four men and six women, y old; caffeine-containing beverage group, four men and six women, y old). All subjects were healthy, nonsmoking adults with no history of heart disease or hemostatic disorders. Women were premenopausal and were not taking estrogens. Participants were instructed to abstain from nonsteroidal anti-inflammatory medication for at least 4 d, from alcoholic beverages for at least 2 d and from flavonoid-rich plant foods, caffeine or theobromine-containing foods for at least 24 h before and during the test day. Subject compliance and medical history were evaluated via a questionnaire. One female subject was not present for the 6-h blood draw after caffeine beverage consumption. All participants gave written informed consent before participation in the study, which was approved by the University of California, Davis, Human Subjects Review Committee. Effect of DRW on platelet activation in vitro. Venous blood was obtained from each subject between 0800 and 1000 h in two 5-ml evacuated tubes containing 0.5 ml of 3.8% (0.129 mol/l) buffered sodium citrate solution (Becton Dickinson, Franklin Lakes, NJ) using a 21-gauge needle. We took extra care that no samples were obtained as the result of traumatic venipuncture and that none contained obvious clots. If either of these conditions had occurred, we would have excluded the sample. Whole blood was incubated with and without dealcoholized Cabernet Sauvignon (1996), donated by the Department of Viticulture and Enology, University of California, Davis. The wine was dealcoholized by vacuum evaporation to 1% alcohol. The total phenol content of DRW was determined in gallic acid equivalents by the Folin-Ciocalteau assay (Sigma Chemical Co., St. Louis, MO). Whole blood was also incubated with the trimer and pentamer cocoa procyanidins purified from Cocoapro cocoa (Mars Incorporated, Hackettstown, NJ), according to Adamson et al. (1999). Within 10 min of draw, whole blood was incubated in polystyrene tubes for 5 min at room temperature with DRW, cocoa procyanidin trimers or pentamers. Final polyphenol concentrations were 3 and 10 mol/l. Samples were then incubated for an additional 5 min with HEPES buffer (ph 7.4, unstimulated control), ADP (final concentrations 20 or 100 mol/l) or epinephrine (final concentration 20 mol/l; BioData, Horsham, PA) in the presence or absence of the peptide Arg-Gly-Asp-Ser (Sigma). After 5 min, samples were suspended in 1 ml of HEPES buffer. One hundred microliters of sample was transferred to tubes containing saturating concentrations (20 l) of each of the following fluorescent labeled monoclonal antibodies: PAC-1-fluorescein isothiocyanate (FITC), anti-cd62p-phycoerythrin (PE) and anti- CD42a-peridinin chlorophyll protein (PerCP). PAC-1 recognizes the activated conformation of the fibrinogen-binding receptor GPIIb-IIIa, and anti-cd62p recognizes P-selectin, present on the surface of activated platelets. Anti-CD42a recognizes GPIb-IX, present on the membrane surface of both activated and resting platelets. Mouse IgG 1 FITC and mouse IgG 1 PE were used as isotype controls. The Arg-Gly-Asp-Ser peptide was used as a blocking control for PAC-1 binding. Antibodies and isotype controls were purchased from Becton Dickinson Immunocytometry Systems (San Jose, CA). Whole blood samples in the presence and absence of the agonists ADP and epinephrine were incubated with monoclonal antibodies or isotype control for 20 min in the dark at room temperature. Samples were then fixed in filtered 1% paraformaldehyde (ph 7.2) and stored in the dark at 2 8 C. All samples were analyzed within 24 h on a FACScan flow cytometer using LYSYS II or CellQuest software. The flow cytometer performance was verified using 1-, 2- and 10- m calibration beads (Becton Dickinson Immunocytometry Systems and Flow Cytometry Systems, Research Triangle Park, NC). Twenty thousand events were collected in list mode with all lightscatter and fluorescence parameters in logarithmic mode. Platelets were gated on

