Inhibitory effect of alcohol on cyclic GMP accumulation in human platelets

Thrombosis

Pergamon

Research, Vol. 80. No. 2, pp. 143-151, 1995 Coovrieht 0 1995 Elsevier Science Ltd Primed m the USA. All rights reserved 0049-3848/95 $9.50 + .OO

0049-3848(95)00160-3

INHIBITORY EFFECT OF ALCOHOL ON CYCLIC GMP ACCUMULATION IN HUMAN PLATELETS Quan Sheng Dong, Barbara Wroblewska and Adam K. Myers Department of Physiology & Biophysics and Department of Biology, Georgetown University, Washington, DC 20007

(Received 14 March 1995by Editor R. Kinlough-Rathbone;revised/accepted 2 August 7995)

Abstract

The effects of ethanol on cyclic GMP (cGMP) in washed human platelets were studied in the presence and absence of sodium nitroprusside (SNP), a nitric oxide donor which stimulates guanylate cyclase. SNP stimulated cGMP accumulation in a dose-dependent fashion. After 1 min exposure to 100 FM SNP, the level of cGMP was approximately four-fold that in vehicle-treated platelets. Alcohol had no effect on basal cGMP, but inhibited SNP-induced cGMP accumulation at 17, 85 and 170 mM. In further experiments, platelets were incubated for 0,0.5, 1, 2 or 5 min with 10 FM SNP, with or without 100 yM zaprinast, a selective cGMPphosphodiesterase (PDE) inhibitor and 85 mM ethanol. In the presence of zaprinast but not alcohol, cGMP levels rose continuously, to IO-fold the basal level at 5 min. Without zaprinast, cGMP levels were lower and reached a plateau by 2 min. Accumulation of cGMP was attenuated by alcohol 2 and 5 min after SNP addition, both in zaprinast-treated platelets and those without zaprinast. Thus, alcohol inhibits platelet cGMP accumulation stimulated by a nitric oxide donor. Its mechanism probably does not involve a major effect on PDE, because the inhibition was observed in the presence or absence of zaprinast. We hypothesize that alcohol inhibits guanylate cyclase, contributing to its complex functional effects in platelets.

Interest in alcohol consumption and its role in cardiac and cerebral vascular diseases has grown due to reports that moderate or light alcohol consumption reduces risk of myocardial infarction, stroke and associated mortality (l-7). In the “French paradox”, regular alcohol consumption is a possible factor in the low incidence of coronary heart disease in the French, whose intake of saturated fats and cholesterol is high (6). -----------Key words: alcohol, ethanol, cyclic GMP, zaprinast, M&B22948, platelets Corresponding author: Adam K. Myers, Ph.D., Department of Physiology & Biophysics, Georgetown University Medical Center, 3900 Reservoir Rd. NW, Washington, DC 20007 USA 143

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A proposed mechanism for the apparent beneficial effect of alcohol is inhibition of platelet function. Platelets have a central role in the pathology of myocardial infarction and stroke, and many studies have demonstrated that alcohol can inhibit in vitro platelet aggregation (8-13). Inhibition of arachidonic acid release and metabolism apparently has a role in this effect of alcohol on aggregation (10,ll). However, a broad literature shows both stimulatory and inhibitory effects of alcohol on platelets (8-19). When early events in the platelet activation cascade have been examined, some studies have found that alcohol is stimulatory, although high concentrations of alcohol are generally required. For example, alcohol, added at concentrations of up to 300 mM to platelets in ~litr’o, stimulates phospholipase C and production of inositol phosphates through a G-protein dependent mechanism, mobilizes intracellular Ca++, and stimulates platelet shape change (18,19). The nitric oxide-cGMP signaling pathway has been extensively studied in many tissues such as brain and blood vessels (20), but has received less attention in platelets. The presence of guanylate cyclase (GC) and cGMP in platelets has been recognized for some time, and studies have demonstrated that stimulation of cGMP accumulation in platelets by compounds such as sodium nitroprusside (SNP) results in inhibition of platelet aggregation (21-23). This elevation in cGMP results in inhibition of agonist-induced increases in free intraplatelet Ca++ (24), an effect similar to that produced by CAMP (25). In addition, cGMP and CAMP have important synergistic actions in platelets, apparently due to inhibition of CAMP breakdown by cGMIP (26). Radomski et al. (27) recently reported that platelets can synthesize NO from L-arginine, and this pathway has a potentially important regulatory role in the platelet activation cascade. Agonist-induced stimulation of platelet aggregation produces a rise in platelet cGMP but not CAMP (27,28), and experiments in platelets and platelet cytosol demonstrate that this elevation in cGMP is related to NO generation from L-arginine (27). Radomski et al. (27) hypothesize that this might be an intra-platelet negative feedback mechanism which regulates platelet reactivity. In addition, the CC-cGMP system might respond to NO produced by the vascular wall; in this regard, synergism between NO and substances which stimulate adenylate cyclase (prostacyclin, for example) (26,29) could be important. The effects of alcohol on this platelet signal transduction pathway involving NO, GC and cGMP have not been studied. Based on the complex effects of alcohol on the platelet activation cascade, we hypothesized that alcohol might affect this signaling pathway, and as a first step, we examined its effects on cGMP production stimulated by the NO donor sodium nitroprusside in washed human platelets. METHODS Preparatiorl

