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*
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
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • polyphenols • red wine • cocoa beverage • platelet activation • GPIIb-IIIa complex • P-selectin • thrombosis • whole blood flow cytometry
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 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
).
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 agonist-stimulated 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
).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ten subjects (five men and five women, 21–49 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, 21–49 y old; water group, four men and six women, 24–50 y old; cocoa beverage group, four men and six women, 24–49 y old; caffeine-containing beverage group, four men and six women, 26–50 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 µmol/L. Samples were then incubated for an additional 5 min with HEPES buffer (pH 7.4, unstimulated control), ADP (final concentrations 20 or 100 µmol/L) or epinephrine (final concentration 20 µmol/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 µl) 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 IgG1 FITC and mouse IgG1 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-µm 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 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 18.75 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. 1998
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 µmol/L; BioData, Horsham, PA) or ADP (final concentrations 20 or 100 µmol/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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Nonstimulated platelets (in whole blood) incubated with DRW or
procyanidin trimer (both at 3 and 10 µmol/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 µmol/L)
also stimulated P-selectin in nonstimulated cells (Fig. 1C
). DRW (10 µmol/L) reduced P-selectin expression in
response to epinephrine (Fig. 1D
). Procyanidin trimer (3
µmol/L) and pentamer (3 µmol/L) inhibited PAC-1 binding response to
epinephrine (Fig. 1B)
. Procyanidin trimer (both at 3 and 10 µmol/L)
reduced P-selectin expression in response to epinephrine (Fig. 1D
).
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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 µmol/L ADP, whereas DRW consumption did increase PAC-1
binding in response to 100 µmol/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 µmol/L ADP, 61.6, 64.3 and
64.3%; 100 µmol/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 µmol/L ADP, 38.7, 37.6 and 36.9%; 100 µmol/L ADP
stimulated, 60.3, 60.0 and 59.1%, at 0, 2 and 6 h
postconsumption, respectively).
|
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 µmol/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 µmol/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 6 h postconsumption). Cocoa consumption decreased 20 µmol/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 µmol/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 µmol/L ADP, 51.9, 50.5 and 50.4%; 100 µmol/L ADP stimulated, 56.8, 57.6 and 60.7%) at 0, 2 and 6 h postconsumption, respectively).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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,
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 µmol/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 µmol/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 0.2–1
µmol 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 ADP-induced 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 membrane protein P-selectin on the platelet surface. The consumption of DRW did not suppress these platelet activation markers. In vitro tests showed that polyphenolic components of these foods affect platelet activation. Additional research is needed to determine the exact food components that are responsible for the observed antiplatelet effects, the amounts needed to reach effective plasma levels and the clinical significance of these findings in the context of CVD and thrombosis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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