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Effects of smoking on platelets and on plasma thromboxane-prostacyclin balance in man
Prostaglandins Leukotrienes and Medicine 9:
141-150, 1982
EFFECTS OF SMOKING ON PLATELETS AND ON PLASMA TBROMBOXANE-PROSTACYCLIN BALANCE IN MAN. Paulette Mehta and Jawahar Mehta, Departments of Pediatrics and Medicine, Divisions of Hematology-Oncology and Cardiology, University of Florida, College of Medicine, Gainesville, Florida 32610, U.S.A.
ABSTRACT To determine the influence of brief period of smoking on platelet release and platelet-vessel wall prostaglandins, we studied 13 nonsmokers and 15 habitual smokers. Blood samples were collected before and immediately after smoking. Before smoking plasma Bthromboglobulin levels were similar in the two groups. Plasma levels of thromboxane B2 (TXB2), stable metabolite of TXA2 .were slightly higher (124 + 43 vs 101 + 36 pg/ml) and of 6-keto-PGFl, (stable metabolite of PGI2) lower (39 + 23 vs 63 + 23 pg/ml) in smokers compared to non-smokers. Immediately after smoking, plasma TXB2 levels increased 197% (PxO.05) and plasma 6-keto-PGFl, 44% (PNS) in non-smokers. Similar increases were not observed in the smoker population, although plasma S-thromboglobulin levels increased 40%. Platelet TXA2 generations induced by arachidonic acid and thrombin were similar in the two groups. These data show that brief period of smoking stimulates both TXA2 and PG12 release in the nonsmokers. Increase in TXA2 is more than in PGI2. Episodic increase in TXA2 may be a mechanism of vascular injury. Absence of increase in TXA2 and PGI2 with smoking in habitual smokers may reflect tolerance to effects of smoking.
INTRODUCTION Vascular disease and its complications are more prevalent in individuals who smoke than in those who do not (1). Various mechanisms of smoking-induced vascular disease have been suggested, i.e. increased blood coagulation (2), and release of free fatty acids and catecholamines (3,4). Nicotine and carbon monoxide also have direct toxic effects on vascular endothelium (5,6). However, the precise mechanism by which smoking causes damage to the vessel wall is still
141
not clear. It is now believed that a balance between platelet and vessel wall activity mediated through certain prostaglandins, mainly thromboxane A2 (TXA2) and prostacyclin (PGI2), is of key importance in maintaining vascular tone and integrity (7). If smoking or its metabolic effects were to alter this balance, vascular damage could ensue (8). Accordingly, this study was designed to evaluate the effects of smoking upon platelet function parameters and TX.A2-PGI2equilibrium in habitual smokers and non-smokers. MATERIALS AND MBTRODS Study Subjects: The study subjects consisted of 13 non-smokers and 15 habitual smokers. All subjects were between the ages of 18 and 35 years (mean age 312 3 years for smokers and 25 t 3 years for non-smokers). The habitual smokers had smoked for an average of one pack of cigarettes for a mean of 6 years. None of the study subjects had any acute or chronic illness, and none had taken medications known to alter platelet function in the preceding 10 days. Informed consent was obtained from each individual prior to the study. Study Protocol: Each individual refrained from smoking for at least 2 hours before the study. All subjects were well rested and had not consumed any food for a minimum of 6 hours. Blood was taken after 15 minutes rest in a chair. Subjects then smoked two cigarettes (nicotine 19 mg, tar 23 mg per cigarette) in succession by deep inhalation. Peripheral venous blood samples were collected immediately after smoking the second cigarette. Blood Collection: Blood was collected from a previously untraumatised vein, Bach time a different vein was used, and care was taken that blood was freely flowing. No tourniquet was used to occlude blood flow to avoid trauma as it might influence platelet and/or vessel wall prostaglandin activity. Blood was immediately collected into polypropylene tubes containing 3.8% sodium citrate (9:1, v/v) for platelet counts and thromboxane generation studies, EDTA-theophylline (2.5:0.5, v/v> for i3thromboglobulin measurements, and aspirin-EDTA (2.0:0.5, v/v) for measurement of TXB2 (stable metabolite of TXA2) and 6-keto-PGFl, (stable metabolite of PGI2), Platelet and Prostaglandin Studies: Platelet counts were performed in duplicate by manual chamber counting. Red blood cells were lysed with procaine hydrochloride prior to counting. Plasma $-thromboglobulin was measured as previously described as index of platelet a-granule release using a commercially available radioimmunoassay kit (9). Blood samples were immediately placed in 142