3 2122S SUPPLEMENT the basis of lightscatter and CD42a expression. Activated platelets were defined as the percentage of CD42a-positive events coexpressing the activated conformation of GPIIb-IIIa (PAC-1 binding) or P-selectin. Effect of polyphenol ingestion on ex vivo platelet activation. Test subjects (n 10) drank 300 ml of DRW, water, a beverage containing g of procyanidin-enriched cocoa powder and 12.5 g of sucrose mixed with distilled water or water containing 17 mg of caffeine and 12.5 g of sucrose. The DRW provided approximately 2.0 mmol of gallic acid equivalents/300 ml determined by the Folin- Ciocalteau assay. The cocoa beverage provided 897 mg of total epicatechin and oligomeric procyanidins (Adamson et al. 1999), plus 17 mg of caffeine and 285 mg of theobromine. The cocoa dose was selected to provide 1.5 times (18.75 g) the amount of a usual serving of cocoa in an 8-oz (240 ml) hot beverage. Blood was drawn as described above for the in vitro study, with the exception that 5-ml evacuated tubes containing 0.5 ml of 3.2% buffered sodium citrate solution were used (Becton Dickinson). We (Wun et al and 1997) have previously shown that this method of collection does not lead to significant artifactual ex vivo platelet activation in this test system. Additional blood samples were obtained 2 and 6 h after consumption of the beverages. Within 10 min of the blood draw, whole blood was incubated with HEPES buffer (ph 7.4, unstimulated control), epinephrine (final concentration 20 mol/l; BioData, Horsham, PA) or ADP (final concentrations 20 or 100 mol/l), stained with monoclonal antibodies and analyzed by flow cytometry as described above. Statistical analysis. Data from each treatment or control group were analyzed for differences using Friedman s repeated measures ANOVA on ranks (SigmaStat Version 2.0 for Windows, SPSS, Richmond, CA). The Tukey all-pairwise comparison test was used to identify differences between baseline (defined as 0 h) and 2 and 6 h postconsumption results. P-values of 0.05 were considered statistically significant. RESULTS Platelet activation in response to in vitro incubation with polyphenols. Nonstimulated platelets (in whole blood) incubated with DRW or procyanidin trimer (both at 3 and 10 mol/l) were activated, as indicated by increased PAC-1 binding (Fig. 1A) and platelet P-selectin expression (Fig. 1C). The procyanidin pentamer (both at 3 and 10 mol/l) also stimulated P-selectin in nonstimulated cells (Fig. 1C). DRW (10 mol/l) reduced P-selectin expression in response to epinephrine (Fig. 1D). Procyanidin trimer (3 mol/l) and pentamer (3 mol/l) inhibited PAC-1 binding response to epinephrine (Fig. 1B). Procyanidin trimer (both at 3 and 10 mol/l) reduced P-selectin expression in response to epinephrine (Fig. 1D). Platelet activation in response to consumption of DRW and water. Neither DRW nor water consumption had a significant effect on either the baseline unstimulated or epinephrine-induced PAC-1 binding (Fig. 2). Similarly, DRW and water consumption did not affect PAC-1 binding in response to 20 mol/l ADP, whereas DRW consumption did increase PAC-1 binding in response to 100 mol/l ADP (P 0.033, median values were 81.4, 85.8 and 88.6% at 0, 2 and 6 h postconsumption, Fig. 2). Neither DRW nor water changed unstimulated or epinephrine- or ADP-induced P- selectin expression at 2 or 6 h postconsumption (DRW median values: unstimulated, 1.6, 1.2 and 1.3%; epinephrine, 6.5, 7.3 and 7.5%; 20 mol/l ADP, 61.6, 64.3 and 64.3%; 100 mol/l ADP stimulated, 65.9, 74.5 and 72.8%; water median values: unstimulated, 1.1, 1.5 and 1.5%; epinephrine, 5.2, 4.9 and 4.9%; 20 mol/l ADP, 38.7, 37.6 and 36.9%; 100 mol/l FIGURE 1 Platelet activation in response to in vitro exposure to DRW and cocoa procyanidins. (A) Expression of the activated conformation of GPIIb-IIIa on platelets (expressed as PAC-1 binding) after in vitro exposure to HEPES buffer (control) or to the agonists DRW and cocoa procyanidins. *P 0.05 compared with the HEPES control (repeated measures ANOVA on ranks, Tukey s all-pairwise comparison). (B) Expression of PAC-1 on platelets that were first incubated in vitro with HEPES or the indicated agonist for 5 min followed by an additional 5-min room temperature incubation with 20 mol/l epinephrine. *P 0.05 compared with platelets stimulated with epinephrine alone compared with epinephrine plus the indicated agonists (n 10 in each study group). (C) Expression of the activation antigen CD62 after in vitro exposure to agonists. (D) Expression of CD62 after in vitro exposure to 20 mol/l epinephrine in the presence and absence of the indicated agonist. White and gray boxes correspond to 3 and 10 M concentrations of the indicated agonists, respectively. Data are presented as Tukey plots. Boxes represent the 25th 75th percentile, and horizontal lines represent the median and the 10th 90th percentile.

