Haemostasis 5 : 14-20 (1976)
A Comparative Study of the Effect of Oral Contraceptives and Cigarette Smoking on Platelet Adhesiveness M. S t r o l in -B en ed etti , D. G utty and P. S tr o lin Battelle Research Center, Geneva
Key Words. Platelet adhesiveness • Oral contraceptives • Cigarette smoking • Throm bosis Abstract. The aim of this work was to discover to what extent cigarette smoking and oral contraceptives, either separately or in association, are responsible for platelet adhe siveness changes in humans. Platelet adhesiveness has been measured in 77 subjects, smokers and nonsmokers. 47 of these subjects were women both using and not using oral contraceptives. Cigarette smoking significantly increases platelet adhesiveness both in men and women. On the contrary, if no distinction is made in the smoking habits of female volunteers and they are only divided into two groups according to the use or nonuse of oral contraceptives, no significant change in platelet adhesiveness occurs following intake of oral contraceptives.
Introduction
Received: 18 August, 1975; accepted: September 16, 1975.
Downloaded by: Stockholm University Library 130.237.165.40 - 12/17/2018 10:00:34 PM
Several reports suggest an association between cigarette smoking and coronary heart disease; in particular it appears that cigarette smoking could be an important factor in the development of coronary thrombosis [1]. On the other hand, the possibility that the use of oral contraceptives may be related to the occurrence of thrombosis has been discussed for more than 10 years, and a number of reports are now available, although the results are often contradictory [13, 24], Oral contraceptives have been implicated partic ularly in venous thromboembolism and cerebral thrombosis [24]. The asso ciation between oral contraception and coronary thrombosis seems to be less definite [13, 14], although two recent investigations clearly indicate that the frequency increases in takers [16, 17],
Strolin -B enedetti/G utty/S trolin
15
F rederiksen and R a v en h o lt [8] have raised the question whether ciga rette smoking may potentiate the possible etiological role of oral contra ceptives in thromboembolism. Following data retabulation of V essf.y and D o ll [23] to evaluate possible separate or combined effects of smoking and use of oral contraceptives, they noted that users of oral contraceptives who were also heavy smokers had an incidence of thromboembolism 23 times greater than those being neither users nor smokers. As blood platelets appear to play the central role in the initiation of a thrombus, platelet parameters such as adhesiveness, aggregation, electro phoretic mobility and survival time have been studied by different authors in order to examine the effect either of cigarette smoking or of oral contra ceptives. The adhesiveness and aggregation of platelets appear to be increased by cigarette smoking [3, 9, 10], the platelet survival time is shortened [18], and the electrophoretic mobility seems to decrease [10, 22]. The same platelet parameters, following treatment with oral contraceptives, show more contra dictory results. In fact in the majority of reports [12, 19, 25] the platelet adhesiveness does not change significantly, whereas in others an increase is reported [2], The same contradictory results have been found for aggrega tion [6, 7, 20]. Concerning platelet electrophoretic behaviour, an alteration has been shown by B o lto n el al. [4] in both men and women receiving oral estrogens; however, according to them this alteration is probably due to changes in plasma lipids and not to an abnormality in the platelets them selves. The aim of this work was to investigate to what extent smoke and contra ceptives, either separately or combined, can be responsible for changes of platelet adhesiveness in humans. For this we measured platelet adhesiveness in volunteers with different histories of cigarette smoking and oral contra ception.
Platelet adhesiveness has been studied in 47 apparently healthy women aged 20-46. No drug had been ingested the day prior to the experiment, with the exception of subject No. 18 who received a dose of 300 mg papaverine some hours before. A blood sample of 5 ml was taken on the morning of the 7th day of the cycle from volunteers who had had no breakfast. The 47 women were divided into three groups: nonsmokers (16), light smokers (13) ( < 10 cigarettes/day), and heavy smokers (18) ( ^ 20 cigarettes/day). This investigation has not been performed blindly. Light and heavy smokers were requested to smoke a cigarette a few minutes immediately before the blood sampling. Of the 47 women
Downloaded by: Stockholm University Library 130.237.165.40 - 12/17/2018 10:00:34 PM
Material and Methods
16
Strolin-B enedetti/G utty/S trolin
26 used oral contraceptives (Eugynon, Neogynon, Gynovlar, Menoquens, Ethalontin, Combiquens, Stediryl, Ortho-novum, Anycycline) and 21 did not. For comparison the effect of smoke on platelet adhesiveness was also measured in men. 30 apparently healthy volunteers, aged 21-51, having ingested no drug the day prior to the experiment, participated in the study and were divided into three groups of 10, nonsmokers, light and heavy smokers, using the same criteria. They were fasting, and the light and heavy smokers were requested to smoke a cigarette immediately before the blood sampling. The blood (4 ml) was anticoagulated with trisodium citrate 5.5 H ,0 , 106 him (1 ml), then 2 ml of citrated blood were introduced into a paraffined syringe and forced through a siliconized needle into a column of glass beads [a piece of polyethylene tubing filled with 2.5 g of glass beads (Reflex Perlen, Bayreuth, FRG) of 0.5 mm diameter] with a constant rate of 1.07 ml/ min. All the glassware were siliconized with siliclad (Becton, Dickson, Parsippany, N. Y.) except the glass beads. The time lapse between blood collection and running through the column was carefully standardized at 15 min. The test is essentially the one described by H ellem [II] with some modification [21], The use of an amount of citrate higher than the one used in Hellem’s method was due to the desire of keeping the values of adhesiveness rather low, as smoke and contraceptives were expected to increase them. The number of platelets was counted before and after passage on the glass beads in a hemocytometer with a phase contrast microscope, and the adhesiveness is expressed as the percentage of adhered platelets of the total platelet count. In order to know whether the smoking of one cigarette immediately before the experiment could have an acute effect on adhesiveness, blood was collected from 18 light and heavy smokers (8 men and 10 women) immediately before and 2-5 min after the first cigarette of the day and the corresponding adhesiveness measured.
