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Passive smoking and platelet thromboxane
Thrombosis Research, Vol. 81, No. 4, pp. 451460, 1996 Copyright 0 1996 Elsevier Science Ltd Printed in the USA. All rights reserved 0049-3848/96 $12.00 + 00
Pergamon
PII s0049-3848(%)ooo17-5
PASSIVE SMOKING AND PLATELET Peter Schmid”,
THROMBOXANE
Georg Karanikas, Harald Kritz’), Christian Pirich2’, Yannis Bernhard A. Peskar3’, and Helmut Sinzinger2’4’
Stamatopoulos”,
“Cardiovascular Rehabilitation Center Bad Schallerbach, Upper Austria,“Department of Nuclear Medicine, University of Vienna, Austria,3’Department of Pharmacology and Toxicology, Atherosclerosis Research Group, Ruhr-University, Bochum, Germany, 4, Wilhelm-Auerswald Vienna, and 5,Rehabilitation Center “Engelsbad-Melanie”, Baden, Lower Austria, Austria
(Received 20 July 7995 by Editor E. Ernst; revised/accepted
Abstract
28 December 1995)
While active smoking is known to enhance platelet thromboxane production, no data on passive smoking is available yet. The influence of single and repeated exposure to passive smoke for 60 minutes in a 18 mi room was assessed in non-smokers as compared to sex and age matched smokers. All the evaluated measures (malondialdehyde, plasma thromboxane B,, 1 1-dehydro-thromboxane B?, serum thromboxane B, , conversion of exogenous arachidonic acid to thromboxane B3 and to hydroxy-5, 8, lo-heptadecatrienoic acid) were higher in smokers than non-smokers at baseline, immediately and 6 hours after passive exposure to cigarette smoke. Repeated exposure of non-smokers rendered their platelets more activated becoming close to the behaviour of smokers, These results indicate that passive smoking may activate thromboxane 4 release from the platelets, contributing to the development of hemostatic imbalance.
Active smoking is a well known risk factor for the development of atherosclerosis (1, 2) and in particular coronary heart disease (3) and peripheral vascular disease (4). The negative effects demonstrated on platelet function (5, 6) the eicosanoid system (7) and platelet thromboxane A1 generation (8) may contribute to the hemostatic imbalance (9, 10) reported. Recently, the problem of passive smoking as a health risk has widely been discussed (8, 1I- 14). Detailed information on the role of passive smoking on hemostatic parameters, however, is still very limited (1 S- 18). We thus assessed the influence of acute single and repeated passive smoking Key words: Passive smoking, platelet thromboxane; Corresponding author: Helmut Sinzinger, MD, Prof., Wilhelm-Auerswald Atherosclerosis Research Group (ASF) Vienna, Nadlergasse 1, A-1090 Vienna, Austria, Phone: 43-l-4082633, Fax: 43-l-4081366. 451
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under standardized conditions on platelet thromboxane formation assessed by different tests in non-smokers and smokers.
MATERIAL
and METHODS
Study design. The study was done as a stratified intervention study comparing the reactions of the different strata to a common exposure. It contains of three parts; first the comparisons between smokers and non-smokers, second the comparisons among repeated exposures on days 1, 2, 3,4, 5 and 12, and third the comparisons among pre-exposure-, immediate post-exposure-, and 6-hour post-exposure periods. Subjects. 12 healthy non-smokers (7 males, 5 females; aged 20-32 years) and 12 smokers (6 males, 6 females; aged 23-33 years, smoking more than 20 cigarettes/day) were examined. All volunteers were students recruited from the University of Vienna. They were not taking any medication during a period of at least 2 weeks prior entering and throughout the entire study. The experiments took place in the morning before work to avoid exposure to environmental smoke before the experimental periods began. The experimental phase started at 8 a.m. and was finished at 3 p.m. During this period as well as between 10 p.m. and 8 a.m. smokers abstained from active smoking. Between 3 p.m. and 10 p.m. they were allowed to continue their smoking behaviour Therefore, the wash-out period from active smoking lasted 10 hours. All the participants had normal weight and no risk factors for the development of atherosclerosis. They were exposed to the smoke of 30 cigarettes (GitanesR , nicotine 1.5mg, tar-contents 25mg) for 60 minutes in a 18 m3 room (room temperature 20°C, 60% relative air moisture) while sitting in an arm chair. Blood was drawn immediately prior to the exposure, immediately thereafter, as well as 6 hours later. This procedure was repeated on 5 subsequent days and on day 12. Blood was removed without venous occlusion using a 1.2 mm diameter needle and processed as follows (19). Laboratory Methods. Malondialdehyde @DA) Venous blood anticoagulated 3:7 with acid citrate dextrose (ACD) was drawn. After sedimentation at 18°C for 15 minutes platelet rich plasma (PRP) was prepared by centrifugation (150 x g for 7 minutes at 1W). 0.5 mL of PRP was centrifuged (700 x g for 25 minutes at 18°C) to get the platelet pellet which was washed twice in buffer (pH 7.2) containing 0.9 % NaCl, 2% Na-EDTA and 50 mM Tris-HCl (20: 1:2). The pellet was resuspended in 0.5 mL 50 mM Tris-HCI (pH 7.4) and incubated with 5 U/100 uL thrombin (Topostatin, Hofmann La Roche, Basle, Switzerland) for 30 minutes in a shaking water bath at 37°C. Additional 0.4 mL trichloracetic acid was added. The reaction was stopped at 4°C. After homogenization by means of ultrasonics the protein was separated by centrilugation (1500 x g for 10 minutes at 4°C). Thiobarbituric acid was added to the supernatant and heated to 100°C for 15 minutes. Thereafter, MDA was determined photometrically at 532 nm. The intraassay variation amounted 3.8*1.0%, the interassay variation 5.911. %. Values are given in &I/lo9 platelets. Serum ihromboxane B, (s-TXB,). 0.5 mL blood was collected. After clotting for exactly 60 min at 37°C in a shaking water bath the sample was centrifuged at 2000 x g for 15 minutes at 4°C. The supernatant was removed and stored at -70°C in Eppendorf-vials for not longer than 2 weeks. The concentration of TXB, was
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determined using a commercial RIA-kit (Amersham, Buckinghamshire, UK).The variation was 3.3*0.7%, the interassay variation 6.1&0.9%. Values are given in ng/mL.