of washed hltmurr platelets

Venous blood was collected from healthy, aspirin- and alcohol-free volunteers into plastic tubes containing l/10 volume of 3.8% sodium citrate. Blood was centrifuged at 170 x g for 15 min at room temperature. The supernatant, platelet-rich plasma (PRP), was collected and centrifuged at 645 x g for 15 min to sediment the platelets. The platelet pellet was washed by resuspension with a buffer (pH 7.4) containing [mM] NaCl 113, Na2HP04 4, NaH2P04 24, KH2P04 4, and glucose 5, supplemented with 50 nM PGEl, and 50 pglml apyrase. After centrifugation again at 645 x g for 15 min, platelets were finally resuspended with HEPES-buffered Tyrode solution (pH 7.4) composed of [mM]

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NaCl 134, NaHC03 12, KC1 2.9, CaCl2 2, NaH2P04 0.36, MgC12 I, HEPES 5, and glucose 5, supplemented with 0.5 mg/ml bovine serum albumin and 50 Kg/ml apyrase. Platelets were counted by phase contrast microscopy. The mean (& SEM) platelet count obtained in the washed platelet suspensions was 273,000 + 32,000/@. Experimental Desigtl To determine effects of drugs on platelet cGMP, 175 ~1 aliquots of the washed platelet suspension were added to siliconized cuvettes and equilibrated at 37OC for 8 min. Subsequently, they were placed in the well of a Chrono-log aggregometer (Model 560VS) and stirred at 1000 rpm with a magnetic stir bar for 4 min at 37OC before the addition of any drug or vehicle. Next, two series of protocols were carried out. In the first series, platelet aliquots were incubated with alcohol alone (25 pl of ethanol solution, to produce a final concentration of 0, 17, 85, or 170 mM), 25 ~1 SNP alone (final concentration 0, 1, 10, or 100 FM), or the same concentrations of alcohol added 0.5 min before 10 FM SNP. The reaction was terminated 1 min after the last drug was added by the addition of 28 11 1 N HCl. After centrifugation at 12,400 x g for 1 min, 150 ~1 of supernatant was collected and neutralized with 15.3 ~1 1 N NaOH. Following brief vortexing, 50 ~1 of this preparation was used for the cGMP assay. In the second series of protocols, 25 1.11of the selective cGMP phosphodiesterase inhibitor zaprinast (25) (final concentration 100 yM), ethanol (final concentration 85 mM), SNP (final concentration 10 PM) or their vehicle (platelet resuspension buffer) were added sequentially to 175 ~1 washed platelet aliquots at 0.5 min intervals in the above sequence. The reaction was stopped at 0,0.5, 1, 2, or 5 min after SNP treatment by addition of 1 N HCl. The rest of the procedures were the same as above. cGMP assay cGMP levels in the samples were measured by RIA using a well-characterized commercial kit (Amersham) routinely used in this laboratory, with proper quality control. The assay uses [ ‘25I]-cGMP as the tracer. The antiserum has less than 0.0075% crossreactivity with other guanine and adenine nucleotides. Separation of bound and unbound cGMP is based on a magnetic separation technique, but the assay is otherwise a standard RIA. Data analysis cGMP content in platelets was standardized according to the actual platelet count and expressed as pmol cGMP/lOg platelets. Levels were compared between groups by Student’s t test for unpaired data, or by ANOVA with multiple comparisons to the control group by Dunnett’s t, as appropriate. All data are expressed below as mean + SEM. Materials Pure ethyl alcohol (Warner-Graham Co.) was diluted on the day of use with HEPESbuffered Tyrode solution to the appropriate concentrations for the experiments above. Sodium nitroprusside (Sigma) was prepared with the same buffer as a 5 mM stock solution which was stored at -2OOC and diluted with buffer daily. Zaprinast (M&B22948; Sigma) was dissolved in 0.1 N NaOH at a concentration of 1 mM; aliquots were stored at -8OOC until the day of use. [ lZsI]-Cyclic GMP assay kits were purchased from Amersham. PGEl was obtained from Biomol and HEPES from Calbiochem; all other reagents were purchased from Sigma.

