THROMBOSIS RESEARCH 63; 481-489,199l 0049-3848/91 $3.00 + .OOPrinted in the USA. Copyright (c) 1991 Pergamon Press pk. All rights reserved.

MECHANISM OF ETIIANOL INDUCED .;ZGGRECATION IN WHOLE BLOOD

Sl,lvie

A. Abi Younes,

Georgetown

Department Llniversity

(Received 28.2.1991

accepted

Michael

L. A),ers

and .4dam K. ?il)‘ers

of Physiolog) & Biophysics Medical Center. Washington. in revised

form 28.6.1991

by Editor

DC 20007 E.I.B.

Peerschke)

ABSTRACT Effects of ethanol on blood clotting and platelet aggregation have been reported in man)’ models. but its in vitro actions in whole blood. impedance aggregometrl’ have not been reported. We investigated the effect of ethanol in vitro in whole blood and platelet rich plasma of humans and rats. as measured b>, impedance aggregometry. Ethanol (39 to I”0 mM) induced concentration-dependent aggregation in whole blood but not platelet rich plasma. In further studies in rats. aggregation was inhibited by pretreatment of whole blood with the prostacyclin analog iloprost or the enzyme apl’rase. which degrades ADP to AMP. Levels of ethanol which produced aggregation in whole blood were also associated with concentration -dependent hemolysis. Based on the requirement for whole blood for ethanol-induced aggregation. the inhibitor), effect of apyrase and our observation of hemolysis. and previous studies whicn hdve demonstrated the potential contribution of ADP from I\,sed red hlgjod cells to platelet aggregation. we conclude that ethanol-induced aggregation in whole blood is mediated h\, er)‘throcyte I\,sis and the ADP released from these cells.

JNTRODL’CTION Several techniques have been used to stud!, effects of ethanol on platelet function. both in vivo as well as ex vivo cl-‘). Ethanol treatment generalI>, inhibits platelet aggregation and secretion in vitro in response to several agonists. including collagen. thrombin. platelet activating factor and ADP (1.2.5). The mechanisms b!, which ethanol deranges cell function remain unclear. Ethanol alters membrane fluidity and can affect several membrane-associated enzymes (8.9) and signal transduction pathways (10). Different studies have focused on the effect of ethanol vn adenylate cyclasc and phospholipase A2 and C (1.2.1 1 13).

platelet

Although studies of platelet function preparations have given invaluable

Ke\. words:

XDP. apyrase.

ethanol.

in platelet information.

hcmol),sis. 481

platelet

rich plasma (PRP) and washed it could prove useful to

aggregation.

whole

blood

ETHANOL-INDUCED

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AGGREGATION

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study platelet responses to ethanol in whole blood (IL~J In whole blood impedance aggregometry. effects of handling and centrifugation during pt’eparation 01 PltP :I’ I_ eliminated. and potentialI), important changcb in platelet function due ta! l!rhci Red blood cells could influence platelet bchavlol formed elements arc preserved through ADP and nucleotide release (15 1’). and by modification of prostac\cIin metabolism (I 8.19). uptake of adenosine 120) and inhibitiljn i)f the action, of cndu thelium derived relaxing factor- !EDRFi I 2 1 ,721, for example. Lcukoc),te> can als~~ potentially influence platelet responses through their capacit)’ to product ar‘achi donic acid metabolites (23,241. platelet activating factor !_‘Sl. and EDRF i.!O! I’hub impedance based platelet aggrcgomctry in whole blood ha\ been used in attempts to mimic the in viva situation more closeI\. C14.2’) Mikhailidis et al. (28) investigated the effect uf ethanol on whole blood platelet aggregation. and observed that ethanol significantly inhibited collagen and ADP induced aggregation 30 min after ethanol ingestion. In contrast, during the course of other whole blood aggregation studies. we observed that addition of ethanol to heparinized whole blood of rats induced a large. reversible aggregation response Thus. we further investigated this effect of ethanol in whole blood of rats and humans in an attempt to determine its mechanism. S