4 DIETARY POLYPHENOLS AND PLATELET FUNCTION 2123S FIGURE 2 Effects of wine, water, cocoa and caffeine ingestion on platelet activation. A blood sample was drawn before, 2 h after, and 6 h after ingestion of wine, water, cocoa or a caffeine-containing beverage. Whole blood from each time point was incubated in vitro with and without 20 mol/l epinephrine, 20 mol/l ADP or 100 mol/l ADP, before staining with PAC-1 FITC, anti-cd62 PE and anti- CD42a PerCP. The percentage of CD42a-positive platelets coexpressing activation markers (PAC-1 binding or CD62) was calculated from the flow cytometric analysis. Results are shown for PAC-1 binding only. White, light gray and dark gray boxes represent the preingestion and 2- and 6-h postingestion time points. There were 10 study subjects in each group. Data are presented as Tukey plots. Boxes represent the 25th 75th percentile, and horizontal lines represent the median and the 10th 90th percentile. The preingestion and 2- and 6-h postingestion time points were compared using repeated measures ANOVA on ranks, Tukey s all-pairwise comparison. *P 0.05 compared with the preingestion time point in each group (n 10 in each study group). ADP stimulated, 60.3, 60.0 and 59.1%, at 0, 2 and 6 h postconsumption, respectively). Platelet activation in response to consumption of cocoa and caffeine beverages. Consumption of a cocoa beverage was associated with decreased unstimulated (P 0.035, Fig. 2) and ex vivo epinephrine-induced (P 0.008, Fig. 2) PAC-1 binding at 2 and 6 h after ingestion. The median percentages of platelets binding PAC-1 without epinephrine or ADP stimulation were 0.9, 0.5 and 0.3%, and in response to epinephrine, the values were 9.6, 6.8 and 3.3% at 0, 2 and 6 h postconsumption, respectively. Similarly, cocoa consumption decreased 20 mol/l ADP-induced activated PAC-1 binding to platelets 2 and 6 h postconsumption (P 0.001, median values were 58.5, 44.2 and 38.8% at 0, 2 and 6 h postconsumption, Fig. 2). In contrast, there was an increase in epinephrine-stimulated PAC-1 binding in the caffeine beverage group (P 0.048, median values were 5.3, 6.5 and 7.5% at 0, 2 and 6 h postconsumption, Fig. 2). There was a trend that suggested decreased PAC-1 binding on platelets after cocoa consumption when activation was induced by 100 mol/l ADP (P 0.067, median values were 76.5, 68.7 and 57.6% at 0, 2 and 6 h postconsumption, Fig. 2). We did not observe any changes in ADP-induced PAC-1 binding after the consumption of the caffeine-containing beverage. There was a trend toward decreased P-selectin expression after cocoa consumption in nonstimulated platelets (P 0.053, median values were 1.6, 1.8 and 0.7% at 0, 2 and 6 h postconsumption) but not in epinephrine-stimulated platelets (median values were 3.6, 4.5 and 3.2% at 0, 2 and

5 2124S SUPPLEMENT 6 h postconsumption). Cocoa consumption decreased 20 mol/l ADP-induced P-selectin expression 2 and 6 h postconsumption (P 0.007, median values were 45.2, 38.9 and 36.4% at 0, 2 and 6 h postconsumption). Cocoa consumption also decreased 100 mol/l ADP-induced P-selectin expression 6 h postconsumption (P 0.025, median values were 56.1, 54.7 and 41.8% at 0, 2 and 6 h postconsumption). Again, there was no evidence of changes in platelet P-selectin expression after the consumption of the caffeine-containing beverage (median values: nonstimulated, 1.0, 1.1 and 1.1%; epinephrine, 3.8, 4.2 and 5.2%; 20 mol/l ADP, 51.9, 50.5 and 50.4%; 100 mol/l ADP stimulated, 56.8, 57.6 and 60.7%) at 0, 2 and 6 h postconsumption, respectively). DISCUSSION The association of the regular intake of foods rich in polyphenols with a reduced risk for CVD has been attributed to a significant extent to the potent antioxidant effects of specific polyphenols in these foods (German et al. 1997, Hillbom 1999). However, clinical confirmation of the physiological significance of these antioxidant effects is limited. The interaction of absorbed and metabolized dietary polyphenols with blood platelets may contribute a significant mechanism in the prevention of excessive thrombosis and CVD (Maalej et al. 1997). In the described set of experiments, we investigated the effects of cocoa and DRW on platelet activation. In vitro, cocoa procyanidins and DRW incubated with whole blood-stimulated activation marker expression on platelets but suppressed weak agonist-induced platelet activation. In vivo, cocoa consumption suppressed epinephrine-induced platelet activation. This in vivo effect was not observed after the consumption of DRW, caffeine and water. In our in vitro experiments, we chose levels of wine and cocoa polyphenols (3 and 10 mol/l) that are 10-fold above the range obtained with the habitual consumption of these foods to determine whether we could find any effect of these compounds on platelet activation. However, plasma levels of catechin, and of other phenols or their metabolites, can reach up to 100 mol/l after consumption (Caccetta et