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RESULTS Incubation of washed human platelets with 1 to 100 pM SNP produced a dosedependent increase in cGMP levels after 1 min (fig. 1). This increase was statistically significant at 10 and 100 pM SNP (~~0.05 and 0.01, respectively, compared to the vehicle-treated control platelets). The cGMP level in vehicle-treated platelets was 1.4f0.3 pmol/lOa platelets; cGMP in the 100 pM SNP group was approximately fourfold this basal level.

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Effect of SNP on platelet cGMP accumulation. Washed human platelets were incubated for 1 min with SNP or its vehicle and cGMP was assayed by RIA. Levels were significantly higher in the 10 and 100 uM SNP groups compared to the controls (0 yM SNP). *p
17

85

Ethanol

(mM)

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FIG. 2 Effects of alcohol, added in dilute form (see text) on human platelet cGMP content after 1 min of incubation. No significant differences were observed between cGMP content of ethanol-treated platelets and vehicle-treated platelets (0 mM ethanol group).

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When washed platelet samples were incubated for 1 min with 17, 85 or 170 mM ethanol, no effect on basal cGMP was observed, although there was a statistically non-significant tendency toward depression of cGMP by ethanol (see Fig. 2). Basal cGMP in this experiment was 1.4kO.2 pmol/108 platelets. Pretreatment of platelets with 17, 85 or 170 mM ethanol before addition of 10 pM SNP resulted in significant inhibition of SNP-stimulated cGMP accumulation after I min (~~0.01; fig. 3). When platelets were treated with SNP alone, a 2.0-fold higher cGMP level was observed, compared to the basal level of 1.7 + 0.2 pmol/lOs platelet. In the final series of experiments (fig. 4), the time-dependent effects of SNP and ethanol on cGMP accumulation in platelets were studied, in the presence and absence of 100 pM zaprinast. The basal level of cGMP in the absence of ethanol or zaprinast, before SNP treatment, was 1.0 f 0.1 pmol/lO8 platelets. Exposure of platelets to 10 p.M SNP, in the absence of other drugs, resulted in a time-dependent elevation of cGMP, which reached a plateau within 2 min. In platelets pretreated with 85 mM ethanol 30 set before SNP exposure, cGMP accumulation was reduced compared to samples treated only with 10 pM SNP, with the difference reaching statistical significance after 2 and 5 min of SNP incubation (~~0.05 and ~~0.01, respectively). When platelets were pretreated with 100 l.tM zaprinast, followed by 10 pM SNP, cGMP levels continued to rise over the 5 min incubation with SNP, attaining levels approximately lo-fold those in the 0 min zaprinast treatment group (controls). In the presence of PDE inhibition, 85 mM ethanol again inhibited cGMP accumulation at 2 and 5 min of SNP exposure (~~0.05).

Ethanol

WW

SNP

WV:0

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::::. :::;_ ::::. ::::_ .‘.....‘. ::::. * * ::::. ::::, . . . ..,._. :.:.:.:.: ::::. ,... ...,._. :.:.:.:.I . . . . ... ,. . . ., . 1; . 0

17

10

10

li0

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FIG. 3 Effect of alcohol on SNP-stimulated cGMP accumulation. Dilute ethanol (see text) or its vehicle were added 1 min before SNP (final concentration, 10 PM) or its vehicle. Samples were incubated for an additional 1 min, and cGMP was assayed by RIA. SNP alone produced a significant accumulation of cGMP, this was significantly attenuated by pretreatment with ethanol. **p
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ZaprinestfSNP EthanollZaprinastlSNP SNP Ethanol/SNP

iI123456 time

(min)