453
intraassay
Plosmn thromboxane B, (p-TXB,). EDTA (2%) was used as anticoagulant and aspisol (1 mg/mL) for cyclooxygenase inhibition. The samples were centrifuged for 30 minutes at 4” C Thereafter, the plasma was removed and stored at -70°C for no longer than 2 weeks. The concentration of TXB, in unextracted samples was determined using a specific radioimmunoassay. Separation of free- and antibody-bound ligand was performed by means of a double-antibody (OTOP 15/16, Behring, Marburg, Germany). The intraassay variation was 3.8* 1 I%, the interassay variation 6.9* 1.5%. Values are given in pg/mL I I-dehydro-thromboxane B, (I I-DH TXB,). Blood was taken with 2% sodium EDTA as anticoagulant. After short sedimentation at room temperature, the samples were centrifuged (1500 x g at 4°C) for platelet poor plasma. This was transferred in small volumes into Eppendorf-vials and stored there at -70°C for not longer than 2 weeks. 1 l-DH-TXB? was determined with a commercial RIA-kit (Amersham, Buckinghamshire, UK). The intraassay variation was 2.7*0.5%, the interassay variation 5.1*0.8%. Values are given in pg/mL. Radiothinlayer Chromatography-‘4C -arachidonic acid conversion to TXB,and HHT. Anticoagulated blood (3:7 with ACD) was collected. PRP was obtained by centrifitgation and divided into 0.5 mL aliquots. After centrihgation (700 x g for 25 minutes at 18°C) the supernatant was removed with a vacuum pump. Afterwards the isolated platelets were resuspended in 0.5 mL 50 mM Tris-buffer (pH 7.4) and incubated with 0.25 pCi [‘“Cl arachidonic acid (AA) for 5 minutes at 37°C in a shaking water bath. The reaction was stopped with HCI (resulting in a pH of 3). After centrifirgation (5000 x g for 10 minutes at 4°C) the supernatant was extracted with ethyl-acetate. The organic phase was dried under nitrogen, dissolved in 100 uL ethanol and stored at -20°C. After a chromatography on silicagel, the labelled arachidonic acid metabolites (TXB, and HHT) were analysed with a TLC linear scanner, Radiolabeled standards (New England Nuclear, Dreieich, Germany) were used for identification. Values are given in %.
STATISTICAL
ANALYSIS
Comparing relations between smokers and non-smokers, parametric tests of significance were used. Data are given as mean f standard error of the mean (SEM). Mean values shown in Figures l-2 were calculated for each day and both groups, and differences between them on day 1, 2,3,4, 5 and 12 were evaluated.
RESULTS I Single exposure to passive smoke All the evaluated measures were higher in smokers than in non-smokers at baseline conditions, after acute exposure and 6 hours after maximal exposure to passive smoke. Comparison of mean values, calculated from the values estimated at day 1, 2, 3, 4, 5 and 12, for both groups for all the investigated measures before and after exposure to cigarette smoke is shown in Table 1 (see
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p-level 3). Differences at baseline conditions, immediately after and 6 hours after maximal passive exposure to cigarette smoke between non-smokers and smokers were statistically significant for plasma TXEJ,, 1 I-DH TXEJ, and conversion to TXB, and HHT, respectively. MDA and serum TXB, mean values steadily increased in smokers, but differences were statistically significant only at baseline for MDA and directly after exposure, respectively 6 hrs after exposure, for serum T=$. As compared to the basal values only MDA and plasma TXB, in non-smokers showed still significantly higher levels 6 hours after smoke exposure. Ail the other measures investigated reached 6 hours after exposure to passive cigarette smoke their baseline values again (Table I).
TABLE
1
Measures of Platelet Function for Non-smokers and Smokers prior, immediately after and 6 hours after Passive Exposure to Cigarette Smoke. Pre-value
Acute
6 hrs later
p-level 1
non-smoker smoker n-level 3
3.50&O. 12 3.86*0.02 *
4.20&0.05 4.343tO.16 ns.
3.66%0.09 3.87kO.02 n.s.
* *
ns
s-TXB,
non-smoker smoker p-level 3
218.3+1.40 220.9*0.78 *
215.9kl.10 220.9*0.61 *
215.UO.52 219.6i0.74 *
ns. ns.
n.s. n.s.
P-TX&
non-smoker smoker p-level 3
2.16&O. 10 3.30*0.10 2.93*0.02 3.83kO.03 * *
2.33&O. 11 3.01&0.02 *
* *
* ns.
1I-DH TXl$
non-smoker smoker o-level 3
26.68*0.61 31.31*0.25 *
30.26*0.82 33.38kO.19 *
27.21*0.38 3O.lUO.95 *
* *
n.s. n.s.
TXE$-conv.
non-smoker smoker p-level 3
25.45hO.84 30.85*0.18 *
28.0811.37 32.61i0.17 *
25.8 lztO.62 30.85&0.12 *
* *
ns. ns.
HHT-conv.
non-smoker smoker p-level 3
25.63hO.94 31.30&0.15 *
28.01*0.47 32.25kO.18 *
25.65*0.75 31.00*0.09 *
* *
n.s. n.s.