ice after collection and centrifuged at 1500 g for 30 minutes at O°C. Plasma TXB2 was measured by radioimmunoassay as previously described (10). Multiple dilutions of the TXB2 standards, tracer solution (3H TXB2) and antiserum raised in rabbits were obtained from Raw England Nuclear, Boston, MA. The specificity using this technique is 100% for TXB2, 0.2% for PGE2, less than 0.2% for PGA2, PGF20 and 6-keto-PGFl,. The duplicate values showed a variation of 10%. The minimum detectable TXB2 levels in our laboratory are 50 pg/ml. The values less than 50 pg/ ml were considered as zero for the purpose of calculations in this study. Arachidonic acid-induced TXA7 generation:Platelet rich plasma was obtained from whole blood by centrifugation at 150 g for 15 minutes. Samples of platelet rich plasma (0.45 ml) were incubated with arachidonic acid (1 mM, 0.05 ml) (Sigma Chemical Co., St. Louis, MO) for 20 minutes at 370C in a shaking water bath. The plasma samples were stored in aspirin-EDTA mixture and TXB2 measured as described above. Thrombin-induced TXA2 generation:Platelet rich plasma was obtained as for arachidonic acid-induced TXA2 generation. Samples of platelet rich plasma (0.45 ml) were incubated with thrombin (lOD/ml, 0.05 ml) for 20 minutes at 370C in a shaking water bath. These samples were stored and TXB2 measured as described above. Plasma 6-keto-PGFI, was measured as a stable metabolic product of PGI2 by radioimmuno3ssay (11). Blood collection was same as for TXB2 assay. 6-keto-PGFlo, H tracer, and antibody to 6-keto-PGFl, raised in rabbits were obtained from New England Nuclear, Boston, MA. The specificity using this technique is 100% for 6-keto-PGFlo, 2.7% for PGF2,, 2.0% for PGE2, less than 0.1% for TXB2 and PGA2,and less than 0.1% for PGAl. The duplicate values obtained in each individual were again very similar (mean variation 10%). The minimum detectable 6-keto-PGFlo levels in our laboratory are 50 pg/ml. Values less 50 pg/ml were considered as zero for the purpose of calculations in this study. Calculations: All measurements were made in duplicate and an average of the duplicate values used to compute the mean. The data are expressed as mean + standard error. Student's t-test (paired and unpaired data) and Fisher Exact Test were used for statistical calculations. A P value less than 0.05 was considered significant. RESULTS The results of the platelet and prostaglandin studies are surnaarized in the Table. Platelet Counts: Platelet counts before smoking were not significantly different in non-smokers (417,000 + 50,000/mm3) compared to habitual smokers (348,000 + 34,OOO/mm3, P-NS). Smoking caused an increase in platelet
143
E
Plasma 6-keto-PGFlo (pg/ml)
Mean + SE Range-
Mean + SE Range-
Mean + SE Range-
AA-induced thromboxane B2 generation (pg/lO8 platelets)
Thrombin-induced thromboxane B2 generation( fig/lo8platelets)
Mean + SE Range
Mean+ SE Range-
Mean + SE Range-
Plasma thromboxane B2 (pg/ml)
Plasma 8-thromboglobulin (ng/ml)
Platelet Counts (xlOOO/mm3)
Smokers
90 + 34 (0 -295)
63 + 23 (0 z 225)
39 + 23 ( 0 -265)
970 + 247 ( 88-1500)
413 + 121 (88 -1500)
439 + 144 ( 30 - 3000)
996 + 303 ( 30 - 3000)
124 + 44 ( 0 = 420)
70 + 13 ( 31 -222)
348 + 34 (205 - 406)
Before
300 + 114 ( 0 -1245)
45 + 8 ( 24 = 97)
480 + 68 (203 -785)
1240 + 290 (85 - 3000)
470 + 148 (85 13000)
1012 36 ( 0 - 360)
62 + 15 ( 18 - 97)
417 + 50 (156 1625)
Non-Smokers Before After
AND 6-KETO-PGFlu
EFFECT OF ACUTE SMOKING ON PLATELETS AND PLASMA THKOMBOKANE B,
TABLE I
26 + 19 (0 = 260)
965 + 211 (20 11500)
355 + 117 (20-1500)
75 + 27 ( 0 Z335)
99 + 24 ( 41 1198)
419 + 42 (308 -600)
After
count in both non-smokers (15%) and smokers (20%). The increase in platelet count was similar in both groups. Plasma B-thromboglobulin: Resting plasma f3-thromboglobulinlevels were similar in non-smokers (62 f- 15 ng/ml) and habitual smokers (70 + 13 ng/ml, P-NS). After smoking, plasma S-thromboglobulin levels were unchanged in non-smokers as well as in smokers. However plasma b-thromboglobulin levels after smoking were higher in the habitual smokers group than in the nonsmokers group (mean 99 + 24 vs 45 + 8 ng/ml, PcO.02). Plasma TXB7: Habitual smokers had slightly higher resting levels of plasma TXB2 compared to non-smokers (mean 124 + 43 and 1012 36 pg/ml, respectively), but the difference was not significant. With brief period of smoking in the non-smoker group (n-13), plasma TXB2 levels increased in eight, declined in one and remained undetectable in four (Figure 1). The mean increase, 197%, was significant (PcO.05). In contrast, in the smoker group (n-14), plasma TXB2 levels increased in four, decreased in four and remained undetectable in the other six. Mean change was not significant. Plasma TXB2 levels after smoking were significantly higher in the non-smoker group than in the smoker group (mean 300 + 114 vs 75 + 27 pg/ml, respectively, PcO.02).
