- Email: [email protected]
Inhibition of platelet aggregation in whole blood by alcohol
Thrombosis Research, Vol. 78, No. 2, pp. 107-115, 1995 Copyright Q 1995 Elsevier Science Ltd Printed in the USA. All rights re.serwd 0049-3848/95 $9.50 + .oO
Pergamon 0049-3848(95)ooo39-9
INHIBITION
OF PLATELET
AGGREGATION
IN WHOLE
BLOOD
BY ALCOHOL
Armida P. Torres Duarte, Quan Sheng Dong, Jose Young, Sylvie Abi-Younes and Adam K. Myers Department of Physiology & Biophysics, Georgetown University Medical Center, Washington, DC 20007
(Received 7 July 1994by Editor R. Kinlough-Rathbone;revised/accepted 6 February 1995)
Abstract Our previous studies have demonstrated that addition of moderate volumes of absolute alcohol (34 - 170 mM final concentration) to whole blood produces concentration-dependent platelet aggregation, due to release of adenosine diphosphate (ADP) from erythrocytes. We have now investigated the effects of exposure of blood to ethanol by a more “physiologic” protocol, in which 7.8% (w/v) alcohol is added to achieve a final concentration of 1 to 85 mM in human and rat blood or platelet rich plasma (PRP). The effects of short incubation with alcohol on platelet aggregation induced by ADP, collagen and arachidonic acid were examined by the impedance method of aggregometry. Aggregation induced by collagen in PRP of either species was significantly inhibited by 85 mM ethanol, with concentrations as low as 4.25 mM inhibiting the response to collagen in rat whole blood. ADP stimulated only primary, reversible aggregation in rat PRP and whole blood, and these responses were unaffected by alcohol. Human platelets responded to ADP with irreversible aggregation, which was significantly attenuated by 85 mM ethanol in whole blood but not PRP. Arachidonic acid evoked irreversible platelet aggregation in all four preparations; this was significantly inhibited by the high dose ethanol in human and rat PRP, but not whole blood. In contrast to our earlier studies with absolute ethanol, there was no evidence of hemolysis (and therefore, ADP release from red blood cells) using the current protocol. The results of these experiments show that alcohol, at physiologically relevant concentrations, has an inhibitory effect on secondary platelet aggregation responses to some agonists in whole blood as well as PRP, possibly by its previously demonstrated effects on arachidonic acid release by phospholipases. The possibility remains to be considered that other blood cells might contribute to the effects of alcohol on platelet aggregation in whole blood. Epidemiologic studies suggest that alcohol consumption can alter the incidence of hemorrhagic and thrombotic disorders and their sequelae, including stroke and coronary heart disease (l-4). Recent reports have continued to associate moderate consumption of alcohol in some forms (wine, for example) with reduced risk of coronary heart disease and myocardial infarction (S-6). Earlier studies indicated that consumption of alcohol can be an independent risk factor for both thrombotic and hemorrhagic stroke (3,7,8). Key words: alcohol, ethanol, platelet aggregation, arachidonic acid Corresponding author: Adam K. Myers, Ph.D., Department of Physiology & Biophysics, Georgetown University Medical Center, 3900 Reservoir Rd. NW, Washington, DC 20007 UsA 107
108
ALCOHOL
AND PLATELET AGGREGATION
Vol. 78, No. 2
Among the mechanisms whereby alcohol could affect hemostatic function, effects on platelet aggregation have received significant attention. Consistent with the potential pro- and antithrombotic effects suggested by the epidemiologic studies, alcohol also has stimulatory and inhibitory effects on platelets (9-13). Activation of platelets, as measured by platelet shape change, is provoked by in vitro exposure to alcohol (9,lO); this activation is associated with a rise in intra-platelet free Ca ++ level. On the other hand, most in vitro and ex vivo studies of platelet aggregation in platelet rich plasma (PRP) or platelets resuspended in buffer solutions have found that relatively low concentrations of alcohol inhibit agonist-induced aggregation (ll13), with a variety of potential biochemical mechanisms, including inhibition of eicosanoid formation (13). In contrast to this inhibitory effect of alcohol in PRP or resuspended platelets, we have shown that direct addition of absolute alcohol (34-170 mM final concentration, by bolus injection) to whole blood causes aggregation of platelets (14). Exposure to alcohol produces lysis of red blood cells and release of ADP, which subsequently stimulates platelet activation. Thus, in the milieu of whole blood, a more physiologic setting for platelet aggregation than PRP, alcohol can induce platelet aggregation through initiation of indirect interactions between erythrocytes and platelets. The degree to which our findings in whole blood relate to the effects of alcohol consumption is unknown. In our experiments, bolus addition of alcohol presumably produces locally high concentrations of alcohol, which blood elements are not exposed to under normal circumstances, and it is apparently such exposure which causes the hemolysis and thus provokes platelet aggregation. The purpose of the present work was to further evaluate the effects of alcohol on platelet function in whole blood. We hypothesized that exposure of platelets to alcohol, in whole blood, by methods which prevent exposure of formed elements to concentrated alcohol (by the use of diluted alcohol) would produce inhibition of aggregation as occurs in PRP. In addition to testing this hypothesis, we examined the extent to which responses to alcohol differed in whole blood vs. PRP, to evaluate whether presence of formed elements besides platelets would affect responses. METHODS Blood Collection and Preparation
of PRP
Human and rat blood samples were collected as previously reported (14). Briefly, human venous blood was collected from drug-free male and female volunteers into syringes containing heparin (10 U/ml final concentration). Arterial blood was collected from pentobarbital-anesthetized Wistar rats into syringes containing heparin (20 U/ml final concentration). PRP was prepared by centrifugation of blood at 100 x g for 15 min as previously reported (14). Platelet counts were determined by phase contrast microscopy. Mean platelet counts f SEM in human and rat whole blood were 242,OOO/ltl + 27,000 (n=9) and 775,000/@ f 74,000 (n=l8), respectively. In human and rat PRP, platelet counts were 350,000 + 3O,OOO/ltl (n=12) and 778,000 f 70,OOO/l~l (n=18). Drugs and Chemicals