The individual results have been grouped according to smoking habits and contraceptive history. The average results obtained with the correspond ing standard deviations are presented in table I. In the same table are given the p values obtained by comparing the different groups by the Student’s t test. If the female volunteers are divided into two groups only, according to the use or nonuse of oral contraceptives, no significant difference in platelet adhesiveness has appeared. If only the women taking oral contraceptives are considered, and they are divided into three groups according to their smoking habits, the difference in platelet adhesiveness between nonsmokers and light smokers as well as between light and heavy smokers is not statistically significant. The increase between nonsmokers and heavy smokers is probably significant (table I). If only the women not taking oral contraceptives are considered and divided as above, no significant change in platelet adhesiveness appears between
Downloaded by: Stockholm University Library 130.237.165.40 - 12/17/2018 10:00:34 PM
Results and Discussion
Oral Contraceptives, Cigarette Smoking and Platelet Adhesiveness
17
Table I. Platelet adhesiveness, % Smoking habit
I II III IV
Nonsmokers Light smokers Heavy smokers Nonsmokers + light smokers + heavy smokers
Men, n = 30
Women using oral contraceptive n = 26
not using oral contra ceptive n = 21
total, n = 47
16.3+6.3 20.4+4.7 23.7+6.1
18.1+4.8 17.2+5.7 16.6+4.0 18.4+4.7 23.0+5.8 23.4+6.0
20.7+6.7
19.0+5.5
Men and women, n = 77
16.1+8.0 ( n = 10) 16.8 ±6.7 (n = 26) 20.2+4.7 ( n = 10) 19.16+4.8 (n = 23) 25.4+5.3 ( n = 10) 24.2 ±5.8 (n = 28)
p >0.05 p values [ I and II: p> 0.05 between II and III: p> 0.05 groups ( l a n d III: 0.01 < p 0.05 p > 0.05 p > 0.05
p > 0.05 0.01 0.05 p< 0.01 p
A Comparative Study of the Effect of Oral Contraceptives and Cigarette Smoking on Platelet Adhesiveness M. S t r o l in -B en ed etti , D. G utty and P. S tr o lin Battelle Research Center, Geneva
Key Words. Platelet adhesiveness • Oral contraceptives • Cigarette smoking • Throm bosis Abstract. The aim of this work was to discover to what extent cigarette smoking and oral contraceptives, either separately or in association, are responsible for platelet adhe siveness changes in humans. Platelet adhesiveness has been measured in 77 subjects, smokers and nonsmokers. 47 of these subjects were women both using and not using oral contraceptives. Cigarette smoking significantly increases platelet adhesiveness both in men and women. On the contrary, if no distinction is made in the smoking habits of female volunteers and they are only divided into two groups according to the use or nonuse of oral contraceptives, no significant change in platelet adhesiveness occurs following intake of oral contraceptives.
Introduction
Received: 18 August, 1975; accepted: September 16, 1975.
Downloaded by: Stockholm University Library 130.237.165.40 - 12/17/2018 10:00:34 PM
Several reports suggest an association between cigarette smoking and coronary heart disease; in particular it appears that cigarette smoking could be an important factor in the development of coronary thrombosis [1]. On the other hand, the possibility that the use of oral contraceptives may be related to the occurrence of thrombosis has been discussed for more than 10 years, and a number of reports are now available, although the results are often contradictory [13, 24], Oral contraceptives have been implicated partic ularly in venous thromboembolism and cerebral thrombosis [24]. The asso ciation between oral contraception and coronary thrombosis seems to be less definite [13, 14], although two recent investigations clearly indicate that the frequency increases in takers [16, 17],
Strolin -B enedetti/G utty/S trolin
15
F rederiksen and R a v en h o lt [8] have raised the question whether ciga rette smoking may potentiate the possible etiological role of oral contra ceptives in thromboembolism. Following data retabulation of V essf.y and D o ll [23] to evaluate possible separate or combined effects of smoking and use of oral contraceptives, they noted that users of oral contraceptives who were also heavy smokers had an incidence of thromboembolism 23 times greater than those being neither users nor smokers. As blood platelets appear to play the central role in the initiation of a thrombus, platelet parameters such as adhesiveness, aggregation, electro phoretic mobility and survival time have been studied by different authors in order to examine the effect either of cigarette smoking or of oral contra ceptives. The adhesiveness and aggregation of platelets appear to be increased by cigarette smoking [3, 9, 10], the platelet survival time is shortened [18], and the electrophoretic mobility seems to decrease [10, 22]. The same platelet parameters, following treatment with oral contraceptives, show more contra dictory results. In fact in the majority of reports [12, 19, 25] the platelet adhesiveness does not change significantly, whereas in others an increase is reported [2], The same contradictory results have been found for aggrega tion [6, 7, 20]. Concerning platelet electrophoretic behaviour, an alteration has been shown by B o lto n el al. [4] in both men and women receiving oral estrogens; however, according to them this alteration is probably due to changes in plasma lipids and not to an abnormality in the platelets them selves. The aim of this work was to investigate to what extent smoke and contra ceptives, either separately or combined, can be responsible for changes of platelet adhesiveness in humans. For this we measured platelet adhesiveness in volunteers with different histories of cigarette smoking and oral contra ception.