Fig. 1.
Plasma thromboxane B2 levels in non-smokers and habitual smokers before and fmmediately after smoking.
145
Platelet TXA2 Generation: At rest arachidonic acid-induced platelet TXA2 generation was not different in non-smokers and smokers (470 + 148 and 413 + 121 pg/lOg platelets, respectively). After smoking there was no significant change in platelet TXA2 generation in either non-smokers or in habitual smokers. Thrombin-induced platelet TXA2 generation similarly was not different in non-smokers and in smokers (1240 + 290 and 970 + 274 pg/108 platelets, respectively) at rest. There was no significant change after smoking in either group. Plasma 6-keto-PGFlo Plasma 6-keto-PGF levels were undetectable in 12 of 15 habitual smokers, but only'?n seven of 13 non-smokers tested. The mean value at rest was slightly lower in the smokers compared to the non-smokers (39 2 23 vs 63 + 23 pg/ml). In the non-smoker group, with brief period of smoking plasma 6-keto-PGFle levels increased in five, declined in two and were undetectable in the remaining eight (Fig. 2). Thus, there was a trend towards an increase in plasma 6-keto-PGFlo (mean change + 43%, from 63 + 23 to 90 + 34 pg/ml). In the habitual smoker group, plasma 6-keto-PGFl, levels were essentially unchanged (-33%, from 39 + 23 to 26 f. 19 pg/ml). Plasma 6-keto-PGFl, levels after smoking were significantly higher in the non-smokers compared to smokers (90 + 34 and 26 + 19 pg/ml, respectively, P
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3
lOO-
O-
Fig. 2.
Plasma 6-keto-PGFle levels in non-smokers and habitual amokers before and immediately after smoking.
146
DISCUSSION Increase in platelet-derived TXA2 (platelet aggregant and vasoconstrictor) and/or decrease in vessel wall generated PG12 (platelet aggregation inhibitor and vasodilator ) may be important factors in increased vascular tone, in vivo thrombosis and vascular injury (12). Recently, techniques have become available to measure plasma concentrations of metabolites of TXA2 and PGI2 (13,14). Measurement of these prostaglandins at rest and during provoked or spontaneous stress may provide information of patbophysiologic significance. Using a standard radioimmunoassay technique, we found that plasma TXB2 and C-keto-PGFl, (stable metabolites of TXA2and PGI respectively) concentrations in the non-smoker and smoker populations I' n steady state are in the similar range. However in the normal non-smoker volunteers, brief period of smoking results in stimulation of both TXA2 and PG12. Increase in TXA2 is significantly more than in PGI2. In contrast, similar increases after smoking in TXA2 and PG12 arenot observed in habitual smokers. Some of the previous studies on platelet aggregation and other function parameters have suggested platelet activation after smoking (2,15,16), although it is not a consistent phenomenon (17). In contrast, nicotine in vitro generally decreases platelet aggregation (18). Platelet aggregation studies performed in vitro are subject to several technical and environmental factors, which could influence these parameters. Measurement of platelet-release product, plasma 8-thromboglobulin, may provide an index of platelet activity (8) and survival (19). We observed increased plasma 8-thromboglobulin levels in habitual smokers before emoking, and a further increase after smoking as compared to non-smokers. Consistent with these observations was slight increase in plasma TXB2 prior to smoking in habitual smokers suggestive of a continuous in vivo platelet activation. However, plasma TXB2 levels increased 197% in non-smokers implying direct effect of smoking or an ingradient of tobacco and/or biochemical alterations caused by smoking (3,4), while plasma &thromboglobulin levels were unchanged. Recently, synthesis of TXA2 in addition to PG12 in the endothelial cells has been documented (20,21). Endothelial TXA2 synthesis increases with stressful stimuli (21). It is conceivable that increase in plasma TXB2 following smoking in nonsmokers is of vascular origin. This may also help explain the apparent discordance between plasma $-thromboglobulin and TXB2 concentrations. No change in plasma TXB2 in habitual smokers following smoking suggests either vascular tolerance to stress caused by smoking or inability of the endothelium to generate TXA2. Increase in plasma $-thromboglobulin reflects some increase in platelet a-granule release. The observations of similar arachidonic acid and thrombin-induced platelet TXA2 generation before and after smoking in our study subjects also suggests that platelet TXA2 generation is not significantly altered with prolonged smoking. This lends further support to the concept of increased TXA2 following smoking being of vascular origin. With brief period of smoking, plasma 6-keto-PGFla concentration was observed to increase in non-smokers representing stress to the endothelium (22). In addition, relative hypoxia (23) and non-specific stress could be factors in PG12 release from vessel wall. In contrast, absence of any change in 6-keto-PGFl, in response to smoking in habitual smokers may be due to: a) tolerance to the hemodynamic effects 147