MDA
p-level 2 *
Changes in the mean values and their level of significance are shown in the right columns. The column titled with “p-level 1” represents the level of significance of changes from baseline values to the values immediately after acute exposure to passive cigarette smoke, the column titled with “p-level 2” represents the one from baseline conditions to the values 6 hrs after maximal exposure. The line titled with “p-level 3” represents the level of significance between the mean values of non-smokers and smokers at the time points prior, immediately after and 6 hours after passive exposure to cigarette smoke. p
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455
2. Repeated exposure to passive smoke
Mean values for each day and both groups were calculated and differences between them on day 1, 2, 3, 4, 5 and 12 were evaluated. The plasmaTXB, levels in non-smokerswere at day 1 for all time points (baseline, immediately after and 6 hours after passive exposure to cigarette smoke) statistically significant lower than in smokers. On and after day 2 values of non-smokersbecame more close to the behaviour of smokers. A similar behaviour could be demonstrated in TXBZ-conversion and I-U-IT-conversion, although changeswere not so distinct. MDA formation in non-smokerscompared to smokerswas lower at day 1, 2 and 3. From day 4 onwards non-smoker values exceeded MDA levels of smokers. Serum TXE3, levels in non-smokerswere again very close to the values of smokers,but changeswere not as distinct than for other measures. Altogether results indicate that repeated exposure to cigarette smoke renders platelet function measuresof non-smokersmore activated and closeto the behaviour of smokers(Figures l-2)
FIG. 1. Figure 1. 1I-DH TX& and MDA for smokers (n=12) and non-smokers (n=l2) prior, immediately after and 6 hours after passiveexposure to cigarette smoke for day 1, 2, 3, 4, 5 and 12 are shown. Differences for each value between both groups are statistically significant at a level of p < 0.05 (*); n.s.=not significant;
FIG. 2 Figure 2. Serum TXB,, plasmaTXB,, TX&conversion and HHT-conversion for smokers (n=lZ) and non-smokers(n=12) prior, immediately after and 6 hours after passiveexposure to cigarette smoke for day 1, 2, 3, 4, 5 and 12 are shown. Differences for each value between both groups are statistically significant at a level of p
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DISCUSSION It is well known that active smoking is associated with an increased overall morbidity and mortality (1, 20, 21). Active smokers are at a greater risk for coronary artery disease and peripheral vascular disease. Only few data about the health effects of passive smoking in non-smokers and smokers are available (3, 4, 12) but there is evidence that passive smoking is associated in adults with an increased incidence of lung cancer and possibly greater mortality rates from coronary heart disease. There is a lack of data about the influence of passive smoking on the hemostatic balance, platelet hmction and the development of atherosclerotic lesions (22, 23). It is difficult to speculate on the role of platelets in the development of arterial disease in smokers without taking into consideration the vessel wall on the one hand and the plenty of platelet fimction tests available. For all the tests available positive and negative data can be found in the literature (24-27). Our findings indicate an activated platelet function upon passive exposure to cigarette smoke. Apparently, the acute response is more pronounced in non-smokers than in smokers. Repeated exposure to cigarette smoke seems to render platelet function measures of non-smokers more close to the behaviour of smokers. In an earlier study we demonstrated (8) the relation between passive smoking and the higher risk for hemostatic imbalance. Acute exposure to passive smoke with identical design induced in non-smokers even after 15 minutes exposure to passive cigarette smoke a short-lasting activation of platelet function and the prostglandin-system, followed by a quick recovery 6 hours after exposure no changes could be found. Repeated exposure of non-smokers to passive smoke, however, resulted in a continuous change of the basal values for platelet aggregation. The trends for platelet migration, platelet adhesion, circulating endothelial cells und microaggregates were similar but less severe. Platelet proteins were less altered. Changes were comparable to those seen in smokers, characterized by an activation of platelet function and a decrease in platelet sensitivity and binding sites to the antiaggregatory PGI,. Furthermore, a decrease in platelet sensitivity is a major determinant of haemostatic regulation and may thus be responsible for early vascular changes e.g. endothelial dysfunction which is the initial event of atherosclerosis preceeding the stage of plaque formation (28). The role of platelets in the initiation of the atherosclerotic process is somewhat controversial. In reviewing this field it becomes more and more apparent that atherosclerosis is a stereotypical response to any form of endothelial injury, either physical or immunological. Hyperlipidemia may be both a modifying or an initiating factor. Platelets play a significant part in the initiation of lesions and have a major role in the later complications which are expressed as clinical evidence of the disease process (29). Among the mechanisms thought to be involved in atherosclerosis and arterial thrombosis both platelet activation and endothelial damage are prominent, Davis et al. showed in ten healthy male non-smokers that brief passive exposure to tobacco smoke under naturally occuring environmental conditions has consistent acute effects on the endothelium and platelets (30). Although the group investigated was small, data may be representative for the general population since the same authors observed similar effects of active smoking on healthy male and female native smokers (31) healthy male habitual smokers (32) and male habitual smokers with coronary artery disease (33, 34). In atherosclerotic lesions there is a close functional association of monocytesimacrophages with blood platelets. Both cell types can activate the coagulation cascade (35). Thrombin, platelet factor 4, fibrin and fibronectin act as chemoattractants for monocytes. The early migration of monocytes into thrombi can cause a liberation of fibrinolytic activity. In addition, monocytes express a tissue factor and prothrombinase activity. Monocytes secrete coagulation factors (36)
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and plasminogen activator, Macrophages are also able to phagocytize platelets (37) and can huther activate platelet aggregation by releasing arachidonic acid metabolites. MDA, a secretory product of platelets, is known to induce a chemical modification of LDL to MDA-LDL, which is recognized by the scavenger receptor of macrophages (38). Active LDL can also be oxidized and is than taken up much more rapidly by the scavenger receptor. Thus, platelets may indirectly favor foam cell formation via modification of LDL (39). The possible influence of smoking on LDL and its biological activity was investigated by Scheffler et al ( 39). Plasma LDL was prepared from healthy male smokers and non-smokers, and oxidized with Cu”” as prooxidant. Oxidized LDL from smokers generated significantly more lipid peroxidation products, so-called thiobarbituric acid reactive substances (TBARS), when compared to oxidized non-smokers LDL. Analysis of vitamin E levels in LDL obtained from bof2;1 smokers and non-smokers revealed that the vitamin E content of smoker LDL was significantb less than that of non-smoker LDL. The amounts of cholestetyl esters formed in cultured P388.D. 1 macrophages were greater in the presence of smoker LDL than with non-smoker LDL. These results suggest that some of the proatherogenic effects of smoking may be related to oxidative modification of LDL and alteration of its biological activity. There is no doubt that inhaling passive smoke has an influence on platelet function for smokers and non-smokers as well. The influence of active cigarette smoking on thromboxane A, formation was recently published by Dotevall et al. (41). In female volunteers (18 smokers and I7 non-smokers, aged 20-46 years) platelet life-span and indices of platelet activity were determined, together with plasma levels of plasminogen activator inhibitor-l (PAI-I), fibrinogen, peripheral blood cell counts and hematocrit. The urinary excretion of TX-M was higher in smokers than in non-smokers (361 versus 204 pg/mg creatinine, respectively, p
Acknowledgment The authors gratefully acknowledge and Susanne Granegger, T A.
the valuable help of Y. Efthimiou,
MD, Y. Iliopoulos.
