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Platelet Sensitivity to Prostacyclin in Smokers and Non-smokers'` O: C. BurgFtuber, M.D.; Ch. hunzengruber, M.D.;, H. Sin_inger, 4f.D.;. P Haber, M.D.; and K Silberbauer M.D. Platelet activating effect of t:igarettir smoking appears to be important td the development of adserosckeosis. We previ- ousfy demonstrated a redlsced sesuitivity of platelets to - e:ogenosu prostacyclin (PCL) in citro from patients with proven atherosclerotic tiisease, indicating a possible role of altered platelet function in the development of atiservsckto- sis. We now hypothesize that cigarette smokusg might be an important cause of altered platelet sensitivity to PGLr observed in patients with at4servsclerosis. To test this hy- pothesis. the response of platelets to exogenous PGI, was tested' in chronic smokers and ntm-smokers, prior to and Smoking has been incriminatedas a pathogenetic fac- tor in cardiovascular disease."Tobacco smoking is associated with an increased risk of myocardial irtfarc- tion'sudd'en death and arterial thrombosis occur more frequently in cigarette smokers.'" Because blood platelets appear to play a central role in the initiation of arterial thrombosis, the difference in aggregation be- havior in platelets from smokers and non-smokers seems to be important. The influence of nicotine andl or other cigarette constituents on platelet function has been investigated in several' in ciro and in t:itro studies. From these studies it is known that smoking induces enhancement-of platelet function." Evidence supporting this idea includes an association of smoking with increased ADP induced platelet aggregation in platelet-rich plasma,' an enhanced tendency of plate- lets to aggregate in blood;- a shortening of platelet survival'° and' increased thromboxane synthesis.'t'' In addition there is some evidence that smoking might exert its action by reducing vascular prostaeyctin (PGI,) synthesis." Since we have previously demon• strated reduced platelet sensitivity to exogenous PGI, in vitro in patients with atherosclerosis," we wondered whether cigarette smoking might decrease platelet sensitivity to PGIx. Because of recent observationsts'" that passive smoking increases the incidence of various diseases primarily associated with active smoking, we also wondered whether even passive smoking could influence platelet sensitivity to PGIr. If platelet sensi- tivity to PGI, were suppressed by active or passive 'Frvm the Second Department of Internal Medicine. University of. Vientsa- ViennZ Austria. Manuscript received Uctober 21.I985: tsvisbn accepted January 20. Reprint repueru: Dr. 8u huber, Carnuongaire !3. ll' Medical Deportnsnu. Urus:ersity of~ienna. Viersna, Auatna A•1090 Ko,TuC E thls mat°~zl' m_y be Orotectsd by ct-~-right law ftiUQ 17 U.S. Ccde), after smofdng t+vo cigarettes (active smoidng) and prior to and after exposure to a tobacco smoke-contamitsated atmo- sphere (passive smoldng). 'lflhis sts.dy indicates tliatplatelets of chsvnic smokers are less sensitiwe to esogeoout PGI, than platelets of aoQ-smokers. In addition, active as well as psssive si<uo+dng decreases plaeidet sensitivity to PCl, in taon-smokers, whereas ebevnic smokers esbibit no further decline. We conclude tlut decreased platelet sensitivity to PGI,, might be an important contributing factor to the altered platelet function observed in patients with athetv- sdesosis. smoking, then we could consider it an additional important mechanism of arterial thrombosis. MATERIAL AND METHODS Actiec Smo/ring The subjects of this study were 14 healthy male volunteers whose :utes ranged fnrm38 toJ6 years. Seven were non-smokers and'seven were moderate-to-hea.y smokers lat least one pack a day for at )east ten vrarsl. Standard commercial brands containing 1.5 mg nicotine and 3; m¢ tu per p,run of cipiette were used: The smokers re- frwned from smoking for at least four hours pnor to the test prncedures, and no mediutton was allowed for tKo weeks prior to the .tudies. A 19 ¢autce plutic cynnulL was inserted into the antecubital vein 15 min pnor to baseline mersurtments to avoid repeated %enuus paunctures.,Then the patients were told to smoke tsAn cia.rrttes, one .ftrr the other. %ithin 10 min. Immediately hefiore And 1.5 min After smukinA t•+n cip,.rettes, blood pressure, pulse rate. and!