of smoking, or b) presence of subclinical vascular disease and inability of damaged endothelium to generate PGI2 (24), or c) absence of platelet activation as the stimulus, or d) a combination of any of these factors. Increase in plasma 6-keto-PGF levels after brief period of smoking in non-smokers and lower rest$o ng levels in chronic smokers are in agreement with ex vivo experiments done in our laboratory. In these experiments, perfusion of umbilical vein with nicotine in levels achieved in smokers resulted in immediate PG12 release. After repeated perfusion of umbilical vein, PG12 release declined to levels lower than the control values (25). Steel et al (26) observed a similar decrease in PG12 release from the umbilical artery perfused with nicotine for prolonged time. In summary,our study shows that brief period of smoking in nonsmokers leads to marked release of TXA2, but only a slight increase in PG12. In contrast, similar brief period of smoking in habitual smokers is not accompanied by increases in TEA2 or PGI2. Smoking, perse, does not appear to affect platelet TEA2 generation. It can be hypothesized that frequent smoking causes episodic TEA2 release, while the protective effect of PG12 are limited. This episodic imbalance between TEA2 and PG12 may be in part a mechanism of vasospasm and subsequent vascular injury. REFERENCES 1. Friedman GD, Petitti DB, Bawol RD, Siegelaub AB. Mortality in cigarette smokers and quitters. N Engl J Med 304: 1404-10, 1981. 2. Rawkins. Smoking, platelets and thrombosis. Nature (London) 236: 450-2, 1972. 3. Eonttinen A, Rajasalmi M. Effect of heavy cigarette smoking on postprandial triglycerides, free fatty acids and cholesterol. Br Med J 1: 850-2, 1963. 4. Watts DT. The effect of nicotine and smoking on the secretion of epinephrine. Ann N Y Acad Sci 90: 74-80, 1960. 5. Shimamoto T. Damage to silicon-like properties of vascular endothelial cells and prevention by monoamine oxidase inhibitor nialamide. Asian Med J 3: 479-84, 1960. 6. Davies RF, Topping DL, Turner DM. The effect of intermittent carbon monoxide exposure on experimental atherosclerosis in the rabbit. Atherosclerosis 24: 572-536, 1976. 7. Moncada S, Vane JR. Arachidonic acid metabolites and the interaction between platelets and blood vessel walls. N Engl J Med 300: 1142-7, 1979.
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8. Gertz SD, Uretsky G, Wajnberg RS, Navott N, GotraurnMS. Endothellal cell damage and thrombus formation after partial arterial constriction. Relevance to the role of coronary artery spasm in the pathogenesis of myocardial infarction. Circulation 63: 476-8, 1981. Mehta,J, Mehta P. Platelet function in hypertension and effect 9. of therapy. Am J Cardiol 47: 331-4, 1981. 10. Mehta P, Mehta J: Potentiation of endoperoxide analog-induced platelet aggregation by heparin. Thromb Res 25: 91-9, 1982. 11. Mehta P, Mehta J. Prostacyclin and platelet aggregation in sickle cell disease. Pediatrics, September, 1982. 12. Mehta J, Mehta P. Role of blood platelets and prostaglandins in coronary artery disease. Am J Cardiol 48: 366-73, 1981. 13. Lewy RI, Wiener L, Walinsky P, Lefer AM, Silver NJ, Smith JB. Thromboxane release during pacing-induced angina pectoris: Possible vaso constrictor influence on the coronary vasculature. Circulation 61: 1165-71, 1980. 14. Hirsh PD, Hillis LD, Campbell WB, Firth BG, Willerson JT. Release of prostaglandins and thromboxane into the coronary circulation in patients with ischemic heart disease. N Engl J Med 304:685-91, 1981. 15. Levine PH. An acute effect of cigarette smoking on platelet function as possible link between smoking and arterial thrombosis. Circulation 43: 619-29, 1973. 16. Mustard JF, Murphy EA. Effect of smoking on blood coagulation and platelet survival in man. Br Med J 1: 846-9, 1963. 17. Rogers WR, Bass RL III, Johnson DE, et al. Atherosclerosis related responses to cigarette smoking in the baboon. Circulation 61: 1188-93, 1980.
