1524-4539 Copyright © 1973 American Heart Association. All rights reserved. Print ISSN: 0009-7322. Online ISSN:Circulation is published by the American Heart Association. 7272 Greenville Avenue, Dallas, TX 72514

1973;48;619-623 CirculationPETER H. LEVINE

Smoking and Arterial ThrombosisAn Acute Effect of Cigarette Smoking on Platelet Function: A Possible Link Between

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An Acute Effect of Cigarette Smokingon Platelet Function

A Possible Link Between Smoking and Arterial Thrombosis

By PETER H. LEVINE, M.D.

SUMMARYIn a controlled, double blind study, the smoking of a single cigarette has been shown to in-

crease the platelet's response to a standard aggregating stimulus. This phenomenon appears to

be specifically related to the inhaling of tobacco smoke; it does not follow the smoking of let-tuce leaf filled cigarettes. The platelet effect seems independent of the rise in plasma free fattyacids which follows cigarette smoking. Smoking-induced potentiation of platelet aggregation may

help to explain the increased incidence of arterial thrombi and/or atheromatous plaques incigarette smokers.

Additional Indexing Words:Free fatty acids Platelet aggregation

ARTERIAL THROMBOSIS occurs with in-creased frequency in cigarette smokers.'

Myocardial infarction,2 occlusive peripheral arterialdisease,3 and late occlusion of aortofemoral bypassgrafts4 are examples of this phenomenon. Becausethe blood platelet appears to play the central role inthe initiation of arterial thrombi,5 definition of therelationship between smoking and platelet functionseems important.The lifespan of platelets has been found to be

reduced during periods of heavy smoking.6 Plateletadhesiveness has been measured in cigarettesmokers,7 and conflicting results have emerged. Intwo independent studies,8 9 platelet aggregationwas chronically increased in heavy smokers whencompared with a population of non-smokers. Acuteeffects of smoking on the electrophoretic mobility ofplatelets was suggested in one of these studies,9 butno acute effect on platelet aggregation could beshown in either study. That any of the abovechanges in platelet function are a result of some

From the Blood Coagulation Laboratory of the NewEngland Medical Center Hospital, and the Department ofMedicine, Tufts University School of Medicine, Boston,Massachusetts.

Supported in part by a grant from the American MedicalAssociation Education and Research Foundation.

Address for reprints: Peter H. Levine, M.D., 171 HarrisonAvenue, Boston, Massachusetts 02111.

Received March 5, 1973; revision accepted for publicationMay 4, 1973.Circulation, Volume XLVIII, September 1973

constituent of tobacco smoke remains unproven. Apossible specific effect of smoking on plateletaggregation is the subject of this paper.The measurement of platelet aggregation in vivo

suffers from lack of reproducibility. Several con-tributing factors have now been identified. 1) Ag-gregation varies depending on emotional stress,recent exercise, and plasma lipid levels.10' 11 2) Theaggregation of platelets in response to a fixed doseof adenosine diphosphate (ADP), for example,varies predictably depending on the age in minutesof the sample.'2 3) With control of these variables,platelet aggregation will still vary significantly in ahealthy man studied from one day to the next.13We have attempted to control the above

variables in a search for a possible acute effect ofcigarette smoking on platelet aggregation. Byvarying the content of the cigarette smoked, in adouble blind fashion, we have also studied whethercigarette smoke itself is responsible for the changesobserved. Finally, we have observed the increase inplasma free fatty acids induced by smoking,14 andstudied its relationship to changes in plateletfunction.

Materials and MethodsPlatelet aggregation studies were performed by the

optical density method of Born,'5 as modified byMustard et al.'6 Blood was collected in plastic syringesand immediately mixed (9:1) with Ware's anticoagu-lant (6 parts 0.1 M sodium citrate to 4 parts 0.1 Mcitric acid) in plastic tubes. The blood was centrifugedat 200 C for 6 min at 22° C and the platelet rich plasma

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LEVINE

(PRP) was removed. The remaining blood was thencentrifuged at 850 G for 15 min to yield platelet poorplasma (PPP). The platelet count of the PRP wasadjusted to 200-300x1W3/cu. mm by dilution with PPP.This material was placed in the cuvette of a Chrono-Log aggregometer and the light transmittence wasrecorded on a moving strip chart recorder. The blankfor each study was a similarly treated sample of PPP.When the addition of ADP caused the isolated plateletsto form aggregates, light transmittence increasedproportional to the size of the aggregates.