Collagen suspension, ADP and arachidonic acid for platelet aggregation were obtained from Chronolog, Sigma and Nu Chek Prep, respectively. ADP was dissolved in 0.9% NaCl; arachidonic acid was dissolved in 100 mM sodium carbonate. Heparin was obtained from Organon and was diluted to the appropriate concentration in 0.9% NaCl. Alcohol was obtained from Warner-Graham Co. Platelet Aggregation
Platelet aggregation studies were performed with a 560VS Chronolog aggregometer, by the impedance method (14); by using this method, aggregation can be quantified either in whole blood or in PRP. Whole blood or PRP was incubated at 37oC in 1 ml aliquots until time of use; during aggregation testing, samples were kept at 37OC and stirred at 1000 rpm. After equilibration in the aggregation well, diluted alcohol (1 part alcohol: 9 parts 0.9% NaCl) or its vehicle (0.9% NaCl) was added at concentrations indicated below. The dilution of alcohol was
Vol. 78, No. 2
ALCOHOL
AND PLATELET AGGREGATION
109
based on preliminary experiments which showed that this solution had no pro-aggregatory effect in whole blood. The incubation time for blood or PRP with alcohol was also based on preliminary experiments which showed that the action of alcohol on platelet aggregation was nearly immediate. After incubation of the whole blood or PRP sample with ethanol solution for 1 min, an appropriate dose of collagen, ADP or arachidonic acid was added to initiate aggregation of platelets. Responses, in terms of change in impedance (Ohms) were generally measured for 5 minutes; ADP caused a rapid, reversible aggregation in rat platelets which was recorded for its entire duration, usually less than 1 min. Aggregation was quantified as change in impedance (Ohms) and expressed as mean + SEM, for various control or experimental groups. Each data point illustrated below consists of 6 or more observations. Statistical significance was assessed by analysis of variance followed by Dunnett’s t test, to determine significance of differences between groups. Henzolysis studies Measurement of the degree of hemolysis caused by addition of ethanol to heparinized rat whole blood samples was performed by a previously published method (14). In this case, effects of dilute ethanol (1:9 in 0.9% NaCl) were tested, as in the platelet aggregation studies, above. In brief, the dilute alcohol was added to samples of whole blood, at the same concentrations used in the platelet aggregation experiments; after the tubes were inverted several times, they were centrifuged in an Eppendorf microfuge for 5 min. The supernatant was collected and absorbance was determined at 410 nm, for measurement of hemoproteins. The degree of hemolysis in these samples was determined by comparison to reference values obtained from supernatants from blood subjected to 100% hemolysis in distilled water (after appropriate correction for dilution factors). RESULTS Rat Platelet Aggregatiorr The effects of dilute alcohol (7.8% w/v; 1:9 dilution in 0.9% NaCl) on platelet aggregation induced by ADP, collagen and arachidonic acid were studied in rat whole blood and PRP (figs. l-3). The final concentration of alcohol in whole blood or PRP ranged from 1.06 to 85 mM.
0
1.06
4.25
17
85 Ethanol
” (mM)
0
1.06
4.25
17
85
FIG. 1 Effect of alcohol, added in dilute form (7.8% w/v), on platelet aggregation induced by 0.2 I.IM ADP in rat blood or PRP. ADP caused primary aggregation in both types of sample and preincubation with alcohol (1 min) had no statistically significant effect on the aggregation. Each bar represents the mean (+ SEM) of at least 6 observations.
110
ALCOHOL
AND PLATELET AGGREGATION
Vol. 78, No. 2
Addition of ADP to rat whole blood and PRP, to achieve a final concentration of 0.2 PM, resulted in only primary, reversible platelet aggregation. Preincubation of blood or PRP with ethanol, at concentrations up to 85 mM, had no effect on ADP induced aggregation (fig. 1). Collagen, at a final concentration of 3 @ml, induced irreversible platelet aggregation in rat PRP and whole blood. Alcohol inhibited collagen-induced aggregation in both preparations (fig. 2). In whole blood, platelet aggregation was inhibited in a dose-dependent fashion, with the inhibition reaching 100% at 85 mM ethanol (P
Whole blood
PRP 20
10
0
1.06
4.25
17
85 Ethanol
n ” (mM)
0
1.06
4.25
17
85
FIG. 2 Effect of alcohol, added in dilute form (7.8% w/v), on platelet aggregation induced by 3 yg/ml collagen in rat blood or PRP. Collagen produced irreversible aggregation in both types of sample. Each bar represents the mean (+ SEM) of at least 6 observations. “PcO.05, **P
40 30 20 20 10
0
10
0
1.06
4.25
17
85 Ethanol
0
0
1.06
4.25
17
85
(mM)
FIG. 3 Effect of alcohol, added in dilute form (7.8% w/v), on platelet aggregation induced by 0.5 mM arachidonic acid in rat blood or PRP. Arachidonate produced irreversible aggregation in both blood and PRP. Each bar represents the mean (+ SEM) of at least 6 observations. “PcO.05 compared to vehicle control (0 mM ethanol).
ALCOHOL
Vol. 70, No. 2
111
AND PLATELET AGGREGATION
Arachidonic acid, at a final concentration of 0.5 mM, produced irreversible aggregation of rat platelets in the two types of platelet preparation. Aggregation of platelets by arachidonic acid in PRP was significantly inhibited by preincubation with the highest concentration of ethanol, 85 mM (P
studies in rat blood
Addition of dilute alcohol (1:9 dilution in 0.9% NaCl) at concentrations of 1.06,4.25, 17 and 85 mM (or the NaCl solution vehicle) did not produce significant hemolysis of rat whole blood, as measured by spectrophotometric analysis of plasma samples for hemoproteins (data not illustrated). The greatest level of hemolysis observed in any samples represented 0.2% of the total erythrocytes. G E
16
40
5 g aI E
12
30
I3
20
5 % F b
4
10
p
0
0
1.06
4.25
17
65 Ethanol
0
0
1.06
4.25
17
85
(mM)
FIG. 4 Effect of alcohol, added in dilute form (7.8% w/v), on platelet aggregation induced by 6 pM ADP in human blood or PRP. ADP produced irreversible platelet aggregation in both blood and PRP. Each bar represents the mean (+ SEM) of at least 6 observations. “PcO.05 compared to vehicle control (0 mM ethanol).