18. Brinson K, Chakrabarti BK. Effect of nicotine on rabbit blood platelet aggregation. Atherosclerosis 20: 527-31, 1974. 19. Doyle DJ, Chesterman CN, Cade JF, McGready JR, Rennie CG, Morgan FJ. Plasma concentration of platelet-specific protein correlated with platelet survival. Blood 55: 82-4, 1980. 20. Ingerman-Wojenski C, Silver MJ, Smith JB, Maccurak E. Bovine endothelial cells in culture produce thromboxane as well as prostacyclin. J Clin Invest 67: 1292-6, 1981. 21. Ally AI, Horrobin DF. Thromboxane A2 in blood vessels walls and its physiological significance: Relevance to thrombosis and hypertension. Prostaglandins Med 4: 431-8, 1980.
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22. Mehta J, Mehta P, Zipper R, Horalek C. Thromboxane/prostacyclin equilibrium at rest and during exercise in man and its relationship to myocardial ischemia. Clin Res 29: 223 A, 1981. 23. Neri Serneri GG, Masotti G, Poggessi L, Galanti G. Release of prostacyclin into the blood stream and its exhaustion in humans after local blood flow changes (ischemia and venous stasis). Thromb Res 17: 197-208, 1980. 24. Dembinska-Kiec A, Greglewska T, Zmuda A, Greglewski RJ. The generation of prostacyclin by arteries and by coronary vascular bed is reduced in experimental atherosclerosis in rabbits. Prostaglandins 14: 1025-33, 1977. 25. Mehta P, Mehta J. Influence of nicotine on platelet and vessel wall prostaglandins. Clin Res 29: 814 A, 1981. 26. Stoel I, Giessen WJ, Zwolsman E, Quadt JFA, Ten Hoor F, Verheught GWA, Hugenholtz PG. Effect of nicotine on prostacyclin production in human umbilical arteries. Circulation 62: (suppl) III97, 1980.
150
141-150, 1982
EFFECTS OF SMOKING ON PLATELETS AND ON PLASMA TBROMBOXANE-PROSTACYCLIN BALANCE IN MAN. Paulette Mehta and Jawahar Mehta, Departments of Pediatrics and Medicine, Divisions of Hematology-Oncology and Cardiology, University of Florida, College of Medicine, Gainesville, Florida 32610, U.S.A.
ABSTRACT To determine the influence of brief period of smoking on platelet release and platelet-vessel wall prostaglandins, we studied 13 nonsmokers and 15 habitual smokers. Blood samples were collected before and immediately after smoking. Before smoking plasma Bthromboglobulin levels were similar in the two groups. Plasma levels of thromboxane B2 (TXB2), stable metabolite of TXA2 .were slightly higher (124 + 43 vs 101 + 36 pg/ml) and of 6-keto-PGFl, (stable metabolite of PGI2) lower (39 + 23 vs 63 + 23 pg/ml) in smokers compared to non-smokers. Immediately after smoking, plasma TXB2 levels increased 197% (PxO.05) and plasma 6-keto-PGFl, 44% (PNS) in non-smokers. Similar increases were not observed in the smoker population, although plasma S-thromboglobulin levels increased 40%. Platelet TXA2 generations induced by arachidonic acid and thrombin were similar in the two groups. These data show that brief period of smoking stimulates both TXA2 and PG12 release in the nonsmokers. Increase in TXA2 is more than in PGI2. Episodic increase in TXA2 may be a mechanism of vascular injury. Absence of increase in TXA2 and PGI2 with smoking in habitual smokers may reflect tolerance to effects of smoking.
INTRODUCTION Vascular disease and its complications are more prevalent in individuals who smoke than in those who do not (1). Various mechanisms of smoking-induced vascular disease have been suggested, i.e. increased blood coagulation (2), and release of free fatty acids and catecholamines (3,4). Nicotine and carbon monoxide also have direct toxic effects on vascular endothelium (5,6). However, the precise mechanism by which smoking causes damage to the vessel wall is still
141