Disodium ADP was dissolved in veronal buffer (pH7.35) to a concentration of 10 mg % adjusted to pH 6.8,and frozen in aliquots at -40(C. For each subject, adose of ADP was chosen which would just produce asmall reversible primary wave of aggregation, and thisdose was then used throughout. The range in ADPconcentration for this effect was between 1.3 and2.6x10-6M. It was hoped that this relatively small ADPstimulus would unmask pre-existing qualitative plateletchanges, and would also be closer to "physiologic"levels of aggregating stimuli.

Other parameters measured on each sample in-cluded: platelet count, platelet factor 3 release by themethod of Zucker and Peterson,17 and plasma free fattyacids (FFA) by the method of Dole.'8Twenty seven healthy male and female volunteers

were requested to abstain from all medications for 10days and from all caloric intake and cigarette smokingfor at least 12 hours prior to study. Each subiect wasplaced in a reclining chair, and a #19 butterfly-typeneedle inserted into the largest antecubital vein. A slowinfusion of physiologic saline was begun to keep theneedle patent. This avoided the stress of recurrentvenepuncture. Following 30 to 60 min rest, bloodsamples were drawn every 10 min via the butterflyneedle, using a two syringe technique. The first severalblood samples were discarded, until the subjectappeared to be "comfortable" with the protocol. Thetotal blood drawn during the entire study was between250 and 300 ml per patient.

After several baseline measurements were obtained,the subject smoked a single cigarette, and another seriesof studies were done at 10 min intervals. After a periodof 30 to 60 min, a second cigarette was smoked and athird series of studies performed. One of the cigaretteswas a standard commercial filtered brand, containing1.3 mg nicotine. The other was a filtered cigarette inwhich lettuce leaf was substituted for tobacco;19 thesecigarettes have no nicotine content. To precludeobserver bias, those performing and calculating theplatelet aggregation studies were not told whichsamples were pre or post smoking or which cigarettehad been used. To minimize artifacts due to ageing ofplatelet samples, all studies were performed in a rigidtime-controlled manner, and were completed within amaximum of 30 min from time of blood drawing.

ResultsSerial Studies. Serial measurements of platelet

aggregation under "baseline" conditions showedexcellent reproducibility. Variability in 40 sets of

experiments never exceeded 10 percent above orbelow the mean in any subject. On three occasions,difficulties with blood return from the intravenousneedle required repeat venepuncture. Followingthis procedure, there was a significant rise inaggregation response. For this reason, any patientwho required a second venepuncture was excludedfrom the study.

Effect of Emotional Stress. Following baselinemeasurements, 10 healthy volunteers were re-quested to "smoke" a standard cigarette in the usualmanner, without lighting it. During this maneuver,the investigators continuously reinforced the sub-ject's awareness that he was being closely observed.The mean platelet aggregation response measured10 and 20 min before such sham smoking wascompared to the mean response 10 and 20 minafterwards. No significant difference was found.The results are shown in the first column of figure 1.

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Figure 1Maximum platelet aggregation in response to a fixed doseof ADP. Paired experiments before and after sham smoking,non-nicotine cigarette smoking, and standard cigarettesmoking.

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SMOKING AND PLATELET FUNCTION

Results in Non-Smokers. Platelet aggregation wasmeasured before and after cigarette smoking in 6non-smokers. All had significant difficulty in inhal-ing, with resultant paroxysms of coughing. This"stress reaction" precluded meaningful interpreta-tion of data. Results were erratic. Of interest werethe plasma FFA levels, which were not significantlyincreased by smoking in these subjects. Thissuggested that relatively little nicotine had beenabsorbed.Acute Effects in Smokers: Lettuce Leaf. The

mean platelet aggregation responses measured 10and 20 min before and after smoking the lettuceleaf cigarette were compared in 11 subjects. Nostatistically valid difference was noted. Theseresults are shown in the second column of figure 1.Aoute Effects in Smokers: Tobacco. On the same

day, similar studies were performed in the same 11subjects before and after smoking the standardcigarette, under the same experimental conditions.Results are shown in the final column of figure 1.Using the t-test for paired data, the increasedaggregation seen following standard smoking wascompared to the change in aggregation from lettuceleaf cigarette smoking. The difference was signifi-cant at the P <.01 level. To determine whetherprior exposure to lettuce leaf smoking had "sensi-