•entilstor% function teats were performed and blood w.u drawn. Blood pressure waameasured.vith the Korotkoff inethod bc the ume ohaer.er, lentilatorc function was assessed by spirome- try usin¢ a Fleuch pneumuucho¢raph att.uhed to an electronic device (Simgnost FD 10. Siemens Elemic 19) and recorded on an x-y recorder (Hewlett-Paciurd); Vital capacity (VC. L)'was determined: by a slow inspintory e6ort. This was followed by three attempts of FEV; maneuvers (FEV,,, L) After completion of these procedures, forced expiratory flow volume curves were obtained. Forced etpiratorv flows at the moment when 50 percent of the vital capacity had been expelled (FEF„ Lsec) and when 75 percent had been expelled (FEFs, L?sec) were read ditrctly from the flow volume curves. The best-ofthnre attempts was used for calculYtion. Blood withdrawal was performed from the previousli inserted plastic cannuls. Nine.olumes ofblood were miaed with one volume of 3:8 percent trssodium citrate solution to obtmn citrated blood. After centrifuganon at LSO g for 5 min, platelet-rieb pluma (PRP) wu obtained. PRP was then removed and plitelet-poor plasma (PPP) prod'uced: by further centr9fuguion of LS00 g for 15 min. PIiP was adjusted with PPP to give a platelet count of' apprvximitely 250 x 10'lµJi ADP'(in a rather high coneentration of 1 mmoUl) was used to cause irreversible platelet aggregation measured in a Born- 34 Pfosa.cyctn n Snds.n tna Won vnok«s reuqtwo.r W tv)

mm Hg NON SMOKER SMOKER 160, 11.0 ~ Systotlc 120 100 80 Diastolic It BEFORE NS AFTER IZ O Ftcuae L Individual data points and N.S. meansofsrstolicanddiastolicbloodpres- sure (BP: mmHR) before and'after smok- ing 2' cigarettes in smokers and non- BEFORE AFTER smokers. t.pe aggreRometer. On all otrasions. second phase of ADP-induced platelet aggregation was seen. The maximal estent of platelet aggregation c.YTmasl was calculated usuming that PPP was 100 per- cent and PRP was d percent aggregation. !n addition, ADP-induced platelet aggregation was inhibited b}- increasing concentrations of PCh (1:2.3 ng/ml) being added 60 sec prior to ADP From this the sensitivity index of PCI, (S1m,t) was calcullted' (Slrca, - 11ID,,; IDb = the concentration of PGIy necessar-y to inhibit ADP-induced piatelet aggregation to 50 percent). Fbasiue Smoking Another 22 healthv male volunteers. 13 smokers and nine non- smokers. whose ages ranged from 25 to 40 ,vears, were e:posed to cigarette smoke. Smokers refrained from active smoking for at kast four hours before studiedl, Volunteers were kept for 20 min in an 18 m' room in which testers smoked 30 hea.v brand cigarettes just prior to the exposure penod. This concentration was calculated to be that occurring in discos, restaurants etc. Again: blood was drawn before and 15 min after the passive s,no!Ring period and aggregation studies were performed'as previouslY described:. Statisti csl ',1'r!al'ysis PairrdanJ u, pured'Student'st-testswereusedtocompateresults HR' beah/min 100 ~ 90, 70 -~ 60 ~ Ftcutu: 2. Individual data points and I w•ithin and between groups respectivel>. Differences were consid- ered significant when p<0.05 RESC'LTS Active Smoking. Prior to smoking hw cigarettes. neither smokers nor non-smokers exhibited any difference in either systolic or diastolic blood~ pressure (Fig 1) or in heart rate (Fig 2)! After smoking two cigarettes, blood pressure re- mained unchanged (Fig 1), whereas a significant in- crease in heart rate could be observed in ~ both groups (Fig 2). There was no difference in VC and FEV, prior to or after smoking between smokers and non-smokers (Tablt? 1). However, smokers had lower forced ex- piratorti• 9ouw rates compared to non,smokers, before as well as after smoking two cigarettes (Table 1). Smoking two cigarettes did not alter ant•ventilatory parameters studied in either group. Prior to smoking two cigarettes, the aggregation of platelets in response to ADP was the same in: smokers NON SMOKER means of heart rate (hIR. beats/minute) ~i ~ before and after smoking 2 cigarettes in smokers and non-smokers. BEFORE . /b AFTER pc -05 ill BEFORE AFTER QiEST r Yo i t i JuLr, ttas6 ]6