not clear. It is now believed that a balance between platelet and vessel wall activity mediated through certain prostaglandins, mainly thromboxane A2 (TXA2) and prostacyclin (PGI2), is of key importance in maintaining vascular tone and integrity (7). If smoking or its metabolic effects were to alter this balance, vascular damage could ensue (8). Accordingly, this study was designed to evaluate the effects of smoking upon platelet function parameters and TX.A2-PGI2equilibrium in habitual smokers and non-smokers. MATERIALS AND MBTRODS Study Subjects: The study subjects consisted of 13 non-smokers and 15 habitual smokers. All subjects were between the ages of 18 and 35 years (mean age 312 3 years for smokers and 25 t 3 years for non-smokers). The habitual smokers had smoked for an average of one pack of cigarettes for a mean of 6 years. None of the study subjects had any acute or chronic illness, and none had taken medications known to alter platelet function in the preceding 10 days. Informed consent was obtained from each individual prior to the study. Study Protocol: Each individual refrained from smoking for at least 2 hours before the study. All subjects were well rested and had not consumed any food for a minimum of 6 hours. Blood was taken after 15 minutes rest in a chair. Subjects then smoked two cigarettes (nicotine 19 mg, tar 23 mg per cigarette) in succession by deep inhalation. Peripheral venous blood samples were collected immediately after smoking the second cigarette. Blood Collection: Blood was collected from a previously untraumatised vein, Bach time a different vein was used, and care was taken that blood was freely flowing. No tourniquet was used to occlude blood flow to avoid trauma as it might influence platelet and/or vessel wall prostaglandin activity. Blood was immediately collected into polypropylene tubes containing 3.8% sodium citrate (9:1, v/v) for platelet counts and thromboxane generation studies, EDTA-theophylline (2.5:0.5, v/v> for i3thromboglobulin measurements, and aspirin-EDTA (2.0:0.5, v/v) for measurement of TXB2 (stable metabolite of TXA2) and 6-keto-PGFl, (stable metabolite of PGI2), Platelet and Prostaglandin Studies: Platelet counts were performed in duplicate by manual chamber counting. Red blood cells were lysed with procaine hydrochloride prior to counting. Plasma $-thromboglobulin was measured as previously described as index of platelet a-granule release using a commercially available radioimmunoassay kit (9). Blood samples were immediately placed in 142
ice after collection and centrifuged at 1500 g for 30 minutes at O°C. Plasma TXB2 was measured by radioimmunoassay as previously described (10). Multiple dilutions of the TXB2 standards, tracer solution (3H TXB2) and antiserum raised in rabbits were obtained from Raw England Nuclear, Boston, MA. The specificity using this technique is 100% for TXB2, 0.2% for PGE2, less than 0.2% for PGA2, PGF20 and 6-keto-PGFl,. The duplicate values showed a variation of 10%. The minimum detectable TXB2 levels in our laboratory are 50 pg/ml. The values less than 50 pg/ ml were considered as zero for the purpose of calculations in this study. Arachidonic acid-induced TXA7 generation:Platelet rich plasma was obtained from whole blood by centrifugation at 150 g for 15 minutes. Samples of platelet rich plasma (0.45 ml) were incubated with arachidonic acid (1 mM, 0.05 ml) (Sigma Chemical Co., St. Louis, MO) for 20 minutes at 370C in a shaking water bath. The plasma samples were stored in aspirin-EDTA mixture and TXB2 measured as described above. Thrombin-induced TXA2 generation:Platelet rich plasma was obtained as for arachidonic acid-induced TXA2 generation. Samples of platelet rich plasma (0.45 ml) were incubated with thrombin (lOD/ml, 0.05 ml) for 20 minutes at 370C in a shaking water bath. These samples were stored and TXB2 measured as described above. Plasma 6-keto-PGFI, was measured as a stable metabolic product of PGI2 by radioimmuno3ssay (11). Blood collection was same as for TXB2 assay. 6-keto-PGFlo, H tracer, and antibody to 6-keto-PGFl, raised in rabbits were obtained from New England Nuclear, Boston, MA. The specificity using this technique is 100% for 6-keto-PGFlo, 2.7% for PGF2,, 2.0% for PGE2, less than 0.1% for TXB2 and PGA2,and less than 0.1% for PGAl. The duplicate values obtained in each individual were again very similar (mean variation 10%). The minimum detectable 6-keto-PGFlo levels in our laboratory are 50 pg/ml. Values less 50 pg/ml were considered as zero for the purpose of calculations in this study. Calculations: All measurements were made in duplicate and an average of the duplicate values used to compute the mean. The data are expressed as mean + standard error. Student's t-test (paired and unpaired data) and Fisher Exact Test were used for statistical calculations. A P value less than 0.05 was considered significant. RESULTS The results of the platelet and prostaglandin studies are surnaarized in the Table. Platelet Counts: Platelet counts before smoking were not significantly different in non-smokers (417,000 + 50,000/mm3) compared to habitual smokers (348,000 + 34,OOO/mm3, P-NS). Smoking caused an increase in platelet