MD
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REFERENCES 1. US PUBLIC HEALTH SERVICE. The Health Consequences of Smoking: Cardiovascular Disease: A Report of the Surgeon General. DHHS (PHS), 84-50204, 1983. 2. ROSS, R. and GLOMSET, J.A. The pathogenesis of atherosclerosis. N Engl J Med 295, 369-374, 1976. 3. STEENLAND, K. Passive smoking and the risk of heart disease. JAMA 267, 94-99, 1992. 4. KRUPSKI, W.C. The peripheral vascular consequences of smoking. Ann Vast Surg 5, 291-304, 1991. 5. BURGHUBER, O.C., PUNZENGRUBER, C., SINZINGER, H., HABER, P., and SILBERBAUER, K. Platelet sensitivity to prostacyclin in smokers and non-smokers. Chest 90, 34-38, 1985. 6. SINZINGER, H. The effect of cigarette smoking on platelet mnction and hemostatic balance in healthy people. Thromb Haemost 51, 296, 1984. 7. HAWKINS, P. Smoking, platelets and thrombosis. Nature 236, 450-462, 1972. 8. SINZINGER, H. and VIRGOLINI, I. Besitzen Passivraucher ein erhohtes Thromboserisiko ? Wr Klin Wochenschr IOI, 694-698, 1989. 9. FITZ GERALD, G.A., OATES, J.A. and NOWAK, J. Cigarette smoking and hemostatic function. Am Heart J 11.5, 267-271, 1988. 10. DAVIS, J.W. and ARNOLD, J. Time course of some effects of cigarette smoking on platelets. J Intern Med 231, 3 l-36, 1992. 11. GLANTZ, ST. A. and PARMLEY, W.W. Passive smoking and heart disease epidemiology, physiology and biochemistry. Circulation 83, 1- 12, 199 1. 12. SIEGEL, M. Involuntary smoking in the restaurant workplace-a review of employee exposure and health effects. JAMA 2 70,490-493, 1993. 13. CHAPMAN, S. Unravelling gossamer with boxing gloves-problems in explaining the decline in smoking. BMJ 307, 429-432, 1993. 14. BOS, R.P. and HENDERSON, P.T. Genotoxic risk of passive smoking. Rev Environ Health 4, 161-178, 1984. 15. LESMES, G.R. and DONOFRIO, K.H. Passive smoking: the medical and economic issue. Am J Med 15, Suppl I, 38-42, 1992. 16. LEONE, A. Cardiovascular damage from smoking: a fact or belief? Int J Cardiol 38, 113-117, 1993. 17. GARLAND, C., BARRETT-CONNOR, E., SUAREZ, L., CRIQUI, M.H. and WINGARD, D.L. Effects of passive smoking on ischemic heart disease mortality of non-smokers, A prospective study. Am J Epidemiol 121, 645650, 1985. 18. SHAHAM, J., RIBAK, J. and GREEN, M. The consequences of passive smoking: an overview. Public Health Rev 20, 15-28. 1992- 1993. 19. SINZINGER, H., REITER, S. and PESKAR, B.A. Removal, preparation and storage of human plasma for radioimmunological detection of prostaglandins. In: Prostaglandins and Other Eicosanoids in the Cardiovascular System. K. Schror (ed.), pp. 62-67, Karger, Base1 (1985). 20. STERNBY, N.H. Atherosclerosis, smoking and other risk factors. In: Atherosclerosis .V A.M. Gotto Jr, L.C. Smith and B. Allen (eds.), pp. 67-70, Springer Verlag, New York (1980). 2 1. LAKIER, J.B. Smoking and cardiovascular disease. Am J Med 93, Suppi 1, 8- 12, 1992. 22. SINZINGER, H. and KEFALIDES, A. Passive smoking severely decreases platelet sensitivity to antiaggregatory prostaglandins. Lancet ii, 392-393, 1982.
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23. ZHU, B.Q., SUN, Y.P., SIEVERS, R.E., ISEIWERG, WM., GLANTZ, ST.A. and PARMLEY, W,W. Passive smoking increases experimental atherosclerosis in cholesterol-fed rabbits, J Am Co11 Cardio121, 225-232, 1993. 24 PUMPHREY, C.W. and DAWES, J. Plasma beta-thromboglobulin as a measure of platelet activity. Effect of risk factors and findings in ischemic heart disease and after acute myocardial infarction. Am J Cardiol 50, 1258-126 1, 1982. 25. MARASINI, B., BIONDI, M.L., BARBESTI, S., ZATTA, G. and AGOSTONI, A. Cigarette smoking and platelet function. Thromb Res 31, 85-94, 1986. 26. DOTEVALL, A., KUTTI, J., TEGER-NILSSON, A.C., WADENVIK, H. and WILHELMSEN, L. Platelet reactivity, fibrinogen and smoking. Eur J Haemato138, 55-59, 1987. 27 WENNMALM, A. Interaction of nicotine and prostaglandins in the cardiovascular system. Prostaglandins 23, 139-144, 1982. 28. SINZINGER, H., SCHERNTHANER G. and KALIMAN, J. Sensitivity of platelets to prostaglandins in coronary heart disease and angina pectoris. Prostaglandins 22, 773-776, 1981. 29 MOORE, S. The role of platelets in the early stages of atherosclerosis. In: P&e&~ ff& Atherosclerosis. Ch. Kessler (ed.), pp. l-9, Springer Verlag, Berlin, Heidelberg (1990). 30 DAVIS, J.W., SHELTON, L., WATANABE, T.S. and ARNOLD, J. Passive smoking affects endothelium and platelets. Arch Intern Med I-/9, 386-389, 1989 3 I DAVIS, J.W., SHELTON, L., EIGENBERG, D.A., HIGNITE, C. and WATANABE, I. Effects of tobacco and non-tobacco cigarette smoking on endothelium and platelets. Clin Pharmacol Ther 37, 529-533, 1985. 32. DAVIS, J.W., SHELTON, L., HARTMAN, C.R., EIGENBERG, D. and RUTTINGER, H. Smoking induced changes in endothelium and platelets are not affected by hydroxyethyh-utosides. Br J Exp Path01 67, 765-77 1, 1986. 33 DAVIS, J.W., SHELTON, L. and EIGENBERG, D.A. Lack ofeffect of aspirin on cigarette smoke-induced increase in circulating endothelial cells. Haemostasis i7, 66-69, 1987. 34. DAVIS, J.W., HARTMANN, C.R., LEWIS JR, H.D., SHELTON, L., EIGENBERG, D., HASSANEIN, K., HIGNITE, C. and RUTTINGER, H. Cigarette smoking-induced enhancement of platelet function: Lack of prevention by aspirin in men with coronary artery disease. J Lab Clin Med 105, 479-483, 1985. 35. RIVERS, R.P.A., HATHAWAY, W.E. and WESTON, W.L. The endotoxine-induced coagulant activity of human monocytes. Br J Haematol 30, 3 1 l-3 16, 1975 36 OSTERYUD, B., LINDAHI., U. and SELJELID. R. Macrophages produce coagulant factors FEBS Lett 120, 4 I-43, 1980. 37 CHANDLER, A.B. and HAND, R.A. Phagocytized platelets: a source of lipids in human thrombi and atherosclerotic plaques. Science 131, 946-949, 196 1. 38. FOGELMAN, A.M., SHECHTER, I., SEAGER, J., HOKOM, M., CHILD, J.S. and EDWARDS P.A. Malondialdehyd alteration of low density lipoprotein leads to cholesteryl ester accumulation in human monocyte-macrophage. Proc Nat1 Acad Sci USA 77, 269-271, 1980. 39. HODENBERG VON, E., HEINEN, S., HAUPTMANN, M., KREUZER, J., HENNINGSEN, H. and KUBLER, W. The role of macrophages in atherogenesis-platelet/monocyte interactions. In: Platelets anu’Atheroscl~rosi.s. Ch. Kessler (ed.), pp. 19-24, Springer Verlag, Berlin, Heidelberg (I 990). 40. SCHEFFLER, E., WIEST, E., WOEHRLE, J., OTTO, I., SCHULZ, I., HUBER, L., ZIEGLER, R. and DRESEL, H.A. Smoking influences the atherogenic potential of low-density lipoprotein Clin Investig 70, 263-268, 1992.