Blood Collection

and Platelet

. : PreDm

Human blood was collected from the antecubital veins of healthy. male and female. aspirin-free volunteers into syringes containing heparin (10 U/ml final concentration). Adult male Wistar rats were purchased from Charles River Breeding Laboratories and maintained on standard rodent diet and water ad libitum. .4fter anesthesia of rats with sodium pentobarbital (50 mg/kg. ip). a midline abdominal incision was made and blood was collected from the abdominal aorta into syringes containing sodium citrate (final concentration 0.38%) or heparin (20 U/ml). PRP was prepared by centrifugation of whole blood at IO0 x g for 15 min: platelet poor plasma (PPP) was prepared by centrifugation of the pellet (I200 x g for 15 min) after PRP was decanted. INas

and Chemical3

Collagen suspension for platelet aggregation (I mg/ml) was obtained from Chronolog. Ethanol was obtained from the Warner Graham Co. Apyrase from potato (4.1 ATPase and 3.8 ADPase U/mg protein) was purchased from Sigma Chemical Co and dissolved in 0.9% NaCI. Iloprost (Berlex Laboratories). a prostacyclin analog. was also dissolved in 0.9% NaCI. Heparin from porcine intestinal mucosa (20.000 U/ml) was purchased from Organon and diluted in 0.9% NaCl to ).icld a concentration of 400 U/ml prior to use in blood. Sodium citrate (Sigma) was prepared as a 3.8% solution in water.

Aggregation experiments were performed using a Chronolog aggrcgometer (Model 56OVS) capable of measuring aggregation by either impedance (whole blood or PRP aggregation studies) (14) or turbidometric methods (PRP aggregation studies, (29). In impedance aggregometry studies, whole blood or PRP was incubated at 37°C: 1 ml aliquots were stirred at 1000 rpm. In some experiments. the samples were incubated with various drugs (iloprost. I min: apyrase. _ZJmin) or vehicle (0.9% NaCI) prior to addition of the aggregating stimulus. Collagen or ethanol was added to induct

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483

ichange in impedance (Ri within 5 aggregation, which was measured as maximum min after stimulation. In the turbidomctric aggregomctr\. experiments. 0.5 ml aliquots of PRP were incubated at 37OC and stirred at 1000 rpm. Various doses of ethanol or collagen were added and aggregation was measured as percent change in optical density. with the instrument calibrated IO y.ield 0% change in optical density, In both for PRP and with PPP as the 100% standard for change in optical density. turbidometric and impedance aggregometry experiments. undiluted ethanol was added to the stirred samples in a bolus injection to achieve the final indicated concentration.

Ethanol was added to achieve the concentrations indicated below to 0.5 ml aliquots of titrated or heparinized whole rat blood at room temperature in Eppendorf tubes. After this bolus addition of ethanol, the tubes were inverted several times and then centrifuged in an Eppendorf microfuge for 5 min and the absorbance of the supernatant was determined at 410 nm. corresponding to the Sore1 peak for hemoproteins. Reference values were obtained from supernatant from the same amount of blood treated with distilled water to effect 100% hemolysis and from untreated blood (0% hemolysis). Hemolysis was then expressed as percent masimum, hased on absorbance.

All data were expressed as mean i SEM. Statistical comparisons of aggregation results between drug treatment groups and controls were made with analysis of variance (ANOVA), followed by Dunnett’s t test for multiple comparisons between experimental and control groups. Hemolysis results were compared between heparinized and titrated whole blood samples treated with equal concentrations of ethanol by Student’s t test for unpaired data. Statistical significance was assumed fat Pi 0.05.