20
10
0
1.06
4.25
17
85 Ethanol
n w (mM)
0
1.06
4.25
17
85
FIG. 5 Effect of alcohol, added in dilute form (7.8% w/v). on platelet aggregation induced by 0.5 pg/rnl collagen in human blood or PRP. Collagen produced irreversible platelet aggregation in both blood and PRP. Each bar represents the mean & SEM) of at least 6 observations. “PcO.05; **P
112
Vol. 78, No. 2
ALCOHOL AND PLATELET AGGREGATION
”
0
1.08
4.25
17
85 Ethanol
” (mM)
0
1.08
4.25
17
85
FIG. 6 Effect of alcohol, added in dilute form (7.8% w/v), on platelet aggregation induced by 0.5 mM arachidonic acid in human blood or PRP. Arachidonic acid produced irreversible platelet aggregation in both blood and PRP. Each bar represents the mean (k SEM) of at least 6 observations. *P
Vol. 70, No. 2
ALCOHOL
AND PLATELET AGGREGATION
dependent inhibition of ADP-induced aggregation in human whole blood, it had no effect on aggregation evoked by ADP in rat whole blood or PRP of either species. The basis for this difference in response is not clear, but might reflect differences in the effects of ADP in various preparations. Whereas ADP produces only primary aggregation in rat platelets (17), it causes irreversible, secondary aggregation and the associated release reaction in citrate-anticoagulated human PRP (18). Responses in heparin-anticoagulated human PRP are variable, perhaps reflecting direct actions of heparin on platelet responses (19). A difference in the extent to which ADP causes secondary aggregation and release reaction in platelet preparations might result in the observed differences in the effects of alcohol, but further studies would be necessary to test this hypothesis. In the present experiments, ADP stimulated irreversible aggregation in human PRP and whole blood anticoagulated with heparin, but we did not measure the platelet release reaction. Consistent with the view that the main inhibitory effect of alcohol is on the secondary phase of platelet aggregation, alcohol significantly inhibited the aggregation caused by collagen in human and rat PRP and whole blood; collagen-induced aggregation involves a secondary, irreversible phase. This speculation, that alcohol affects the second phase of aggregation, as opposed to the first, is in agreement with earlier reports (12,15,20). Previous studies have also demonstrated that alcohol affects secondary aggregation and release reaction of platelets, associated with inhibition of arachidonic acid release from platelet membranes, and thus subsequent metabolism to endoperoxides and thromboxane formation (21,22). Notably, primary aggregation induced by ADP is not dependent on thromboxane formation, and when we observed only reversible, primary aggregation (as in the ADP induced aggregation in rat platelets), alcohol had no effect. Although our data are generally consistent with this mechanism of alcohol action, we did find that 85 mM ethanol significantly inhibited arachidonic acid-induced aggregation in human and rat PRP, but not rat or human whole blood. Because addition of arachidonic acid to platelets bypasses the putative point in the arachidonic acid cascade at which alcohol acts (release of arachidonic acid from membrane phospholipids by phospholipases), this result is in apparent contradiction to that proposed mechanism of alcohol’s action (15). Alcohol probably has multiple effects on different levels of various platelet biochemical pathways which contribute to the overall inhibitory action, with the primary effect being inhibition of arachidonic acid release. Another interesting point regarding the arachidonic acid experiments was the contrast between inhibition of aggregation by alcohol in PRP and the lack of significant effect in whole blood of two species. The inhibition in PRP suggests alcohol interferes with one of the enzymes involved in thromboxane production from arachidonic acid (cyclooxygenase and thromboxane synthetase), because thromboxane is the major product in platelets. In whole blood, the effect of arachidonic acid is certain to be more complex. Arachidonic acid might affect other formed elements which in turn may release factors capable of influencing the platelet aggregation response. It also will be metabolized to numerous products, depending on cell type. Under those circumstances, inhibition of arachidonate metabolism might have less predictable effects than in PRP. In designing these experiments, we incorporated concentrations of alcohol deemed to be “physiologically relevant”. The lowest concentration tested (1.06 mM) is equivalent to a blood alcohol level of 5 mg/dl, which would not be associated with discernable behavioral effects, whereas the highest concentration (85 mM) is equivalent to blood alcohol of 391 mg/dl, which would produce profound intoxication (coma and death can occur above 500 mg/dl) (23). The exposure of blood to dilute alcohol is also more relevant to alcohol consumption than direct addition of absolute ethanol. Based on our present findings it appears that the major effect of alcohol consumption, at moderate doses, is inhibition rather than stimulation of platelet aggregation. This is consistent with the single report of which we are aware regarding whole blood platelet aggregation after ingestion of alcohol (24). Furthermore, because the effects of alcohol, when added in dilute form, are generally similar in PRP and whole blood, alcohol clearly has significant effects on the platelet itself, but we cannot rule out other actions through formed elements besides platelets without further studies. Assuming that inhibition of phospholipase A2 (and thus arachidonic acid release and metabolism) is a major mechanism for
114
ALCOHOL
AND PLATELET AGGREGATION
Vol. 78, No. 2
alcohol’s effect in platelets, it will be interesting to know how modification of arachidonate metabolism in other blood components by alcohol or its consumption (2526) affects platelet aggregation in whole blood. Finally, although it is tempting to speculate on how alcohol consumption might affect risk for thrombotic events such as myocardial infarction and thrombotic stroke, it is important to consider that alcohol consumption and its many effects are associated with other factors (such as diet, smoking, etc.), with complicated biological as well as sociological implications, making any conclusions tenuous. Regarding the experimental use of alcohol, however, it is quite clear that some precautions are necessary: our results suggest that when alcohol is used as a diluent for drugs, either for platelet aggregation studies or intravascular injections in experimental animals, proper controls are necessary, because of the potential for both platelet stimulatory and inhibitory actions. Acknowledgment This work was supported by a grant from the National Institutes of Health (AA09586). REFERENCES
5. 6. 7. 8. 9.