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460 41. DOTEVALL,
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A., WGEMARK, CH., ERIKSSON, E., KUTTI, J., WADENVIK, H. and WENNMALM, A. Cigarette smoking increases thromboxane a, formation without affecting platelet survival in young healthy females. Thromb Haemost 68, 583-588, 1992
Pergamon
PII s0049-3848(%)ooo17-5
PASSIVE SMOKING AND PLATELET Peter Schmid”,
THROMBOXANE
Georg Karanikas, Harald Kritz’), Christian Pirich2’, Yannis Bernhard A. Peskar3’, and Helmut Sinzinger2’4’
Stamatopoulos”,
“Cardiovascular Rehabilitation Center Bad Schallerbach, Upper Austria,“Department of Nuclear Medicine, University of Vienna, Austria,3’Department of Pharmacology and Toxicology, Atherosclerosis Research Group, Ruhr-University, Bochum, Germany, 4, Wilhelm-Auerswald Vienna, and 5,Rehabilitation Center “Engelsbad-Melanie”, Baden, Lower Austria, Austria
(Received 20 July 7995 by Editor E. Ernst; revised/accepted
Abstract
28 December 1995)
While active smoking is known to enhance platelet thromboxane production, no data on passive smoking is available yet. The influence of single and repeated exposure to passive smoke for 60 minutes in a 18 mi room was assessed in non-smokers as compared to sex and age matched smokers. All the evaluated measures (malondialdehyde, plasma thromboxane B,, 1 1-dehydro-thromboxane B?, serum thromboxane B, , conversion of exogenous arachidonic acid to thromboxane B3 and to hydroxy-5, 8, lo-heptadecatrienoic acid) were higher in smokers than non-smokers at baseline, immediately and 6 hours after passive exposure to cigarette smoke. Repeated exposure of non-smokers rendered their platelets more activated becoming close to the behaviour of smokers, These results indicate that passive smoking may activate thromboxane 4 release from the platelets, contributing to the development of hemostatic imbalance.
Active smoking is a well known risk factor for the development of atherosclerosis (1, 2) and in particular coronary heart disease (3) and peripheral vascular disease (4). The negative effects demonstrated on platelet function (5, 6) the eicosanoid system (7) and platelet thromboxane A1 generation (8) may contribute to the hemostatic imbalance (9, 10) reported. Recently, the problem of passive smoking as a health risk has widely been discussed (8, 1I- 14). Detailed information on the role of passive smoking on hemostatic parameters, however, is still very limited (1 S- 18). We thus assessed the influence of acute single and repeated passive smoking Key words: Passive smoking, platelet thromboxane; Corresponding author: Helmut Sinzinger, MD, Prof., Wilhelm-Auerswald Atherosclerosis Research Group (ASF) Vienna, Nadlergasse 1, A-1090 Vienna, Austria, Phone: 43-l-4082633, Fax: 43-l-4081366. 451
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under standardized conditions on platelet thromboxane formation assessed by different tests in non-smokers and smokers.