‘The addition of ethanol (34, 85 or 170 mM. final concentration) to heparinized. whole blood of rats induced a rapid. concentrationdependent and reversible platelet aggregation response, as determined by measurement of impedance (fig. 1.2). The same or higher concentrations of ethanol (up to 340 mM) had no aggregatory effect in rat whole blood anticoagulated with sodium citrate (data not shown). When ethanol was added to heparinized rat PRP (as opposed to whole blood). no aggregation response occurred. measured either as change in impedance (fig. 2) or optical density (data not shown). In contrast to these results. collagen. which, like ethanol. produced concentrationdependent aggregation in whole rat blood. induced aggregation in PRP as well, measured by impedance aggregometry (fig. 2). The addition of ethanol (42. 85 or 170 mM final concentration) also induced concentration dependent aggregation in heparinized human whole blood (fig. 2). The aggregation response in human blood was characterized by a rapid primary phase as seen in rat whole blood. but unlike the rat tracings illustrated in fig. 1. this primary phase in phase of aggregation. human whole blood was followed by a less rapid. irreversible Heparinized PRP from two volunteers was tested and found to have no response (measured as change in impedance) to ethanol ;at concentrations up to 340 mM (data nor shown)

484

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,

5 mln

AGGREGATION

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,

20-

170 mM

o-

Apyrare

Figure 1. Representative tracings illustrating the aggregatory effect of ethanol in whole. hcparinized rat blood, measured as change in impedance (left). and the inhibitory effect of apyrase on ethanol induced (170 mM) aggregation (right).

25 20

--Ef

WB. rat

+

WE, rat

-f-

WE. human

+

PRP. rat

+

PRP. fat

1 T

0

10

100 Ethanol

Figure

1000 (mM)

.l

k 1

Collagen

10

(ug/ml)

2. Platelet aggregatory effects of ethanol (left) or collagen (right) in whole rat blood (WB: open squares), whole human blood (solid triangles) and rat platelet rich plasma (PRP: solid squares). measured by impedance aggregometry (n=6 or more for each point). Collagen induced concentration-dependent aggregation in both whole blood and PRP: ethanol only produced aggregation in whole blood.

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AGGREGATION

0 0

10-g lloprost

10’8 (M)

0

0.06 Apyrase

0.6 (U/ml)

Figure 3. Effects of ilOprcJSt (left. n;6) and aphrase (right: n-5 on ethanols induced l170 rnYv1)apgrcgati )n of vh~~le rat blcx)d, measureJ by, impedance agpregomctry’. “P(O.01 compared to the control rr~~up

When heparinized uhole blood of rats was incubated with either ilopr‘l)st ‘1~ apyrase before addition of ethanol (final concentration of ethanol: 170 m51). the aggregation response (measured by impedance aggreglmetry) was inhibited in A concentration dependent manner by both drugs (fig. 1.3). The inhibitory actrun of iloprost was nearly complete and was statistically, significant at 10 xhl (pc0 01 : tile inhibitory effect of apyrase on ethanol induceaJ aggregation was statisticall\, significant at 0.06 and 0.6 II ml (p,sis uccurred in both the heparinized and titrated samples. and kas concentration dependent over the same range that concentration dependent aggregation was seen in the heparinized blood. However. ethanol had no aggregator)’ action when citrate anticoagulation was used. ‘This apparent conflict in our results is readill, explained b) the fact that ADP has no aggr‘opator)’ effect in titrated whole rat hluod at IO times higher concentrations than those producing a full primar) agpr‘cgatiun response in hepar‘inizcd rat blood (unpublished observations in our laborator),). ‘Thus. we conclucle. based on the results of our hemolysis studies combined with the inhibitur? action of ap)‘rase. that ethanol-induced aggregation in heparinizcd whole blood is mediated b). red blood ccl1 I),sis and release of ADP. Considerable evidence now exists that alcohol consumption is an independent risk factor for both hemorrhagic and ischemic stroke (30 351. among other platelet used in CKJI‘ related cardiovascular disorders. In view of the in vitro approach experiments and the method of ethanol addition to blood, it is inappropriate to attempt to directly correlate our observations to the increased incidence of cardiovascular accidents of various types in alcohol intoxication and alcoholism. llowcver. the data arc of interest due to the marked actions of ethanol observed and the potential for in viva effects. should these be borne out b>, future studies. intravascular administration of alcohol in humans (36) and animals (17) produces hemolysis and thrombocytoptnia, and marked, transient thrombocytopcnia i 1.3) and hemolysis (37) are sometimes observed with high dose alcohol ingestion. Because hcmol)‘sis is known to release ADP (15.16). it is possible that cr)‘throcytc derived r\DP could be a factor in the thrombocytopenia. through its effects on platelet adhesion and aggregation. It should also be noted that ethanol has been ubscrved to haLe both stimulatory and inhibitory actions on platelets in numerous reports 11.2). and man! issues remain to be resolved concerning its effects. In brief. it appears at present that ethanol can have both direct stimulatory (32.38,39.40) and inhibitor). Ci.12.13,38.41) effects on platelet function in PRP or washed platelet: red blood cells. if present. provide an additional pathu’a!, for platelet stimulation b). ethanol. Finall\,. our findings are also of interest from a technical standpoint. Although small volumes of ethanol can sometimes be used in turbidometric aggregation studies without obscuring the actions of ethanol- soluble drugs, investigators should consider the possible contribution of hemol>,sis caused b>, ethanol or other organic solvents