Lbk I-rwd'+oiidYal rsag FUwdiow Pbrosserrs bs`o.r and aRer ss.ohin6 Two eidarsttss m Now-.,.okm snd S.wher, VC (L)' FEV~ (L) FEF. (L/sec) FEFe (Usec)~ Non-smokers Beiore After Before After Before After Before After 1 6,5 6.1 4.3 4.6 4.3 5.4 2.2 2:8 2 7.8 7.6 5.8' 6.2 11.4 9.4 3.7 5:3' 3 6.4 6.5 5.6 5:8 5.6 5.8 2.9 3.3' 4 5.6 5_8 3.7 4.6 5.1 4.8 2.6 2.4 5 3.5 3_6 2.8 2.7 4.3 3.5 2:4 1.4 6 4.5 4.9 4.3 3.7 5.9 5.2 4.5 4.1 7 jp 5.6 4.8 4.0 3.9 5.1 4.1 2.1 2.1 mean S SEM Smokers 5.7 s 0.5 5.6 z 0.5 4.3 _ 0.'4 4.5 s 0:5 6:0 = 0:9 5.5 = 0.7 2:9 z 0.3 3.1 s 0:5 1 5.3 5.5 4:11 4.6 6.2 7.3 3.7 3.9 2 6.4 6.3 5.1 4.5 5:2 5.2 2.4 2.2 3 4.6 4.7 3.2 3.5 3.9 3.1 2.7 1.4 4 6.0 6.5 4.5 4.5 5.0 4.8 2.2 2:7 5 5.8 5.7 4.2 4.2 4.7 4.5 2.6 2:1 6 3.9 4.46 2.9 2.9 3.0 2.6 1.1 1.2 7 4,.6 4_7 3.5 3.2 3.5 3.1 1.6 1.0 mean ~ SEM 5.3c0.4 5.4_0:3' 3.9_0.3 3.9_-0.3 •p<0.1i tp<0.05, smokers vs non-smokers and' non-smokers (48 t4 percent in smokers vs 44 :t4 percent in non-smokers). However, platelet sensitivity to PGI,, expressed'as sensitivity index to PGI„ was sig- nificantly lower in smokers compared to non-smokers (Fig 3). After smoking two cigarettes, ADF ind'uced platelet aggregation did not change in either group (49 _ 2 percent in smokers vs 50 !: 2 percent in non- smokers). In contrast, the sensitivity index to PGI, significantly decreased in non-smokers, almost reach- ing baseline-level: for smokers (Fig 3). In smokers, liowever, no further decrease in platelet sensitivity to PGI, could' be observed. Thus, after smoking two ciga- rettes, no further statistically significant difference between smokers and non-smokers could be iound. Phssiee Smoking Sensitivity index to PGI, again was significantly lower in the smoker group as compared to non-smok- ers (Fig 4). Passive smoking in non-smokers indueed'a SI' 1.07 PGIZ , 0.51 . 0 M ~ER P<AS ~ `P<.08 ` SMOKER I1is ~-- BEFORE AFTER Frcuns 3;, Sensitivky inda oEPCI, (St~'bei'ore and s[!er smok- ing 2 cigarettes in smokers and noo-amobess. ~ 4.5 x 0:4• 4.3 _ 0.6• 2,3 _ 01" 2.0 s 0,4Y significant decrease in platelet sensitivity to PGI,, whereas in smokers no further decrease could be demonstrated. DISCUSSION The main finding of this study is that smokers' plate- lets are less sensitive to the anti-aggregatory action of exogenous PGI, compared to platelets of non-smokers. Further, our results show that only in non-smokers does acute inhalation of tobacco smoke decrease plate- let sensitivity to PGI, in vitro. Finally, a decrease in platelet sensitivity to PGI, was observed in non-smok- ers after active as well as after passive smoking. It is well~-established that platelet sensitivity to PGI, is a reliable and sensitive test to examine platelet func- tion." In , tr experiments, there were two lines of evidence fo the reliability of this procedure. First, platelet sensitivity to PGI, was reproducible in two dif- I.0 Sl PG12 P<.a ~ SMOKER ~'~ ~• , 3 ~ ~.~ AFTER N BEFORE FieuRE4. Sensitivity indes oEPCIs (Slrcy)'before and aRer passis r^ smoidng in smokers and non-smokers W. 36 Prc.heycM n Sma*rs ard Mbn-rfnksn (durpMuWW or so

ferent populations (eg, in 14 volunteers smoking ac- tively and in 21 volunteers smoking passively): Second, platelet sensitivity to PGI, was sensitive in distinguish- ing between the platelet function,of smokers and non- smokers. Thus, it appears that this procedure is of additional~ value in examining platelet function in various circumstances. However, as with other platelet function tests, we do not know if this in vitro procedure accurately reflects platelet function in ciuo. We have usej a rather high concentration of ADP, previously shown to be optimal for measurements of platelet sensitivity to PG1,.'° Therefore„ our results of ADP-induced platelet aggregation cannot be compared with a previous stud}6 using different, lower concen- trations of ADP. This study showe& a significant increase of ADP ind'uced; platelet aggregation after smoking. Nevertheless, even with the high ADP-con- centration used in this study, there was a tendency towards an increase in AD P-induced platelet aggrega- tion after smoking two cigarettes in the non-smoker group. The influence of cigarette smoking on platelet func- tion has been investigated in several in vitro and in L-ivo studies.'