MATERIAL
and METHODS
Study design. The study was done as a stratified intervention study comparing the reactions of the different strata to a common exposure. It contains of three parts; first the comparisons between smokers and non-smokers, second the comparisons among repeated exposures on days 1, 2, 3,4, 5 and 12, and third the comparisons among pre-exposure-, immediate post-exposure-, and 6-hour post-exposure periods. Subjects. 12 healthy non-smokers (7 males, 5 females; aged 20-32 years) and 12 smokers (6 males, 6 females; aged 23-33 years, smoking more than 20 cigarettes/day) were examined. All volunteers were students recruited from the University of Vienna. They were not taking any medication during a period of at least 2 weeks prior entering and throughout the entire study. The experiments took place in the morning before work to avoid exposure to environmental smoke before the experimental periods began. The experimental phase started at 8 a.m. and was finished at 3 p.m. During this period as well as between 10 p.m. and 8 a.m. smokers abstained from active smoking. Between 3 p.m. and 10 p.m. they were allowed to continue their smoking behaviour Therefore, the wash-out period from active smoking lasted 10 hours. All the participants had normal weight and no risk factors for the development of atherosclerosis. They were exposed to the smoke of 30 cigarettes (GitanesR , nicotine 1.5mg, tar-contents 25mg) for 60 minutes in a 18 m3 room (room temperature 20°C, 60% relative air moisture) while sitting in an arm chair. Blood was drawn immediately prior to the exposure, immediately thereafter, as well as 6 hours later. This procedure was repeated on 5 subsequent days and on day 12. Blood was removed without venous occlusion using a 1.2 mm diameter needle and processed as follows (19). Laboratory Methods. Malondialdehyde @DA) Venous blood anticoagulated 3:7 with acid citrate dextrose (ACD) was drawn. After sedimentation at 18°C for 15 minutes platelet rich plasma (PRP) was prepared by centrifugation (150 x g for 7 minutes at 1W). 0.5 mL of PRP was centrifuged (700 x g for 25 minutes at 18°C) to get the platelet pellet which was washed twice in buffer (pH 7.2) containing 0.9 % NaCl, 2% Na-EDTA and 50 mM Tris-HCl (20: 1:2). The pellet was resuspended in 0.5 mL 50 mM Tris-HCI (pH 7.4) and incubated with 5 U/100 uL thrombin (Topostatin, Hofmann La Roche, Basle, Switzerland) for 30 minutes in a shaking water bath at 37°C. Additional 0.4 mL trichloracetic acid was added. The reaction was stopped at 4°C. After homogenization by means of ultrasonics the protein was separated by centrilugation (1500 x g for 10 minutes at 4°C). Thiobarbituric acid was added to the supernatant and heated to 100°C for 15 minutes. Thereafter, MDA was determined photometrically at 532 nm. The intraassay variation amounted 3.8*1.0%, the interassay variation 5.911. %. Values are given in &I/lo9 platelets. Serum ihromboxane B, (s-TXB,). 0.5 mL blood was collected. After clotting for exactly 60 min at 37°C in a shaking water bath the sample was centrifuged at 2000 x g for 15 minutes at 4°C. The supernatant was removed and stored at -70°C in Eppendorf-vials for not longer than 2 weeks. The concentration of TXB, was
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determined using a commercial RIA-kit (Amersham, Buckinghamshire, UK).The variation was 3.3*0.7%, the interassay variation 6.1&0.9%. Values are given in ng/mL.
453
intraassay
Plosmn thromboxane B, (p-TXB,). EDTA (2%) was used as anticoagulant and aspisol (1 mg/mL) for cyclooxygenase inhibition. The samples were centrifuged for 30 minutes at 4” C Thereafter, the plasma was removed and stored at -70°C for no longer than 2 weeks. The concentration of TXB, in unextracted samples was determined using a specific radioimmunoassay. Separation of free- and antibody-bound ligand was performed by means of a double-antibody (OTOP 15/16, Behring, Marburg, Germany). The intraassay variation was 3.8* 1 I%, the interassay variation 6.9* 1.5%. Values are given in pg/mL I I-dehydro-thromboxane B, (I I-DH TXB,). Blood was taken with 2% sodium EDTA as anticoagulant. After short sedimentation at room temperature, the samples were centrifuged (1500 x g at 4°C) for platelet poor plasma. This was transferred in small volumes into Eppendorf-vials and stored there at -70°C for not longer than 2 weeks. 1 l-DH-TXB? was determined with a commercial RIA-kit (Amersham, Buckinghamshire, UK). The intraassay variation was 2.7*0.5%, the interassay variation 5.1*0.8%. Values are given in pg/mL. Radiothinlayer Chromatography-‘4C -arachidonic acid conversion to TXB,and HHT. Anticoagulated blood (3:7 with ACD) was collected. PRP was obtained by centrifitgation and divided into 0.5 mL aliquots. After centrihgation (700 x g for 25 minutes at 18°C) the supernatant was removed with a vacuum pump. Afterwards the isolated platelets were resuspended in 0.5 mL 50 mM Tris-buffer (pH 7.4) and incubated with 0.25 pCi [‘“Cl arachidonic acid (AA) for 5 minutes at 37°C in a shaking water bath. The reaction was stopped with HCI (resulting in a pH of 3). After centrifirgation (5000 x g for 10 minutes at 4°C) the supernatant was extracted with ethyl-acetate. The organic phase was dried under nitrogen, dissolved in 100 uL ethanol and stored at -20°C. After a chromatography on silicagel, the labelled arachidonic acid metabolites (TXB, and HHT) were analysed with a TLC linear scanner, Radiolabeled standards (New England Nuclear, Dreieich, Germany) were used for identification. Values are given in %.
STATISTICAL
ANALYSIS
Comparing relations between smokers and non-smokers, parametric tests of significance were used. Data are given as mean f standard error of the mean (SEM). Mean values shown in Figures l-2 were calculated for each day and both groups, and differences between them on day 1, 2,3,4, 5 and 12 were evaluated.
RESULTS I Single exposure to passive smoke All the evaluated measures were higher in smokers than in non-smokers at baseline conditions, after acute exposure and 6 hours after maximal exposure to passive smoke. Comparison of mean values, calculated from the values estimated at day 1, 2, 3, 4, 5 and 12, for both groups for all the investigated measures before and after exposure to cigarette smoke is shown in Table 1 (see
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p-level 3). Differences at baseline conditions, immediately after and 6 hours after maximal passive exposure to cigarette smoke between non-smokers and smokers were statistically significant for plasma TXEJ,, 1 I-DH TXEJ, and conversion to TXB, and HHT, respectively. MDA and serum TXB, mean values steadily increased in smokers, but differences were statistically significant only at baseline for MDA and directly after exposure, respectively 6 hrs after exposure, for serum T=$. As compared to the basal values only MDA and plasma TXB, in non-smokers showed still significantly higher levels 6 hours after smoke exposure. Ail the other measures investigated reached 6 hours after exposure to passive cigarette smoke their baseline values again (Table I).
TABLE
1
Measures of Platelet Function for Non-smokers and Smokers prior, immediately after and 6 hours after Passive Exposure to Cigarette Smoke. Pre-value
Acute
6 hrs later
p-level 1
non-smoker smoker n-level 3
3.50&O. 12 3.86*0.02 *
4.20&0.05 4.343tO.16 ns.
3.66%0.09 3.87kO.02 n.s.
* *
ns
s-TXB,
non-smoker smoker p-level 3
218.3+1.40 220.9*0.78 *
215.9kl.10 220.9*0.61 *
215.UO.52 219.6i0.74 *
ns. ns.
n.s. n.s.
P-TX&
non-smoker smoker p-level 3
2.16&O. 10 3.30*0.10 2.93*0.02 3.83kO.03 * *
2.33&O. 11 3.01&0.02 *
* *
* ns.
1I-DH TXl$
non-smoker smoker o-level 3
26.68*0.61 31.31*0.25 *
30.26*0.82 33.38kO.19 *
27.21*0.38 3O.lUO.95 *
* *
n.s. n.s.