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AGGREGATION

when used in whole blood platelet aggregation experiments. An interesting specul ation is that our model system of heparinizcd whole blood and impedance aggrepo metr)’ may prove to be a useful method for studying the potential contribution of cr)‘throcytes to platelet aggregation in a ph),siolopical setting. as well as for inbestlRatInK the possible effects of ethanol on er\‘thloc)‘te platelet interactions

LED-

..

‘This work was supported in part by a grant from the National HL43160. lloprost was a gift from Berlcs Laboratories

Institutes

of llealth

I.

MIKHAILIDIS. M.A., BARRADAS .4. and JEREMY. J.Y. The effect of ethanol platelet function and vascular prostanoids. .-l/cohol 7 171-180. 1990.

2.

RUBIN. R. Effects

3,

COWAN’, D.Il. and GRAHAM. R.C.. Jr. Studies on the platelet Thromb. Diathes. Haemorrh. 33. 310 327. 1975.

‘i

NEIMAN. J,. BEVING. H. and MALMCREN. R. Platelet uptake of serotonin (5 III’) during ethanol withdrawal in male alcoholics. Thromb. Res. 46. 803 809. 198~.

5. /,

R.4ND. ML.. PACKIIAM. of ethanol on pathwa),s 383-387. 1988.

6.

RAND. M.L., GROSS, P.L., JAKOWEC. D.M.. PACKHAM. M.A and MLiS’l’XRD 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. Haemosr. 6 1. 254-258. 1989.

-7

HILLBOM. M.. MUURONEN. A., LOWBEER. C., ANGGARD. E.. BEVING. II. and thromboxane formation and bleeding time is influenced KANGASAHO. M. Platelet by ethanol withdrawal but not by cigarette smoking. Thromb. Ilaemosl. 5.3. 419422. 1985.

8.

of Ethanol

on platelets.

Lab. Inr;esl.

63, 729

732. defect

on

1990 in alc~~holism.

M.A., KINLOUGH RATIIBONE. R.L. and MUSTARD. J.F. Effects of platelet aggrenation in vitro. Thromb. Haemosr 59.

TARASCHI. T.F. and RUBIN. E. Effects of ethanol on the chemical and structural properries of biologic membranes. Lab. Intesl. 52. 120. 131, 1985.

9.

CIII. L. M.. WU. W.. SUNG, K.L.P. and CIIIEK‘. S. Biophysical correlates of Iysophosphatidylcholineand ethanol-mediated shape transformation and hemolysis of human erythrocytes. Membrane viscoelasticity and N41R measurement. Biochjm. Bioph),s. .4cta 1027. 163- 17 1, 1990.