al. 2000, Donovan et al. 1999). Chocolate and cocoa consumption can also give rise to polyphenolic metabolites in the plasma, with typical values of mol epicatechin/l being reported (Rein et al. 2000a, Richelle et al. 1999), although the intact absorption of procyanidin dimers has not been described. Flavonoids from other foods, such as tea and onions, are absorbed in the low micromolar range with similar kinetics (Hollman et al. 1996, Lee et al. 1995). Regardless, it would be premature to draw conclusions about the long-term effects of these compounds on platelets from this study, which examined only the acute effects on platelet function. Purified cocoa procyanidins or DRW added to whole blood in vitro increased PAC-1 binding and P-selectin expression on platelets not treated with any agonist (unstimulated platelets). The opposite effect was observed after the polyphenol-incubated platelets were stimulated with a mild platelet agonist. In our in vitro system, we determined the expression of activation-dependent platelet antigens after two consecutive 5-min incubations: first the test component (cocoa procyanidins or DRW) and then an agonist or a buffer. Interestingly, procyanidin trimers and DRW were better stimulants than procyanidin pentamers in our system. This raises the possibility that the chain length of the epicatechin oligomers may affect platelet activation. Because incubation with the test components preceded epinephrine stimulation, we speculate that the cocoa or DRW desensitized the platelets to epinephrine, either by competing with its platelet receptor or by interfering with signal transduction. Considering the large number of potentially active components in foods, these sensitive in vitro tests can provide very specific and rapid information about specific food components. Nevertheless, in vitro results may not reflect what occurs in vivo. There is a dramatic change in polyphenol composition in the peripheral blood circulation after the ingestion of plant polyphenols in food (Donovan et al. 1999, Hackett et al. 1983, Hollman et al. 1996). Thus, we applied a similar test system for postconsumption studies. Platelet effects after DRW consumption did not reflect in vitro measurements, but cocoa consumption showed significant platelet inhibition. As expected, postprandial effects of water consumption on platelet activation were not observed. This group served to control for diurnal changes and for the potential changes in platelet response after fluid intake. Similar to water, DRW consumption did not show effects on platelet PAC-1 binding or P-selectin expression, except for enhancement of PAC-1 after high ADP stimulation. The observed small increase in ADP stimulation after water consumption is unexplained. However, there is evidence for serotonin release after red wine consumption. Serotonin could synergize with ADP for platelet activation. For the current study, we used Cabernet Sauvignon with a total phenol content of 6.7 mmol of gallic acid equivalents/l after removal of the alcohol. This total phenol content corresponds to that of typical Californian red wines (Frankel et al. 1995). The 2.0 mmol that was consumed in the experiment from 300 ml of DRW may have been too low to affect platelet activation. In contrast, cocoa suppressed platelet PAC-1 binding on unstimulated and stimulated platelets, and it suppressed ADPinduced P-selectin expression. The sustained effect of cocoa on markers of platelet activation over 6 h is unlikely to be attributable to the caffeine fraction of the cocoa beverage because the effect of the caffeine-containing beverage was to stimulate, rather than to decrease, epinephrine-induced PAC-1 binding. The polyphenol composition differs between cocoa and red wine. The major difference in the polyphenol composition is in the procyanidin fraction, of which red wine contains only minor quantities (Waterhouse and Walzem 1998). Procyanidins may have specific biological activities, but their absorption has not yet been established. Another compositional difference lies in the high theobromine content of cocoa. Theobromine is efficiently absorbed from chocolate and rapidly metabolized (Richelle et al. 1999). Methylxanthines have been suggested to inhibit platelet aggregation (Ardlie et al. 1967), although the effects on hemostatic and cardiovascular variables of theobromine are controversial (Bak and Grobbee 1990, Baron et al. 1999). Caffeine and related methylxanthines may have weak antithrombotic effects through a competitive inhibition of adenosine receptors (Biaggioni et al. 1991, Daly et al. 1991). In summary, we have shown that the consumption of a cocoa drink results in the inhibition of epinephrine-induced platelet activation. Significantly, this was shown in a controlled clinical setting, using specific antibodies against the activated conformation of GPIIb-IIIa and granular

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