TXE$-conv.
non-smoker smoker p-level 3
25.45hO.84 30.85*0.18 *
28.0811.37 32.61i0.17 *
25.8 lztO.62 30.85&0.12 *
* *
ns. ns.
HHT-conv.
non-smoker smoker p-level 3
25.63hO.94 31.30&0.15 *
28.01*0.47 32.25kO.18 *
25.65*0.75 31.00*0.09 *
* *
n.s. n.s.
MDA
p-level 2 *
Changes in the mean values and their level of significance are shown in the right columns. The column titled with “p-level 1” represents the level of significance of changes from baseline values to the values immediately after acute exposure to passive cigarette smoke, the column titled with “p-level 2” represents the one from baseline conditions to the values 6 hrs after maximal exposure. The line titled with “p-level 3” represents the level of significance between the mean values of non-smokers and smokers at the time points prior, immediately after and 6 hours after passive exposure to cigarette smoke. p
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455
2. Repeated exposure to passive smoke
Mean values for each day and both groups were calculated and differences between them on day 1, 2, 3, 4, 5 and 12 were evaluated. The plasmaTXB, levels in non-smokerswere at day 1 for all time points (baseline, immediately after and 6 hours after passive exposure to cigarette smoke) statistically significant lower than in smokers. On and after day 2 values of non-smokersbecame more close to the behaviour of smokers. A similar behaviour could be demonstrated in TXBZ-conversion and I-U-IT-conversion, although changeswere not so distinct. MDA formation in non-smokerscompared to smokerswas lower at day 1, 2 and 3. From day 4 onwards non-smoker values exceeded MDA levels of smokers. Serum TXE3, levels in non-smokerswere again very close to the values of smokers,but changeswere not as distinct than for other measures. Altogether results indicate that repeated exposure to cigarette smoke renders platelet function measuresof non-smokersmore activated and closeto the behaviour of smokers(Figures l-2)
FIG. 1. Figure 1. 1I-DH TX& and MDA for smokers (n=12) and non-smokers (n=l2) prior, immediately after and 6 hours after passiveexposure to cigarette smoke for day 1, 2, 3, 4, 5 and 12 are shown. Differences for each value between both groups are statistically significant at a level of p < 0.05 (*); n.s.=not significant;
FIG. 2 Figure 2. Serum TXB,, plasmaTXB,, TX&conversion and HHT-conversion for smokers (n=lZ) and non-smokers(n=12) prior, immediately after and 6 hours after passiveexposure to cigarette smoke for day 1, 2, 3, 4, 5 and 12 are shown. Differences for each value between both groups are statistically significant at a level of p
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DISCUSSION It is well known that active smoking is associated with an increased overall morbidity and mortality (1, 20, 21). Active smokers are at a greater risk for coronary artery disease and peripheral vascular disease. Only few data about the health effects of passive smoking in non-smokers and smokers are available (3, 4, 12) but there is evidence that passive smoking is associated in adults with an increased incidence of lung cancer and possibly greater mortality rates from coronary heart disease. There is a lack of data about the influence of passive smoking on the hemostatic balance, platelet hmction and the development of atherosclerotic lesions (22, 23). It is difficult to speculate on the role of platelets in the development of arterial disease in smokers without taking into consideration the vessel wall on the one hand and the plenty of platelet fimction tests available. For all the tests available positive and negative data can be found in the literature (24-27). Our findings indicate an activated platelet function upon passive exposure to cigarette smoke. Apparently, the acute response is more pronounced in non-smokers than in smokers. Repeated exposure to cigarette smoke seems to render platelet function measures of non-smokers more close to the behaviour of smokers. In an earlier study we demonstrated (8) the relation between passive smoking and the higher risk for hemostatic imbalance. Acute exposure to passive smoke with identical design induced in non-smokers even after 15 minutes exposure to passive cigarette smoke a short-lasting activation of platelet function and the prostglandin-system, followed by a quick recovery 6 hours after exposure no changes could be found. Repeated exposure of non-smokers to passive smoke, however, resulted in a continuous change of the basal values for platelet aggregation. The trends for platelet migration, platelet adhesion, circulating endothelial cells und microaggregates were similar but less severe. Platelet proteins were less altered. Changes were comparable to those seen in smokers, characterized by an activation of platelet function and a decrease in platelet sensitivity and binding sites to the antiaggregatory PGI,. Furthermore, a decrease in platelet sensitivity is a major determinant of haemostatic regulation and may thus be responsible for early vascular changes e.g. endothelial dysfunction which is the initial event of atherosclerosis preceeding the stage of plaque formation (28). The role of platelets in the initiation of the atherosclerotic process is somewhat controversial. In reviewing this field it becomes more and more apparent that atherosclerosis is a stereotypical response to any form of endothelial injury, either physical or immunological. Hyperlipidemia may be both a modifying or an initiating factor. Platelets play a significant part in the initiation of lesions and have a major role in the later complications which are expressed as clinical evidence of the disease process (29). Among the mechanisms thought to be involved in atherosclerosis and arterial thrombosis both platelet activation and endothelial damage are prominent, Davis et al. showed in ten healthy male non-smokers that brief passive exposure to tobacco smoke under naturally occuring environmental conditions has consistent acute effects on the endothelium and platelets (30). Although the group investigated was small, data may be representative for the general population since the same authors observed similar effects of active smoking on healthy male and female native smokers (31) healthy male habitual smokers (32) and male habitual smokers with coronary artery disease (33, 34). In atherosclerotic lesions there is a close functional association of monocytesimacrophages with blood platelets. Both cell types can activate the coagulation cascade (35). Thrombin, platelet factor 4, fibrin and fibronectin act as chemoattractants for monocytes. The early migration of monocytes into thrombi can cause a liberation of fibrinolytic activity. In addition, monocytes express a tissue factor and prothrombinase activity. Monocytes secrete coagulation factors (36)
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and plasminogen activator, Macrophages are also able to phagocytize platelets (37) and can huther activate platelet aggregation by releasing arachidonic acid metabolites. MDA, a secretory product of platelets, is known to induce a chemical modification of LDL to MDA-LDL, which is recognized by the scavenger receptor of macrophages (38). Active LDL can also be oxidized and is than taken up much more rapidly by the scavenger receptor. Thus, platelets may indirectly favor foam cell formation via modification of LDL (39). The possible influence of smoking on LDL and its biological activity was investigated by Scheffler et al ( 39). Plasma LDL was prepared from healthy male smokers and non-smokers, and oxidized with Cu”” as prooxidant. Oxidized LDL from smokers generated significantly more lipid peroxidation products, so-called thiobarbituric acid reactive substances (TBARS), when compared to oxidized non-smokers LDL. Analysis of vitamin E levels in LDL obtained from bof2;1 smokers and non-smokers revealed that the vitamin E content of smoker LDL was significantb less than that of non-smoker LDL. The amounts of cholestetyl esters formed in cultured P388.D. 1 macrophages were greater in the presence of smoker LDL than with non-smoker LDL. These results suggest that some of the proatherogenic effects of smoking may be related to oxidative modification of LDL and alteration of its biological activity. There is no doubt that inhaling passive smoke has an influence on platelet function for smokers and non-smokers as well. The influence of active cigarette smoking on thromboxane A, formation was recently published by Dotevall et al. (41). In female volunteers (18 smokers and I7 non-smokers, aged 20-46 years) platelet life-span and indices of platelet activity were determined, together with plasma levels of plasminogen activator inhibitor-l (PAI-I), fibrinogen, peripheral blood cell counts and hematocrit. The urinary excretion of TX-M was higher in smokers than in non-smokers (361 versus 204 pg/mg creatinine, respectively, p
Acknowledgment The authors gratefully acknowledge and Susanne Granegger, T A.