IO

HOEK. J.B. and RUBIN’. E.Alcohol and membrane-associated Alcohol Alcoholism 25. 143. 1990.

11

COWAN.

1’

BENISTANT, C. and RLIBIN. R. Ethanol inhibits thrombin-induced human platelets at a site distinct from phospholipase C or protein Biochem J. 269. 489 497. 1990.

D.H. Effect of alcoholism

on hemostasis.

signal

Scm Hem. 1’. 137

transducrilm

14’.

19811

secretion h?; I;inase C

488

ETHANOL-INDUCED

AGGREGATION

doi 63, No.

14. C:\RDINAL. D.C. and FLOWER. R J Ihc electronic aggregometer nokel dc\icc i I’hdf‘nlac‘c)l. !Merh 3 I ,3s I =JK 19fic1 assessing platelet behavior in blood

trll

it

15. G.-\ARDER. A.. JONSEN .I.. L.4L.ANL) S flELLEM,l. A. and OW’REN. P.A Adcnosrne diphosphate in red cells as a factlir‘ in the adhesiveness of human blood platelets Xaatur-e 192. 53 1 537. 1961. 16. BORS. G.V.R. and WEIIMEIER A. Inhibition of platelet chlorpromazine acting to diminish hacmoly,sis #alure

thrombus formation 282. 2 17 2 1.3 19-9.

by

1’. IIORAK. J.K.. BRANDON. I‘..A.. RIBEIRO. I,.(; I’ WARE. .I..+\ MILLER. R.R.. and SOLIS. R.T. Effects of ethanol and hemoly,sis on in \ivo and in vitro platelet aggregation. ,I, Cardjorasc. Pharmacol. 4. 10.37~ 104 1, 1982 18. WILLEMS. C.. SIEL. f1.V.. \!XN AKEN. Wt.;. and VAN XlOURIK. .I..-\, Binding and inactivation of prostac),clin IPGI?) b\ human crythroc) tcs. Dr. ,I. ffaemaro/. 5-r. 43 52. 1983. 19. J.4CKUBOWSKI. J.A.. ‘I’fIOMPSOS. CD. and DEYKIN, D. Inactivation (PGI2) by crythrocytes. Dr. ,I, ffacmatol. 54. 658 660. 1983

of prostacy~clin

20. GRESELE. P. ARNOUT. J,. DECKMYN, II. and VCRMYLEN, J.Mechanism of the antiplatelet action of dipyridamole in whole blood: hlodulation of adenosine concentration and activity. Tbr-omh. ffaemt1sfa.s. 55, 12 18. 1986. 21

E1’AXS, 1I.G.. RYLEY. f1.C.. fI.4LLE’l“l‘. I. and LEWIS, M.J. fluman inhibit endothelium-derived relasing factor IEDRF) activity.. 16.3. 36 1 -364. 1989.

22. fIOG.Afi, JC..

platelet

red blood cells Eur. .I. Pharmacol

LEWIS. hl.,I. and fIEkDERSON. .-\.fI. In viva EDRF activity. function. Rr-. ./. Pharmacol. 94. 1020 1022. 1988

influences

73. BL.L\CKWELL. G.J.. FLOWER, R.,]., RUSSELL SMI’I’H. N.. S.ALkIOi\‘. .[.A. I‘f~I0ROGOOD, P.B. and V.4NE. J.R. Prostacyclin is produced in whole blood. Dr. .I. Pharmacol. 64. 4.16. 19-8. 14. WEKSLER. B.B. and GOLDSTEIN. 1.M. Prostaglandins: and polymorphonuclear leukocy’tes in hemostasis 68.419-428. 1980.

Interactions with platelets and inflammation, .-lm. J. Med.