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tized" the patient to the second cigarette, the orderof testing was reversed in 3 subjects. The resultswere unchanged; increased aggregation responsefollowed the standard cigarette only.Timing of Response. Serial measurements (fig. 2)

showed no increase in aggregation immediatelyfollowing cigarette smoking. Increase in aggrega-tion was maximum at 10 and 20 min, and at or nearbaseline by 30 min. Two patterns were noted. Thecommonest (fig. 2, top line) was an increase inprimary wave, with loss of complete disaggregation.Less often, the primary wave was of sufficientmagnitude to result in a release reaction, with asecondary wave of aggregation resulting (fig. 2,bottom line).

Free Fatty Acids. None of the above subjectsdeveloped an increase in FFA after the lettuce leafcigarette. After the standard cigarette, all elevendeveloped increased FFA. The mean rise was 134mg% from a mean baseline level of 556 mg%. Thetime of maximal free fatty acid rise was 30 min aftersmoking. The degree of increment for individualsubjects did not correlate with the degree ofenhancement of aggregation. In three subjects, therise in FFA occurred only after the increase inaggregation was noted.

-10 0 lo 20MINUTES AFTER SMOKING ONE CIGARETTE

-lo 0 l0 30MINUTES AFTER SMOKING ONE CIGARETTE

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Serial platelet aggregation curves before and after smoking a standard cigarette. Two typical subjects areshown. Free fatty acid concentration in milli-equivalents per liter of plasma, as shown by dashed line.

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LEVINE

Platelet Factor 3. PF3 measurements did notdiffer significantly in any of the groups studied.

DiscussionThe smoking of a single cigarette results in some

qualitative change in the platelet which renders itmore responsive to exposure to low doses of ADP.This does not appear to be due to the stress of theprotocol, and it does not occur following thesmoking of nicotine-free cigarettes. Nicotine causesa rise in plasma epinephrine levels,20 and this hasbeen shown to occur in cigarette smokers. The risein free fatty acids is thought to be a result of theepinephrine increase.21 The timing and magnitudeof the increase in FFA observed by us is inagreement with the previous data of Kershbaum etal.22 We had expected that the free fatty acidswould in turn be responsible for the observedincrease in platelet aggregability, but our data didnot suggest this.The enhancement of platelet aggregation ob-

served after smoking could be a direct effect ofnicotine, or could be due to the known increase inplasma catecholamines.20 (We were unable tomeasure plasma epinephrine levels in parallel withplatelet function in this study, because of the largevolumes of blood which such determinations wouldhave required.) Catecholamines may play a majorrole in the in vivo platelet aggregation response.Epinephrine is a well-studied stimulus of plateletaggregation and the release reaction.23 24 Moreover,the addition of epinephrine to platelets in vivohas recently been shown to enhance their subse-quent response to ADP-induced aggregation.25

Cigarette smokers have an increased risk ofmyocardial infarction26 as well as sudden death.2 Inexperimental animals also, in vivo induction ofplatelet aggregates can lead to both myocardialinfarction and sudden death.27 If a single cigarettecan sensitize blood platelets to ADP, such plateletswould hypothetically be more ready to participatein platelet plug formation, leading to vascularocclusion.The data presented here suggest a possible direct

causative link between cigarette smoking andarterial thrombotic disease. Moreoever, there isliterature2'31 which suggests that the encrustationof layers of platelets on the arterial wall, at sites ofturbulent blood flow and/or endothelial damage,may be the precursor of the atherosclerotic plaque.If this literature has any validity, the results of thepresent study may also help to explain themechanism by which smoking may accelerate

atherosclerosis. These considerations lend addedimportance to the development and study of agentswhich can effectively modify platelet reactions.

References1. MURPHY EA, MUSTARD JF: Tobacco and thrombosis.