10. 11. 12. 13. 14. 15. 16.
MARMOT, M.G. Alcohol and coronary heart disease. Int J. Epidemiol 13,160-167, 1984. YANO, K., RHOADS, G.G. and KAGAN, A. Coffee, alcohol and risk of coronary heart disease among Japanese men living in Hawaii. N Eng J Med 297,405-409, 1977. GILL, J.S., ZEZULKA, A.V., SHIPLEY, M.J., GILL, S.K. and BEEVERS, D.G. Stroke and alcohol consumption. N Eng J Med 31.5, 1041- 1046, 1986. KLATSKY, A.L., FRIEDMAN, G.D. and SIEGELAUB, A.B. Alcohol consumption before myocardial infarction. Results from the Kaiser-Permanente epidemiologic study of myocardial infarction. Ann Intern Med 81,294-301, 1974. RENAUD, S. and DE LORGERIL, M. Wine, alcohol, platelets, and the French paradox for coronary heart disease. Lancet 339,1523- 1526, 1992. KLATSKY, A.L., ARMSTRONG, M.A. and FRIEDMAN, G.D. Alcohol and mortality. Ann Intern Med ZZ7,646-654, 1992. GILL, J.S., SHIPLEY, M.J, TSEMENTZIS, S.A., HORNBY, R.S, GILL, S.K., HITCHCOCK, E.R. and BEEVERS, D.G. Alcohol consumption- a risk factor for hemorrhagic and non-hemorrhagic stroke. Am J Med 90,489-497, 1991. Hillbom, M.E. What supports the role of alcohol as a risk factor for stroke? Acta Med Stand Suppl 717,93-106,1987, RUBIN, R. and HOEK, J.B. Alcohol-induced stimulation of phospholipase C in human platelets requires G-protein activation. Biochem J 254,147- 153, 1988. RUBIN, R. and HOEK, J.B. Inhibition of ethanol-induced platelet activation by agents that elevate CAMP. Thromb Res X%625-632, 1990. RENAUD, S.C., BESWICK, A.D., FEHILY, A.M., SHARP, D.S. and ELWOOD, P.C. Alcohol and platelet aggregation: The Caerphilly prospective heart disease study. Am J Clin Nutr55,1012-1017, 1992. HAUT, M.J. and COWAN, D.H. The effect of ethanol on hemostatic properties of human blood platelets. Am J Med 56,22-33, 1974. RUBIN, R. Ethanol interferes with collagen-induced platelet activation by inhibition of at-achidonic acid mobilization. Arch Biochem Biophys 270,99-l 13, 1989. ABI-YOUNES, S.A., AYERS, M.L. and MYERS, A.K. Mechanism of ethanol-induced aggregation in whole blood. Thromb Res 63,481-489, 1991. RAND, M.L., PACKHAM, M.A., KINLOUGH-RATHBONE, R.L. and MUSTARD, J.F. Effects of ethanol on pathways of platelet aggregation in vitro. Thromb Haemost 59, 383387, 1988. FENN, C.G. and LITTLETON, J.M. Inhibition of platelet aggregation by ethanol in vitro shows specificity for aggregating agent used and is influenced by platelet lipid composition, Thromb Haemost d&49-53, 1982.
Vol. 78, No. 2
ALCOHOL
AND PLATELET AGGREGATION
115
MACMILLAN, D.C. and SIM, A.K. A comparative study of platelet aggregation in man and laboratory animals. Thromb Diath Haemorrh 24, 385-394, 1970. 18. VARGAFTIG, B.B., CHIGNARD, M. and BENVENISTE, J. Present concepts on the mechanism of platelet aggregation. Biochem Pharmacol30,263-27 1, 198 1. 19. LAGES, B. and WEISS, H.J. Dependence of human platelet functional responses on divalent cations: aggregation and secretion in heparin- and hirudin-anticoagulated plateletrich plasma and the effects of chelating agents. Thromb Haemost 45, 173-179, 1981. 20. BENISTANT, C. and RUBIN, R. Ethanol inhibits thrombin-induced secretion by human platelets at a site distinct from phospholipase C or protein kinase C. Biochem J 269,489497, 1990. 21. RUBIN, R. Ethanol interferes with collagen-induced platelet activation by inhibition of arachidonic acid mobilization. Arch Biochem Biophys 270,99-l 13, 1989. 22. HWANG, D.H. Ethanol inhibits the formation of endoperoxide metabolites in human platelets. Prostaglandins Med 7, 511-513, 1981. 23. DUTTA, S.N. and PRADHAN, S.N. Alcohols. In: Pharmacology in Medicine: Principles and Practice. Pradhan, S.N., Maickel, R.P. and Dutta, S.N. (eds.), pp 285290, SP Press, Bethesda (1986). 24. MIKHAILIDIS, D.P., BARRADAS, M.A., EPEMOLU, 0. and DANDONA, P. Ethanol ingestion inhibits human whole blood platelet impedance aggregation. Am J Clin Path01 88, 342-345, 1987. 25. MEHTA, P., MEHTA, J., LAWSON, D. and PATEL, S. Ethanol stimulates prostacyclin biosynthesis by human neutrophils and potentiates anti-platelet aggregatory effects of prostacyclin. Thromb Res 48,653-661, 1987. 26. MAXWELL, W.J., KEATING, J.J., HOGAN, F.P., KENNEDY, N.P. and KEELING, P.W.N. Prostaglandin E:! and leukotriene B4 synthesis by peripheral leucocytes in alcoholics. Gut 30, 1270-1274, 1989. 17.