FIG. 4 Effect of alcohol (85 mM) on SNP-induced (10 pM) cGMP accumulation in the presence and absence of zaprinast, a cGMP-phosphodiesterase inhibitor. Ethanol pretreatment significantly inhibited cGMP accumulation at 2 and 5 min of SNP incubation, regardless of phosphodiesterase inhibition. apc0.05; **p
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cGMP accumulation in platelets. Our results establish that alcohol has an inhibitory effect on the cGMP accumulation stimulated by the NO donor SNP. SNP induced a dose-dependent rise in platelet cGMP, which was inhibited by concentrations of ethanol as low as 17 mM (78 mg%), a level which can be achieved by moderate drinking. An inhibitory effect has been observed in other tissues, for example, in cultured porcine coronary vascular smooth muscle cells (30). It has been suggested that inhibition of endothelium-dependent vasodilation associated with the NO-cGMP system by alcohol might account for the angina which accompanies alcohol ingestion in some patients (30). Because alcohol inhibited platelet cGMP production in either the presence or absence of the cGMP-phosphodiesterase inhibitor zaprinast, the mechanism for the inhibition of cGMP accumulation apparently does not involve an action (stimulation) on the platelet PDE. Another indication that alcohol does not stimulate PDE is the finding that basal platelet cGMP was not significantly reduced by alcohol treatment. Rather, alcohol appears to either inhibit GC, or enhance the degradation or block the action of NO. A minor inconsistency in our results is that in the studies illustrated in fig. 3, ethanol concentrations of 17, 85 and 170 mM produced inhibition of cGMP in platelets stimulated for 1 min with SNP, while in subsequent studies (fig. 4), inhibition of cGMP accumulation by 85 mM ethanol was not significant until after 2 min of SNP exposure, compared to control platelets. This discrepancy likely reflects differences between groups of subjects (human volunteers), or some difference in experimental design. Nonetheless, relatively low concentrations of alcohol are capable of blocking cGMP accumulation in platelets. This finding, while inconsistent with the widely reported inhibition of platelet aggregation by alcohol (8-14), is consistent with observations that early events in the platelet activation cascade (rise in intraplatelet free Ca ++, IPJ production, platelet shape change) are stimulated by alcohol (17-lo), as discussed above. However, these observations have been usually made at relatively high concentrations of alcohol, while the present experiments have been performed at lower, more physiologically relevant levels. In light of the conflicting evidence, how in V~VO hemostatic balance might be affected by platelet exposure to alcohol is difficult to assess. Platelet aggregation studies are sometimes criticized as being “unphysiological”, representing only the end-event in a complex cascade; early signaling events, while avoiding that criticism, are difficult to assess in terms of functional relevance. Stimulation of early steps of the platelet activation cascade might be directly detrimental, promoting thrombosis, but repeated, low level stimulation might also inhibit platelets through desensitization or related mechanisms. We also do not yet know how alcohol affects the rise in cGMP associated with platelet activation, as opposed to basal or SNP-stimulated cGMP levels. Clearly, more work needs to be done, both in the biochemical and molecular effects of ethanol, and in its effects on platelet physiology. In summary, these studies show a new effect of alcohol in platelets: inhibition of cGMP accumulation. While it is noteworthy that this effect occurs at alcohol concentrations as low as 17 mM, well within the range of blood alcohol levels attained by moderate drinking, more studies are needed to determine how overall, in vivo hemostatic balance will be affected through this mechanism. Additional studies will also be required to define what step in the cGMP second messenger pathway is affected by alcohol, but it appears at present that inhibition of GC is a likely mechanism. Future experiments should also focus on how the whole of the L-arginine-NO-cGMP pathway is affected by

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alcohol, what specific mechanisms are involved, and how alcohol affects the rise in cGMP levels which is associated with platelet aggregation. Acknowledgment This work was supported by a grant from the National Institutes of health (AA09586). REFERENCES 1. ST. LEGER, AS., COCHRANE, A. L. and MOORE, F. Factors associated with cardiac mortality in developed countries with particular reference to the consumption of wine. Lancet i, 1017-1020, 1979. 2. YANO, K., RHOADS, G.C., and KAGAN, A. Coffee, alcohol and risk of coronary heart disease among Japanese men living in Hawaii. New Eng J Med 297, 405 409, 1977. 3. MARMOT, M.G. Alcohol and coronary heart disease. Int J Epidemiol 13, 160-167, 1984. 4. GILL, J.S., ZEZULKA, A.V., SHIPLEY, M.J., GILL, S.K., BEEVERS, D.G. Stroke and alcohol consumption. New Eng J Med 315, 1041-1046, 1986. 5. 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. 6. RENAUD, S. and DE LORGERIL, M. Wine, alcohol, platelets, and the French paradox for coronary heart disease. Lancet 339, 1523-1526, 1992. 7. KLATSKY, A.L., ARMSTRONG, M.A. and FRIEDMAN, G.D. Alcohol and mortality. Ann Int Med I 17,646-654, 1992. RUBIN, R. Effects of ethanol on platelets. Lab Invest 63, 729-732, 1990. ;: 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 59,383-387, 1988. 10. MIKHAILIDIS, D.P., BARRADAS, M.A. and JEREMY, JY. The effect of ethanol on platelet function and vascular prostanoids. Alcohol 7, 171-180, 1990. 11. RUBIN, R. Ethanol interferes with collagen-induced platelet activation by inhibition of arachidonic acid mobilization. Arch Biochem Biophys 270, 99-l 13, 1989. 12. RAND, M.L., GROSS, P.L., JAKOWEC, D.M., PACKHAM, M.A. and MUSTARD, J.F. In vitro effects of ethanol on rabbit platelet aggregation, secretion of granule contents, and cyclic AMP levels in the presence of prostacyclin. Thromb Haemost 61, 254-258, 1989. 13. VEENSTRA, J., KLUFT, C., OCKHUIZEN, T., VAN DER POL, H., WEDEL, M. and SCHAAFSMA, G. Effects of moderate alcohol consumption on platelet function, tissue-type plasminogen activator and plasminogen activator inhibitor. Thromb Haemost 63, 345-348, 1990. 14. TORRES DUARTE, A.P., YOUNG, J., ABI-YOUNES, S.,DONG, Q.S. and MYERS, A.K. Inhibition of platelet aggregation in whole blood by alcohol. Thromb Res (in press), 1995. 15. ABI-YOUNES, S.A., AYERS, M.L. and MYERS, A.K. Mechanism of ethanolinduced aggregation in whole blood. Thromb Res 63,481-489, 1991. H. and 16. HILLBOM, M., KANGASAHO, M., KASTE, M., NUMMINEN, VAPAATALO, II, Acute ethanol ingestion increases platelet reactivity: Is there a relationship to stroke? Stroke 16, 19-23, 1985.