Amer J Pub Health 56: 1061, 19662. SPAIN DM, BRADESS VA: Sudden death from coronary

heart disease. Chest 58: 107, 19703. JUERGENS JL, BARKER NW, HINES EA: Arteriosclerosis

obliterans: review of 520 cases with special referenceto pathogenic and prognostic factors. Circulation 21:188, 1960

4. WRAY WV, DEPALMA RG, HUBAY CH: Late occlusion ofaortofemoral bypass grafts: influence of cigarettesmoking. Surgery 70: 969, 1971

5. DEYKIN D: Thrombogenesis. New Eng J Med 276:622, 1967

6. MUSTARD JF, MURPHY EA: Effect of smoking on bloodcoagulation and platelet survival in man. Brit Med JI: 846, 1963

7. MURCHISON LE, FYFE T: Effects of cigarette smokingon serum lipids, blood glucose, and plateletadhesiveness. Lancet II: 182, 1966

8. GLYNN MF, MUSTARD JF, BUCHANAN MR, MURPHYEA: Cigarette smoking and platelet aggregation.Canad Med Assoc J 95: 549, 1966

9. HAWKINS RI: Smoking, platelets and thrombosis.Nature 236: 450, 1972

10. HARRISON MJG, EMMONS PR, MITCHELL JRA: Thevariability of human platelet aggregation. J Athero-scl Res 7: 197, 1967

11. HASLAm RJ: Role of adenosine diphosphate in theaggregation of human blood platelets by thrombinand fatty acids. Nature 202: 765, 1964

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13. O'BRIEN JR: Variability in the aggregation of humanplatelets by adrenaline. Nature 202: 1188, 1964

14. KERSHBAUM A, BELLET S, DICKSTEIN ER, FEINBERGLJ: Effect of cigarette smoking and nicotine on serumfree fatty acids. Circ Res 9: 631, 1961

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16. MUSTARD JF, HEGARDT B, ROWSELL HC, MACMILLANRL: Effect of adenosine nucleotides on plateletaggregation and clotting time. J Lab Clin Med 64:548, 1964

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18. DOLE VP: A relation between non-esterified fatty acidsin plasma and the metabolism of glucose. J ClinInvest 35: 150, 1956

19. ARANOW WS, SWANSON AJ: Non-nicotinized cigarettesand angina pectoris. Ann Intern Med 72: 1227,1969

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20. WATTS DT: The effect of nicotine and smoking on thesecretion of epinephrine. Ann N Y Acad Sci 90: 74,1960

21. KERSHBAUM A, KHORSANDIAN R, CAPLAN RF, BELLETS, FEINBERG LJ: The role of catecholamines in thefree fatty acid response to cigarette smoking.Circulation 28: 52, 1963

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23. SCHWARTZ CJ, ARDLIE NG: Catecholamines andplatelet aggregation. Circ Res 21 (suppl III): III-187, 1967

24. PACKHAM MA, MUSTARD JF: Platelet reactions. Sem inHemat 8: 30, 1971

25. YAMAZAKI H, KOBAYOSHI I, SANO T, SHIMAMOTO T:Enhancement of ADP-induced platelet aggregationby adrenaline in vivo and its prevention. Thromb etDiath Haemorrh, In Press

26. FRANK CW, WEINBLATT E, SHAPIRO S, SAGER RW:Myocardial infarction in men. JAMA 198: 103,1966

27. JORGENSEN L, ROWSELL HC, Hovic T, GLYNN MF,MUSTARD JF: Adenosine diphosphate-induced plateletaggregation and myocardial infarction in swine. LabInvest 17: 616, 1967

28. MURPHY EA, ROWSELL HC, DOWNIE HG, ROBINSONGA, MUSTARD JF: Encrustation and atherosclerosis:the analogy between early in vivo lesions anddeposits which occur in extracorporeal circulations.Canad Med Assoc J 87: 259, 1962

29. MUSTARD JF, MURPHY EA, ROWSELL HC, DOWNIEHG: Platelets and atherosclerosis. J Atheroscl Res 4:1, 1964

30. HAND RA, CHANDLER AB: Atherosclerotic metamorpho-sis of autologous pulmonary thromboemboli in therabbit. Amer J Path 40: 469, 1962

31. FRENCH JE: Atherogenesis and thrombosis. Sem inHemat 8: 84, 1971

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