143
E
Plasma 6-keto-PGFlo (pg/ml)
Mean + SE Range-
Mean + SE Range-
Mean + SE Range-
AA-induced thromboxane B2 generation (pg/lO8 platelets)
Thrombin-induced thromboxane B2 generation( fig/lo8platelets)
Mean + SE Range
Mean+ SE Range-
Mean + SE Range-
Plasma thromboxane B2 (pg/ml)
Plasma 8-thromboglobulin (ng/ml)
Platelet Counts (xlOOO/mm3)
Smokers
90 + 34 (0 -295)
63 + 23 (0 z 225)
39 + 23 ( 0 -265)
970 + 247 ( 88-1500)
413 + 121 (88 -1500)
439 + 144 ( 30 - 3000)
996 + 303 ( 30 - 3000)
124 + 44 ( 0 = 420)
70 + 13 ( 31 -222)
348 + 34 (205 - 406)
Before
300 + 114 ( 0 -1245)
45 + 8 ( 24 = 97)
480 + 68 (203 -785)
1240 + 290 (85 - 3000)
470 + 148 (85 13000)
1012 36 ( 0 - 360)
62 + 15 ( 18 - 97)
417 + 50 (156 1625)
Non-Smokers Before After
AND 6-KETO-PGFlu
EFFECT OF ACUTE SMOKING ON PLATELETS AND PLASMA THKOMBOKANE B,
TABLE I
26 + 19 (0 = 260)
965 + 211 (20 11500)
355 + 117 (20-1500)
75 + 27 ( 0 Z335)
99 + 24 ( 41 1198)
419 + 42 (308 -600)
After
count in both non-smokers (15%) and smokers (20%). The increase in platelet count was similar in both groups. Plasma B-thromboglobulin: Resting plasma f3-thromboglobulinlevels were similar in non-smokers (62 f- 15 ng/ml) and habitual smokers (70 + 13 ng/ml, P-NS). After smoking, plasma S-thromboglobulin levels were unchanged in non-smokers as well as in smokers. However plasma b-thromboglobulin levels after smoking were higher in the habitual smokers group than in the nonsmokers group (mean 99 + 24 vs 45 + 8 ng/ml, PcO.02). Plasma TXB7: Habitual smokers had slightly higher resting levels of plasma TXB2 compared to non-smokers (mean 124 + 43 and 1012 36 pg/ml, respectively), but the difference was not significant. With brief period of smoking in the non-smoker group (n-13), plasma TXB2 levels increased in eight, declined in one and remained undetectable in four (Figure 1). The mean increase, 197%, was significant (PcO.05). In contrast, in the smoker group (n-14), plasma TXB2 levels increased in four, decreased in four and remained undetectable in the other six. Mean change was not significant. Plasma TXB2 levels after smoking were significantly higher in the non-smoker group than in the smoker group (mean 300 + 114 vs 75 + 27 pg/ml, respectively, PcO.02).
Fig. 1.
Plasma thromboxane B2 levels in non-smokers and habitual smokers before and fmmediately after smoking.
145
Platelet TXA2 Generation: At rest arachidonic acid-induced platelet TXA2 generation was not different in non-smokers and smokers (470 + 148 and 413 + 121 pg/lOg platelets, respectively). After smoking there was no significant change in platelet TXA2 generation in either non-smokers or in habitual smokers. Thrombin-induced platelet TXA2 generation similarly was not different in non-smokers and in smokers (1240 + 290 and 970 + 274 pg/108 platelets, respectively) at rest. There was no significant change after smoking in either group. Plasma 6-keto-PGFlo Plasma 6-keto-PGF levels were undetectable in 12 of 15 habitual smokers, but only'?n seven of 13 non-smokers tested. The mean value at rest was slightly lower in the smokers compared to the non-smokers (39 2 23 vs 63 + 23 pg/ml). In the non-smoker group, with brief period of smoking plasma 6-keto-PGFle levels increased in five, declined in two and were undetectable in the remaining eight (Fig. 2). Thus, there was a trend towards an increase in plasma 6-keto-PGFlo (mean change + 43%, from 63 + 23 to 90 + 34 pg/ml). In the habitual smoker group, plasma 6-keto-PGFl, levels were essentially unchanged (-33%, from 39 + 23 to 26 f. 19 pg/ml). Plasma 6-keto-PGFl, levels after smoking were significantly higher in the non-smokers compared to smokers (90 + 34 and 26 + 19 pg/ml, respectively, P
1300if *& zooto
3
lOO-
O-
Fig. 2.
Plasma 6-keto-PGFle levels in non-smokers and habitual amokers before and immediately after smoking.