Pergamon 0049-3848(95)ooo39-9
INHIBITION
OF PLATELET
AGGREGATION
IN WHOLE
BLOOD
BY ALCOHOL
Armida P. Torres Duarte, Quan Sheng Dong, Jose Young, Sylvie Abi-Younes and Adam K. Myers Department of Physiology & Biophysics, Georgetown University Medical Center, Washington, DC 20007
(Received 7 July 1994by Editor R. Kinlough-Rathbone;revised/accepted 6 February 1995)
Abstract Our previous studies have demonstrated that addition of moderate volumes of absolute alcohol (34 - 170 mM final concentration) to whole blood produces concentration-dependent platelet aggregation, due to release of adenosine diphosphate (ADP) from erythrocytes. We have now investigated the effects of exposure of blood to ethanol by a more “physiologic” protocol, in which 7.8% (w/v) alcohol is added to achieve a final concentration of 1 to 85 mM in human and rat blood or platelet rich plasma (PRP). The effects of short incubation with alcohol on platelet aggregation induced by ADP, collagen and arachidonic acid were examined by the impedance method of aggregometry. Aggregation induced by collagen in PRP of either species was significantly inhibited by 85 mM ethanol, with concentrations as low as 4.25 mM inhibiting the response to collagen in rat whole blood. ADP stimulated only primary, reversible aggregation in rat PRP and whole blood, and these responses were unaffected by alcohol. Human platelets responded to ADP with irreversible aggregation, which was significantly attenuated by 85 mM ethanol in whole blood but not PRP. Arachidonic acid evoked irreversible platelet aggregation in all four preparations; this was significantly inhibited by the high dose ethanol in human and rat PRP, but not whole blood. In contrast to our earlier studies with absolute ethanol, there was no evidence of hemolysis (and therefore, ADP release from red blood cells) using the current protocol. The results of these experiments show that alcohol, at physiologically relevant concentrations, has an inhibitory effect on secondary platelet aggregation responses to some agonists in whole blood as well as PRP, possibly by its previously demonstrated effects on arachidonic acid release by phospholipases. The possibility remains to be considered that other blood cells might contribute to the effects of alcohol on platelet aggregation in whole blood. Epidemiologic studies suggest that alcohol consumption can alter the incidence of hemorrhagic and thrombotic disorders and their sequelae, including stroke and coronary heart disease (l-4). Recent reports have continued to associate moderate consumption of alcohol in some forms (wine, for example) with reduced risk of coronary heart disease and myocardial infarction (S-6). Earlier studies indicated that consumption of alcohol can be an independent risk factor for both thrombotic and hemorrhagic stroke (3,7,8). Key words: alcohol, ethanol, platelet aggregation, arachidonic acid Corresponding author: Adam K. Myers, Ph.D., Department of Physiology & Biophysics, Georgetown University Medical Center, 3900 Reservoir Rd. NW, Washington, DC 20007 UsA 107
108
ALCOHOL
AND PLATELET AGGREGATION
Vol. 78, No. 2
Among the mechanisms whereby alcohol could affect hemostatic function, effects on platelet aggregation have received significant attention. Consistent with the potential pro- and antithrombotic effects suggested by the epidemiologic studies, alcohol also has stimulatory and inhibitory effects on platelets (9-13). Activation of platelets, as measured by platelet shape change, is provoked by in vitro exposure to alcohol (9,lO); this activation is associated with a rise in intra-platelet free Ca ++ level. On the other hand, most in vitro and ex vivo studies of platelet aggregation in platelet rich plasma (PRP) or platelets resuspended in buffer solutions have found that relatively low concentrations of alcohol inhibit agonist-induced aggregation (ll13), with a variety of potential biochemical mechanisms, including inhibition of eicosanoid formation (13). In contrast to this inhibitory effect of alcohol in PRP or resuspended platelets, we have shown that direct addition of absolute alcohol (34-170 mM final concentration, by bolus injection) to whole blood causes aggregation of platelets (14). Exposure to alcohol produces lysis of red blood cells and release of ADP, which subsequently stimulates platelet activation. Thus, in the milieu of whole blood, a more physiologic setting for platelet aggregation than PRP, alcohol can induce platelet aggregation through initiation of indirect interactions between erythrocytes and platelets. The degree to which our findings in whole blood relate to the effects of alcohol consumption is unknown. In our experiments, bolus addition of alcohol presumably produces locally high concentrations of alcohol, which blood elements are not exposed to under normal circumstances, and it is apparently such exposure which causes the hemolysis and thus provokes platelet aggregation. The purpose of the present work was to further evaluate the effects of alcohol on platelet function in whole blood. We hypothesized that exposure of platelets to alcohol, in whole blood, by methods which prevent exposure of formed elements to concentrated alcohol (by the use of diluted alcohol) would produce inhibition of aggregation as occurs in PRP. In addition to testing this hypothesis, we examined the extent to which responses to alcohol differed in whole blood vs. PRP, to evaluate whether presence of formed elements besides platelets would affect responses. METHODS Blood Collection and Preparation
of PRP
Human and rat blood samples were collected as previously reported (14). Briefly, human venous blood was collected from drug-free male and female volunteers into syringes containing heparin (10 U/ml final concentration). Arterial blood was collected from pentobarbital-anesthetized Wistar rats into syringes containing heparin (20 U/ml final concentration). PRP was prepared by centrifugation of blood at 100 x g for 15 min as previously reported (14). Platelet counts were determined by phase contrast microscopy. Mean platelet counts f SEM in human and rat whole blood were 242,OOO/ltl + 27,000 (n=9) and 775,000/@ f 74,000 (n=l8), respectively. In human and rat PRP, platelet counts were 350,000 + 3O,OOO/ltl (n=12) and 778,000 f 70,OOO/l~l (n=18). Drugs and Chemicals