25. BENVENIS’I’E. J., fIENSON. P.M. and COCIIRANE, C.G. Lcukvcy’te dependent histamine release from rabbit platelets: the role of IgEbasophils and platelet activating factor. .I. E.cp. Med. 1.36. 13 j6 1.37’. 19-2. 26. NICOLINI. F.A. and MEfI1‘4, J.L. Inhibitory platelet aggregation by release of a factor relaxing factor (EDRF). Biochem. Pharmacol. 2’.

effect of unstimulated neutrophils similar to endothelium-derived 40. 2265 ~2269. 1990.

RIESS. II.. BRAUN, G.. BREHM. G. and Ililler. E. Critical aggregation in whole human blood. .-lm. .I, C/in. Parhol.

evaluation of platelet 85, 50 56. 1986.

on

Vol. 63, No. 5

ETHANOL-INDUCED

AGGREGATION

489

2s. IIIKIIAILIDIS, D.P.. BARRADAS. MA.. EPE>lOLC. 0. and DANDON.4. P. Ethanol ingestion inhibits human whole blood platelet impedance aggregation. I-Zfn i. C/in. Pathol. 88. 342-345. 1987. 79

BORN, G.V.R. Quantitative investigations Ph\~iol. (London) 162. 6’-68, 1962.

into the aggregation

Yi

IIILLBOM. M AND KXSTE. %I. Does ethanol intoxication in )‘oung adults? Lancer ii. 1 I8 l- 1183. 19-8.

31

GILL. J.S.. ZEZULKA. .4 V.. SIIIPLET. M.J., GILL, S.K. and BEEVERS.D.C alcohol consumption. S EngI. .I. bled. 315. 1041 1046. 1986.

32

IIILLBOM. M.. KANCASAflO. M.. KXS’I‘E, \l. NUMkllNEN. 11. AND V.4PAATXLO Acute ethanol ingestion increases platelet rcactivitl,: Is there a relationshin stroke? Srroke 16. 19 73. 1985.

brain

Strobe

factor

for ischemic

34

IIILLBOM M. and KAS’TE. M Alcohol intosication: .A risk factor subarac hnoid hemorrhage. I’l’euro/og)’ 32, 7O6- 7 1 1. 1982.

for primarv,

35

flILBOh1. hl. and KASTE. kl. Does alcohol intoxication precipitate aneurysmal subarachnoid hemorrhage? ,I. Seur-ol. Senrosur-g. P.c!,ch. 44. 523-526. 1981.

36

POST. RM. and DESFORGES. J.F. Thromboc) Blood 3 I, 344-347. 1968.

3-

SIEVE,

and hemolytic with alcoholic

effect

of ethanol

.I.

infarctton

IlILLBOM. M and KAS’I’C, M. Ethanol infarction. Stroke 14. 694-699. 1983.

topenic

A risk

promote

platelrets.

33

L. Jaundice h).perlipidemia unrecolxiized syndrome associated Int. Med. 48, 471-496, 1958.

intoxication:

of blond

and

!l to

brain

infusion

anemia: A heretofore fatty liver and cirrhosis

.~YI CIl.~BIfSLSKA. E.. MALINOWSKA. B. and BUCZKO, W:. Influence of ethanol scrotonin on rat platelet aggregation. Phar-macologk, 40. 288 292, 1990

Ann.

and

YJ

RUBIN. R.. PONNAPPA, B.C.. TIIOMAS. A.P. and flOEK, J.B. Ethanol stimulates change in human platelets b), activation of phosphoinositide specific Phospholipase C. Arch. Biochem. Bioph!,s 260. 4811 492. 1988.

shape

j(1

RUBIN. R. and HOEK. J.B. Inhibition of ethanol-induced platelet agents that elevate CAMP. Thromh. Res. 58. 625 632. 1990.

hj,

-tj

J.AKUBOWSKI. J.A.. V;\ILLXNCOUR’l’. Ii. AND DETKIN. D. Interaction ProstacYclin. and aspirin in determining human platelet reactivit). ilr-tericlsclerosis 8. 436~441, 1988.

activation

of ethanol, in virrc,