the valuable help of Y. Efthimiou,
MD, Y. Iliopoulos.
MD
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23. ZHU, B.Q., SUN, Y.P., SIEVERS, R.E., ISEIWERG, WM., GLANTZ, ST.A. and PARMLEY, W,W. Passive smoking increases experimental atherosclerosis in cholesterol-fed rabbits, J Am Co11 Cardio121, 225-232, 1993. 24 PUMPHREY, C.W. and DAWES, J. Plasma beta-thromboglobulin as a measure of platelet activity. Effect of risk factors and findings in ischemic heart disease and after acute myocardial infarction. Am J Cardiol 50, 1258-126 1, 1982. 25. MARASINI, B., BIONDI, M.L., BARBESTI, S., ZATTA, G. and AGOSTONI, A. Cigarette smoking and platelet function. Thromb Res 31, 85-94, 1986. 26. DOTEVALL, A., KUTTI, J., TEGER-NILSSON, A.C., WADENVIK, H. and WILHELMSEN, L. Platelet reactivity, fibrinogen and smoking. Eur J Haemato138, 55-59, 1987. 27 WENNMALM, A. Interaction of nicotine and prostaglandins in the cardiovascular system. Prostaglandins 23, 139-144, 1982. 28. SINZINGER, H., SCHERNTHANER G. and KALIMAN, J. Sensitivity of platelets to prostaglandins in coronary heart disease and angina pectoris. Prostaglandins 22, 773-776, 1981. 29 MOORE, S. The role of platelets in the early stages of atherosclerosis. In: P&e&~ ff& Atherosclerosis. Ch. Kessler (ed.), pp. l-9, Springer Verlag, Berlin, Heidelberg (1990). 30 DAVIS, J.W., SHELTON, L., WATANABE, T.S. and ARNOLD, J. Passive smoking affects endothelium and platelets. Arch Intern Med I-/9, 386-389, 1989 3 I DAVIS, J.W., SHELTON, L., EIGENBERG, D.A., HIGNITE, C. and WATANABE, I. Effects of tobacco and non-tobacco cigarette smoking on endothelium and platelets. Clin Pharmacol Ther 37, 529-533, 1985. 32. DAVIS, J.W., SHELTON, L., HARTMAN, C.R., EIGENBERG, D. and RUTTINGER, H. Smoking induced changes in endothelium and platelets are not affected by hydroxyethyh-utosides. Br J Exp Path01 67, 765-77 1, 1986. 33 DAVIS, J.W., SHELTON, L. and EIGENBERG, D.A. Lack ofeffect of aspirin on cigarette smoke-induced increase in circulating endothelial cells. Haemostasis i7, 66-69, 1987. 34. DAVIS, J.W., HARTMANN, C.R., LEWIS JR, H.D., SHELTON, L., EIGENBERG, D., HASSANEIN, K., HIGNITE, C. and RUTTINGER, H. Cigarette smoking-induced enhancement of platelet function: Lack of prevention by aspirin in men with coronary artery disease. J Lab Clin Med 105, 479-483, 1985. 35. RIVERS, R.P.A., HATHAWAY, W.E. and WESTON, W.L. The endotoxine-induced coagulant activity of human monocytes. Br J Haematol 30, 3 1 l-3 16, 1975 36 OSTERYUD, B., LINDAHI., U. and SELJELID. R. Macrophages produce coagulant factors FEBS Lett 120, 4 I-43, 1980. 37 CHANDLER, A.B. and HAND, R.A. Phagocytized platelets: a source of lipids in human thrombi and atherosclerotic plaques. Science 131, 946-949, 196 1. 38. FOGELMAN, A.M., SHECHTER, I., SEAGER, J., HOKOM, M., CHILD, J.S. and EDWARDS P.A. Malondialdehyd alteration of low density lipoprotein leads to cholesteryl ester accumulation in human monocyte-macrophage. Proc Nat1 Acad Sci USA 77, 269-271, 1980. 39. HODENBERG VON, E., HEINEN, S., HAUPTMANN, M., KREUZER, J., HENNINGSEN, H. and KUBLER, W. The role of macrophages in atherogenesis-platelet/monocyte interactions. In: Platelets anu’Atheroscl~rosi.s. Ch. Kessler (ed.), pp. 19-24, Springer Verlag, Berlin, Heidelberg (I 990). 40. SCHEFFLER, E., WIEST, E., WOEHRLE, J., OTTO, I., SCHULZ, I., HUBER, L., ZIEGLER, R. and DRESEL, H.A. Smoking influences the atherogenic potential of low-density lipoprotein Clin Investig 70, 263-268, 1992.
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A., WGEMARK, CH., ERIKSSON, E., KUTTI, J., WADENVIK, H. and WENNMALM, A. Cigarette smoking increases thromboxane a, formation without affecting platelet survival in young healthy females. Thromb Haemost 68, 583-588, 1992