Collagen suspension, ADP and arachidonic acid for platelet aggregation were obtained from Chronolog, Sigma and Nu Chek Prep, respectively. ADP was dissolved in 0.9% NaCl; arachidonic acid was dissolved in 100 mM sodium carbonate. Heparin was obtained from Organon and was diluted to the appropriate concentration in 0.9% NaCl. Alcohol was obtained from Warner-Graham Co. Platelet Aggregation
Platelet aggregation studies were performed with a 560VS Chronolog aggregometer, by the impedance method (14); by using this method, aggregation can be quantified either in whole blood or in PRP. Whole blood or PRP was incubated at 37oC in 1 ml aliquots until time of use; during aggregation testing, samples were kept at 37OC and stirred at 1000 rpm. After equilibration in the aggregation well, diluted alcohol (1 part alcohol: 9 parts 0.9% NaCl) or its vehicle (0.9% NaCl) was added at concentrations indicated below. The dilution of alcohol was
Vol. 78, No. 2
ALCOHOL
AND PLATELET AGGREGATION
109
based on preliminary experiments which showed that this solution had no pro-aggregatory effect in whole blood. The incubation time for blood or PRP with alcohol was also based on preliminary experiments which showed that the action of alcohol on platelet aggregation was nearly immediate. After incubation of the whole blood or PRP sample with ethanol solution for 1 min, an appropriate dose of collagen, ADP or arachidonic acid was added to initiate aggregation of platelets. Responses, in terms of change in impedance (Ohms) were generally measured for 5 minutes; ADP caused a rapid, reversible aggregation in rat platelets which was recorded for its entire duration, usually less than 1 min. Aggregation was quantified as change in impedance (Ohms) and expressed as mean + SEM, for various control or experimental groups. Each data point illustrated below consists of 6 or more observations. Statistical significance was assessed by analysis of variance followed by Dunnett’s t test, to determine significance of differences between groups. Henzolysis studies Measurement of the degree of hemolysis caused by addition of ethanol to heparinized rat whole blood samples was performed by a previously published method (14). In this case, effects of dilute ethanol (1:9 in 0.9% NaCl) were tested, as in the platelet aggregation studies, above. In brief, the dilute alcohol was added to samples of whole blood, at the same concentrations used in the platelet aggregation experiments; after the tubes were inverted several times, they were centrifuged in an Eppendorf microfuge for 5 min. The supernatant was collected and absorbance was determined at 410 nm, for measurement of hemoproteins. The degree of hemolysis in these samples was determined by comparison to reference values obtained from supernatants from blood subjected to 100% hemolysis in distilled water (after appropriate correction for dilution factors). RESULTS Rat Platelet Aggregatiorr The effects of dilute alcohol (7.8% w/v; 1:9 dilution in 0.9% NaCl) on platelet aggregation induced by ADP, collagen and arachidonic acid were studied in rat whole blood and PRP (figs. l-3). The final concentration of alcohol in whole blood or PRP ranged from 1.06 to 85 mM.
0
1.06
4.25
17
85 Ethanol
” (mM)
0
1.06
4.25
17
85
FIG. 1 Effect of alcohol, added in dilute form (7.8% w/v), on platelet aggregation induced by 0.2 I.IM ADP in rat blood or PRP. ADP caused primary aggregation in both types of sample and preincubation with alcohol (1 min) had no statistically significant effect on the aggregation. Each bar represents the mean (+ SEM) of at least 6 observations.
110
ALCOHOL
AND PLATELET AGGREGATION
Vol. 78, No. 2
Addition of ADP to rat whole blood and PRP, to achieve a final concentration of 0.2 PM, resulted in only primary, reversible platelet aggregation. Preincubation of blood or PRP with ethanol, at concentrations up to 85 mM, had no effect on ADP induced aggregation (fig. 1). Collagen, at a final concentration of 3 @ml, induced irreversible platelet aggregation in rat PRP and whole blood. Alcohol inhibited collagen-induced aggregation in both preparations (fig. 2). In whole blood, platelet aggregation was inhibited in a dose-dependent fashion, with the inhibition reaching 100% at 85 mM ethanol (P
Whole blood
PRP 20
10
0
1.06
4.25
17
85 Ethanol
n ” (mM)
0
1.06
4.25
17
85
FIG. 2 Effect of alcohol, added in dilute form (7.8% w/v), on platelet aggregation induced by 3 yg/ml collagen in rat blood or PRP. Collagen produced irreversible aggregation in both types of sample. Each bar represents the mean (+ SEM) of at least 6 observations. “PcO.05, **P
40 30 20 20 10
0
10
0
1.06
4.25
17
85 Ethanol
0
0
1.06
4.25
17
85
(mM)
FIG. 3 Effect of alcohol, added in dilute form (7.8% w/v), on platelet aggregation induced by 0.5 mM arachidonic acid in rat blood or PRP. Arachidonate produced irreversible aggregation in both blood and PRP. Each bar represents the mean (+ SEM) of at least 6 observations. “PcO.05 compared to vehicle control (0 mM ethanol).
ALCOHOL
Vol. 70, No. 2
111
AND PLATELET AGGREGATION
Arachidonic acid, at a final concentration of 0.5 mM, produced irreversible aggregation of rat platelets in the two types of platelet preparation. Aggregation of platelets by arachidonic acid in PRP was significantly inhibited by preincubation with the highest concentration of ethanol, 85 mM (P
studies in rat blood
Addition of dilute alcohol (1:9 dilution in 0.9% NaCl) at concentrations of 1.06,4.25, 17 and 85 mM (or the NaCl solution vehicle) did not produce significant hemolysis of rat whole blood, as measured by spectrophotometric analysis of plasma samples for hemoproteins (data not illustrated). The greatest level of hemolysis observed in any samples represented 0.2% of the total erythrocytes. G E
16
40
5 g aI E
12
30
I3
20
5 % F b
4
10
p
0
0
1.06
4.25
17
65 Ethanol
0
0
1.06
4.25
17
85
(mM)
FIG. 4 Effect of alcohol, added in dilute form (7.8% w/v), on platelet aggregation induced by 6 pM ADP in human blood or PRP. ADP produced irreversible platelet aggregation in both blood and PRP. Each bar represents the mean (+ SEM) of at least 6 observations. “PcO.05 compared to vehicle control (0 mM ethanol).