-" Although the precise effect is not clear, in most studies potentiation of platelet aggregation has been observed. Our findings of a diminished platelet sensitivity to an anti-aggregatory substance in smokers fits into the overall idea of a proaggregatory effect of cigarette smoking. Chronic smoking can desensitize blood platelets to PGI,; such platelets wouldhypothet- ically be more ready to aggregate and participate in plug formation, leading to arterial thrombosis. Our re- sults are thus in-agreement with the well-known clini- cal finding of the increase& incidence of thromboem- bolic diseases in smoken." Since causes other than smoking could conceivably have led to a decrease in platelet sensitivity to PGI, observed' in our smokers, we investigated whether smoking two cigarettes v-vuld acutely influence platelet sensitivity to PGIi,. A signifi- cant decrease in platelet sensitivity to PGI, in non- smokers indicated that cigarette smoking is responsi- ble for the decreased platelet sensitivity to PGI, in smokers. The fact that acute smoking did not alter platelet sensitivity to PGI, in chronic smokers remains to be clarif,ed, One esplanation is that smoking two ciga- rettes exhibits a much lower emotional stress in smok- en than in non-smokers. Since platelet aggregation has been shown to vary withemotional stress,° this could have led to a different platelet behavior after acute smoking. We did not measure plasma epinephrine concentrations parallel to platelet function in this study. However, if one compares blood pressure and heart rate before and after smoking two cigarettes in smokers and non-smokers, one will find: a) no statis- tical significant changes in blood pressure, and b), a similar, marked ~ increase in heart rate in both groups. Despite not being statistically significant, there was an obvious increase in systolic and diastolic blood' pres- sure after smoking two cigarettes in the non-smoking group (Fig 1). This increase could have reached statis- tical significance if more patients had been studied. Nevertheless, it seems unlikely that different adre- nergic stimuli were responsible for the difference in platelet behavior after acute smoking. An alternative explanation is that acute smoking in- fluences lung function parameters diETerently in smok- ers and non-smokers, which could cause differences in platelet function, Since no significant change in lung function-could be demonstrated in either group after smoking two cigarettes, we strongly feel that differ- ences in lung function cannot account for the different behavior in platelet sensitivity to PGI, after acute smoking. It is interesting to note, however, that smok- ers revealed lower forced expiratory flow rates at 50 and 25 percent of vital capacity compared to non-smokers (table 1): These findings confirm previous studies" ° indicating some degree of smalli airways disease in smokers. Finally, platelets of chronic smokers may already, be desensitized to an extent where no further decrease is possible, but this alternative was not clarified. In recent years the possible consequences to the health of non-smokers exposed to cigarette smoke (passive smoking) have been ezaminedl 1- It has been shown thatpassive smoking could lead to deterioration of lung function in adults° and children.r'•" Further, the effect! of passive smoking on the development of lung cancer was studied epidemiologically in ]apan," indicating the possible importance of passive smoking as one of the causal factors of lung cancer. To, our knowledge, there is no evid'ence published so far, indi- cating a higher risk of developing arterial thrombosis in passive smokers. In the present study of healthy male non-smokers, we found a significant decrrase in platelet sensitivity to PGI, after acute passive exposure to tobacco smoke. This finding at least suggests that platelets of non- smokers passively exposed to tobacco smoke might also exhibit a higher tendency to aggregate. Further inves- tigation is needed to elucidate whether this finding is important with respect to a possible increased inci- dence of thromboembolic disease among non-smokers passively exposed to cigarette smoke. In any event, the present study has suggested that active and passive tobacco smoking is primarily re- sponsible for a decrease in platelet sensitivity to PGI, in vitro. Although the results obtained in vitro cannot be directly extrapolated to in vioo situations, they may extend our understanding of the mechanisms by which smoking increases the risk of embolic disease. Q£Sr rf0 r t r JuIY, two 37