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DISCUSSION Increase in platelet-derived TXA2 (platelet aggregant and vasoconstrictor) and/or decrease in vessel wall generated PG12 (platelet aggregation inhibitor and vasodilator ) may be important factors in increased vascular tone, in vivo thrombosis and vascular injury (12). Recently, techniques have become available to measure plasma concentrations of metabolites of TXA2 and PGI2 (13,14). Measurement of these prostaglandins at rest and during provoked or spontaneous stress may provide information of patbophysiologic significance. Using a standard radioimmunoassay technique, we found that plasma TXB2 and C-keto-PGFl, (stable metabolites of TXA2and PGI respectively) concentrations in the non-smoker and smoker populations I' n steady state are in the similar range. However in the normal non-smoker volunteers, brief period of smoking results in stimulation of both TXA2 and PG12. Increase in TXA2 is significantly more than in PGI2. In contrast, similar increases after smoking in TXA2 and PG12 arenot observed in habitual smokers. Some of the previous studies on platelet aggregation and other function parameters have suggested platelet activation after smoking (2,15,16), although it is not a consistent phenomenon (17). In contrast, nicotine in vitro generally decreases platelet aggregation (18). Platelet aggregation studies performed in vitro are subject to several technical and environmental factors, which could influence these parameters. Measurement of platelet-release product, plasma 8-thromboglobulin, may provide an index of platelet activity (8) and survival (19). We observed increased plasma 8-thromboglobulin levels in habitual smokers before emoking, and a further increase after smoking as compared to non-smokers. Consistent with these observations was slight increase in plasma TXB2 prior to smoking in habitual smokers suggestive of a continuous in vivo platelet activation. However, plasma TXB2 levels increased 197% in non-smokers implying direct effect of smoking or an ingradient of tobacco and/or biochemical alterations caused by smoking (3,4), while plasma &thromboglobulin levels were unchanged. Recently, synthesis of TXA2 in addition to PG12 in the endothelial cells has been documented (20,21). Endothelial TXA2 synthesis increases with stressful stimuli (21). It is conceivable that increase in plasma TXB2 following smoking in nonsmokers is of vascular origin. This may also help explain the apparent discordance between plasma $-thromboglobulin and TXB2 concentrations. No change in plasma TXB2 in habitual smokers following smoking suggests either vascular tolerance to stress caused by smoking or inability of the endothelium to generate TXA2. Increase in plasma $-thromboglobulin reflects some increase in platelet a-granule release. The observations of similar arachidonic acid and thrombin-induced platelet TXA2 generation before and after smoking in our study subjects also suggests that platelet TXA2 generation is not significantly altered with prolonged smoking. This lends further support to the concept of increased TXA2 following smoking being of vascular origin. With brief period of smoking, plasma 6-keto-PGFla concentration was observed to increase in non-smokers representing stress to the endothelium (22). In addition, relative hypoxia (23) and non-specific stress could be factors in PG12 release from vessel wall. In contrast, absence of any change in 6-keto-PGFl, in response to smoking in habitual smokers may be due to: a) tolerance to the hemodynamic effects 147
of smoking, or b) presence of subclinical vascular disease and inability of damaged endothelium to generate PGI2 (24), or c) absence of platelet activation as the stimulus, or d) a combination of any of these factors. Increase in plasma 6-keto-PGF levels after brief period of smoking in non-smokers and lower rest$o ng levels in chronic smokers are in agreement with ex vivo experiments done in our laboratory. In these experiments, perfusion of umbilical vein with nicotine in levels achieved in smokers resulted in immediate PG12 release. After repeated perfusion of umbilical vein, PG12 release declined to levels lower than the control values (25). Steel et al (26) observed a similar decrease in PG12 release from the umbilical artery perfused with nicotine for prolonged time. In summary,our study shows that brief period of smoking in nonsmokers leads to marked release of TXA2, but only a slight increase in PG12. In contrast, similar brief period of smoking in habitual smokers is not accompanied by increases in TEA2 or PGI2. Smoking, perse, does not appear to affect platelet TEA2 generation. It can be hypothesized that frequent smoking causes episodic TEA2 release, while the protective effect of PG12 are limited. This episodic imbalance between TEA2 and PG12 may be in part a mechanism of vasospasm and subsequent vascular injury. REFERENCES 1. Friedman GD, Petitti DB, Bawol RD, Siegelaub AB. Mortality in cigarette smokers and quitters. N Engl J Med 304: 1404-10, 1981. 2. Rawkins. Smoking, platelets and thrombosis. Nature (London) 236: 450-2, 1972. 3. Eonttinen A, Rajasalmi M. Effect of heavy cigarette smoking on postprandial triglycerides, free fatty acids and cholesterol. Br Med J 1: 850-2, 1963. 4. Watts DT. The effect of nicotine and smoking on the secretion of epinephrine. Ann N Y Acad Sci 90: 74-80, 1960. 5. Shimamoto T. Damage to silicon-like properties of vascular endothelial cells and prevention by monoamine oxidase inhibitor nialamide. Asian Med J 3: 479-84, 1960. 6. Davies RF, Topping DL, Turner DM. The effect of intermittent carbon monoxide exposure on experimental atherosclerosis in the rabbit. Atherosclerosis 24: 572-536, 1976. 7. Moncada S, Vane JR. Arachidonic acid metabolites and the interaction between platelets and blood vessel walls. N Engl J Med 300: 1142-7, 1979.
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18. Brinson K, Chakrabarti BK. Effect of nicotine on rabbit blood platelet aggregation. Atherosclerosis 20: 527-31, 1974. 19. Doyle DJ, Chesterman CN, Cade JF, McGready JR, Rennie CG, Morgan FJ. Plasma concentration of platelet-specific protein correlated with platelet survival. Blood 55: 82-4, 1980. 20. Ingerman-Wojenski C, Silver MJ, Smith JB, Maccurak E. Bovine endothelial cells in culture produce thromboxane as well as prostacyclin. J Clin Invest 67: 1292-6, 1981. 21. Ally AI, Horrobin DF. Thromboxane A2 in blood vessels walls and its physiological significance: Relevance to thrombosis and hypertension. Prostaglandins Med 4: 431-8, 1980.
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