20
10
0
1.06
4.25
17
85 Ethanol
n w (mM)
0
1.06
4.25
17
85
FIG. 5 Effect of alcohol, added in dilute form (7.8% w/v). on platelet aggregation induced by 0.5 pg/rnl collagen in human blood or PRP. Collagen produced irreversible platelet aggregation in both blood and PRP. Each bar represents the mean & SEM) of at least 6 observations. “PcO.05; **P
112
Vol. 78, No. 2
ALCOHOL AND PLATELET AGGREGATION
”
0
1.08
4.25
17
85 Ethanol
” (mM)
0
1.08
4.25
17
85
FIG. 6 Effect of alcohol, added in dilute form (7.8% w/v), on platelet aggregation induced by 0.5 mM arachidonic acid in human blood or PRP. Arachidonic acid produced irreversible platelet aggregation in both blood and PRP. Each bar represents the mean (k SEM) of at least 6 observations. *P
Vol. 70, No. 2
ALCOHOL
AND PLATELET AGGREGATION
dependent inhibition of ADP-induced aggregation in human whole blood, it had no effect on aggregation evoked by ADP in rat whole blood or PRP of either species. The basis for this difference in response is not clear, but might reflect differences in the effects of ADP in various preparations. Whereas ADP produces only primary aggregation in rat platelets (17), it causes irreversible, secondary aggregation and the associated release reaction in citrate-anticoagulated human PRP (18). Responses in heparin-anticoagulated human PRP are variable, perhaps reflecting direct actions of heparin on platelet responses (19). A difference in the extent to which ADP causes secondary aggregation and release reaction in platelet preparations might result in the observed differences in the effects of alcohol, but further studies would be necessary to test this hypothesis. In the present experiments, ADP stimulated irreversible aggregation in human PRP and whole blood anticoagulated with heparin, but we did not measure the platelet release reaction. Consistent with the view that the main inhibitory effect of alcohol is on the secondary phase of platelet aggregation, alcohol significantly inhibited the aggregation caused by collagen in human and rat PRP and whole blood; collagen-induced aggregation involves a secondary, irreversible phase. This speculation, that alcohol affects the second phase of aggregation, as opposed to the first, is in agreement with earlier reports (12,15,20). Previous studies have also demonstrated that alcohol affects secondary aggregation and release reaction of platelets, associated with inhibition of arachidonic acid release from platelet membranes, and thus subsequent metabolism to endoperoxides and thromboxane formation (21,22). Notably, primary aggregation induced by ADP is not dependent on thromboxane formation, and when we observed only reversible, primary aggregation (as in the ADP induced aggregation in rat platelets), alcohol had no effect. Although our data are generally consistent with this mechanism of alcohol action, we did find that 85 mM ethanol significantly inhibited arachidonic acid-induced aggregation in human and rat PRP, but not rat or human whole blood. Because addition of arachidonic acid to platelets bypasses the putative point in the arachidonic acid cascade at which alcohol acts (release of arachidonic acid from membrane phospholipids by phospholipases), this result is in apparent contradiction to that proposed mechanism of alcohol’s action (15). Alcohol probably has multiple effects on different levels of various platelet biochemical pathways which contribute to the overall inhibitory action, with the primary effect being inhibition of arachidonic acid release. Another interesting point regarding the arachidonic acid experiments was the contrast between inhibition of aggregation by alcohol in PRP and the lack of significant effect in whole blood of two species. The inhibition in PRP suggests alcohol interferes with one of the enzymes involved in thromboxane production from arachidonic acid (cyclooxygenase and thromboxane synthetase), because thromboxane is the major product in platelets. In whole blood, the effect of arachidonic acid is certain to be more complex. Arachidonic acid might affect other formed elements which in turn may release factors capable of influencing the platelet aggregation response. It also will be metabolized to numerous products, depending on cell type. Under those circumstances, inhibition of arachidonate metabolism might have less predictable effects than in PRP. In designing these experiments, we incorporated concentrations of alcohol deemed to be “physiologically relevant”. The lowest concentration tested (1.06 mM) is equivalent to a blood alcohol level of 5 mg/dl, which would not be associated with discernable behavioral effects, whereas the highest concentration (85 mM) is equivalent to blood alcohol of 391 mg/dl, which would produce profound intoxication (coma and death can occur above 500 mg/dl) (23). The exposure of blood to dilute alcohol is also more relevant to alcohol consumption than direct addition of absolute ethanol. Based on our present findings it appears that the major effect of alcohol consumption, at moderate doses, is inhibition rather than stimulation of platelet aggregation. This is consistent with the single report of which we are aware regarding whole blood platelet aggregation after ingestion of alcohol (24). Furthermore, because the effects of alcohol, when added in dilute form, are generally similar in PRP and whole blood, alcohol clearly has significant effects on the platelet itself, but we cannot rule out other actions through formed elements besides platelets without further studies. Assuming that inhibition of phospholipase A2 (and thus arachidonic acid release and metabolism) is a major mechanism for
114
ALCOHOL
AND PLATELET AGGREGATION
Vol. 78, No. 2
alcohol’s effect in platelets, it will be interesting to know how modification of arachidonate metabolism in other blood components by alcohol or its consumption (2526) affects platelet aggregation in whole blood. Finally, although it is tempting to speculate on how alcohol consumption might affect risk for thrombotic events such as myocardial infarction and thrombotic stroke, it is important to consider that alcohol consumption and its many effects are associated with other factors (such as diet, smoking, etc.), with complicated biological as well as sociological implications, making any conclusions tenuous. Regarding the experimental use of alcohol, however, it is quite clear that some precautions are necessary: our results suggest that when alcohol is used as a diluent for drugs, either for platelet aggregation studies or intravascular injections in experimental animals, proper controls are necessary, because of the potential for both platelet stimulatory and inhibitory actions. Acknowledgment This work was supported by a grant from the National Institutes of Health (AA09586). REFERENCES
5. 6. 7. 8. 9.