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17. STUBBS, C.D. and RUBIN, R. Effect of ethanol on platelet phospholipase AZ. Lipids 27,255260, 1992. 1X. RUBIN, R., PONNAPPA, B.C., THOMAS, A.P. and HOEK, J.B. Ethanol stimulates shape change in human platelets by activation of phosphoinositide-specific phospholipase C. Arch Biochem Biophys 260,480-492, 1988. 19. 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. 20. MONCADA, S., PALMER, R.M.J., HIGGS, E.A. Nitric Oxide: Physiology, pathophysiology, and pharmacology. Pharmacol Rev 43, 109-142, 1991. 2 1. ANDERSON, T.L.G. and VINGE, E. Interactions between isoprenaline, sodium nitroprusside, and isozyme-selective phosphodiesterase inhibitors on ADPinduced aggregation and cyclic nucleotide levels in human platelets. J Cardiovasc Pharmacol l&237-242, 199 1. 22. IVANOVA, K., SCHAEFER, M., DRUMMER, D. and GERZER, R. Effects of nitric oxide-containing compounds on increases in cytosolic ionized Ca*+ and on aggregation of human platelets. Eur J Pharmacol244, 37-47, 1993. 23. WATANABE, H., KAKIHANA, M., OHTSUKA, S., ENOMOTO, T., TASUl, K. and SUGISHITA, Y. Platelet cyclic GMP. A potentially useful indicator to evaluate the effects of nitroglycerin and nitrate tolerance. Circulation 86, 29-36, 1993. 24. YAMANISHI, J., KAWAHARA, Y. and FUKUZAKI, H. Effect of cyclic AMP on cytoplasmic free calcium in human platelets stimulated by thrombin: Direct measurement with quin2. Thromb Res 32, 183188, 1983. 25. KAWAHARA, Y., YAMANISHI, J. and FUKUZAKI, H. Inhibiting action of guanosine 3’, S-monophosphate on thrombin-induced calcium mobilization in human platelets. Thromb Res 33, 203-209, 1984. 26. MAURICE, D.H. and HASLAM, R.J. Molecular basis of the synergistic inhibition of platelet function by nitrovasodilators and activators of adenylate cyclase: Inhibition of cyclic AMP breakdown by cyclic GMP. Mol Pharmacol 37, 67 l-681, 1990. 27. RADOMSKI, M.W., PALMER, R.M.J. and MONCADA, S. An L-arginine/nitric oxide pathway present in human platelets modulates platelet aggregation. Proc Nat1 Acad Sci 87, 5 193-5 197, 1990. 2X. SEVERINA, I.S. and BELUSHKINA, N.N. Increase in activating ability of human platelet guanylate cyclase during aggregation. Biochem Int 28, 621-63 1, 1992. 29. RADOMSKI, M.W., PALMER, R.M.J. and MONCADA, S. The anti-aggregating properties of vascular endothelium: interaction between prostacyclin and nitric oxide. Br J Pharmacol 92,639-646, 1987. 30. KlSHI, Y., ONIKI, T., ASHIKAGA, T. and NUMANO, F. Ethanol modulates cyclic GMP metabolism in cultured coronary smooth muscle cells, Angiology 44 fsuppl. I), 62-68, 1993.