10. 11. 12. 13. 14. 15. 16.
MARMOT, M.G. Alcohol and coronary heart disease. Int J. Epidemiol 13,160-167, 1984. YANO, K., RHOADS, G.G. and KAGAN, A. Coffee, alcohol and risk of coronary heart disease among Japanese men living in Hawaii. N Eng J Med 297,405-409, 1977. GILL, J.S., ZEZULKA, A.V., SHIPLEY, M.J., GILL, S.K. and BEEVERS, D.G. Stroke and alcohol consumption. N Eng J Med 31.5, 1041- 1046, 1986. KLATSKY, A.L., FRIEDMAN, G.D. and SIEGELAUB, A.B. Alcohol consumption before myocardial infarction. Results from the Kaiser-Permanente epidemiologic study of myocardial infarction. Ann Intern Med 81,294-301, 1974. RENAUD, S. and DE LORGERIL, M. Wine, alcohol, platelets, and the French paradox for coronary heart disease. Lancet 339,1523- 1526, 1992. KLATSKY, A.L., ARMSTRONG, M.A. and FRIEDMAN, G.D. Alcohol and mortality. Ann Intern Med ZZ7,646-654, 1992. GILL, J.S., SHIPLEY, M.J, TSEMENTZIS, S.A., HORNBY, R.S, GILL, S.K., HITCHCOCK, E.R. and BEEVERS, D.G. Alcohol consumption- a risk factor for hemorrhagic and non-hemorrhagic stroke. Am J Med 90,489-497, 1991. Hillbom, M.E. What supports the role of alcohol as a risk factor for stroke? Acta Med Stand Suppl 717,93-106,1987, RUBIN, R. and HOEK, J.B. Alcohol-induced stimulation of phospholipase C in human platelets requires G-protein activation. Biochem J 254,147- 153, 1988. RUBIN, R. and HOEK, J.B. Inhibition of ethanol-induced platelet activation by agents that elevate CAMP. Thromb Res X%625-632, 1990. RENAUD, S.C., BESWICK, A.D., FEHILY, A.M., SHARP, D.S. and ELWOOD, P.C. Alcohol and platelet aggregation: The Caerphilly prospective heart disease study. Am J Clin Nutr55,1012-1017, 1992. HAUT, M.J. and COWAN, D.H. The effect of ethanol on hemostatic properties of human blood platelets. Am J Med 56,22-33, 1974. RUBIN, R. Ethanol interferes with collagen-induced platelet activation by inhibition of at-achidonic acid mobilization. Arch Biochem Biophys 270,99-l 13, 1989. ABI-YOUNES, S.A., AYERS, M.L. and MYERS, A.K. Mechanism of ethanol-induced aggregation in whole blood. Thromb Res 63,481-489, 1991. RAND, M.L., PACKHAM, M.A., KINLOUGH-RATHBONE, R.L. and MUSTARD, J.F. Effects of ethanol on pathways of platelet aggregation in vitro. Thromb Haemost 59, 383387, 1988. FENN, C.G. and LITTLETON, J.M. Inhibition of platelet aggregation by ethanol in vitro shows specificity for aggregating agent used and is influenced by platelet lipid composition, Thromb Haemost d&49-53, 1982.
Vol. 78, No. 2
ALCOHOL
AND PLATELET AGGREGATION
115
MACMILLAN, D.C. and SIM, A.K. A comparative study of platelet aggregation in man and laboratory animals. Thromb Diath Haemorrh 24, 385-394, 1970. 18. VARGAFTIG, B.B., CHIGNARD, M. and BENVENISTE, J. Present concepts on the mechanism of platelet aggregation. Biochem Pharmacol30,263-27 1, 198 1. 19. LAGES, B. and WEISS, H.J. Dependence of human platelet functional responses on divalent cations: aggregation and secretion in heparin- and hirudin-anticoagulated plateletrich plasma and the effects of chelating agents. Thromb Haemost 45, 173-179, 1981. 20. BENISTANT, C. and RUBIN, R. Ethanol inhibits thrombin-induced secretion by human platelets at a site distinct from phospholipase C or protein kinase C. Biochem J 269,489497, 1990. 21. RUBIN, R. Ethanol interferes with collagen-induced platelet activation by inhibition of arachidonic acid mobilization. Arch Biochem Biophys 270,99-l 13, 1989. 22. HWANG, D.H. Ethanol inhibits the formation of endoperoxide metabolites in human platelets. Prostaglandins Med 7, 511-513, 1981. 23. DUTTA, S.N. and PRADHAN, S.N. Alcohols. In: Pharmacology in Medicine: Principles and Practice. Pradhan, S.N., Maickel, R.P. and Dutta, S.N. (eds.), pp 285290, SP Press, Bethesda (1986). 24. MIKHAILIDIS, D.P., BARRADAS, M.A., EPEMOLU, 0. and DANDONA, P. Ethanol ingestion inhibits human whole blood platelet impedance aggregation. Am J Clin Path01 88, 342-345, 1987. 25. MEHTA, P., MEHTA, J., LAWSON, D. and PATEL, S. Ethanol stimulates prostacyclin biosynthesis by human neutrophils and potentiates anti-platelet aggregatory effects of prostacyclin. Thromb Res 48,653-661, 1987. 26. MAXWELL, W.J., KEATING, J.J., HOGAN, F.P., KENNEDY, N.P. and KEELING, P.W.N. Prostaglandin E:! and leukotriene B4 synthesis by peripheral leucocytes in alcoholics. Gut 30, 1270-1274, 1989. 17.