Platelet adhesiveness has been studied in 47 apparently healthy women aged 20-46. No drug had been ingested the day prior to the experiment, with the exception of subject No. 18 who received a dose of 300 mg papaverine some hours before. A blood sample of 5 ml was taken on the morning of the 7th day of the cycle from volunteers who had had no breakfast. The 47 women were divided into three groups: nonsmokers (16), light smokers (13) ( < 10 cigarettes/day), and heavy smokers (18) ( ^ 20 cigarettes/day). This investigation has not been performed blindly. Light and heavy smokers were requested to smoke a cigarette a few minutes immediately before the blood sampling. Of the 47 women
Downloaded by: Stockholm University Library 130.237.165.40 - 12/17/2018 10:00:34 PM
Material and Methods
16
Strolin-B enedetti/G utty/S trolin
26 used oral contraceptives (Eugynon, Neogynon, Gynovlar, Menoquens, Ethalontin, Combiquens, Stediryl, Ortho-novum, Anycycline) and 21 did not. For comparison the effect of smoke on platelet adhesiveness was also measured in men. 30 apparently healthy volunteers, aged 21-51, having ingested no drug the day prior to the experiment, participated in the study and were divided into three groups of 10, nonsmokers, light and heavy smokers, using the same criteria. They were fasting, and the light and heavy smokers were requested to smoke a cigarette immediately before the blood sampling. The blood (4 ml) was anticoagulated with trisodium citrate 5.5 H ,0 , 106 him (1 ml), then 2 ml of citrated blood were introduced into a paraffined syringe and forced through a siliconized needle into a column of glass beads [a piece of polyethylene tubing filled with 2.5 g of glass beads (Reflex Perlen, Bayreuth, FRG) of 0.5 mm diameter] with a constant rate of 1.07 ml/ min. All the glassware were siliconized with siliclad (Becton, Dickson, Parsippany, N. Y.) except the glass beads. The time lapse between blood collection and running through the column was carefully standardized at 15 min. The test is essentially the one described by H ellem [II] with some modification [21], The use of an amount of citrate higher than the one used in Hellem’s method was due to the desire of keeping the values of adhesiveness rather low, as smoke and contraceptives were expected to increase them. The number of platelets was counted before and after passage on the glass beads in a hemocytometer with a phase contrast microscope, and the adhesiveness is expressed as the percentage of adhered platelets of the total platelet count. In order to know whether the smoking of one cigarette immediately before the experiment could have an acute effect on adhesiveness, blood was collected from 18 light and heavy smokers (8 men and 10 women) immediately before and 2-5 min after the first cigarette of the day and the corresponding adhesiveness measured.
The individual results have been grouped according to smoking habits and contraceptive history. The average results obtained with the correspond ing standard deviations are presented in table I. In the same table are given the p values obtained by comparing the different groups by the Student’s t test. If the female volunteers are divided into two groups only, according to the use or nonuse of oral contraceptives, no significant difference in platelet adhesiveness has appeared. If only the women taking oral contraceptives are considered, and they are divided into three groups according to their smoking habits, the difference in platelet adhesiveness between nonsmokers and light smokers as well as between light and heavy smokers is not statistically significant. The increase between nonsmokers and heavy smokers is probably significant (table I). If only the women not taking oral contraceptives are considered and divided as above, no significant change in platelet adhesiveness appears between
Downloaded by: Stockholm University Library 130.237.165.40 - 12/17/2018 10:00:34 PM
Results and Discussion
Oral Contraceptives, Cigarette Smoking and Platelet Adhesiveness
17
Table I. Platelet adhesiveness, % Smoking habit
I II III IV
Nonsmokers Light smokers Heavy smokers Nonsmokers + light smokers + heavy smokers
Men, n = 30
Women using oral contraceptive n = 26
not using oral contra ceptive n = 21
total, n = 47
16.3+6.3 20.4+4.7 23.7+6.1
18.1+4.8 17.2+5.7 16.6+4.0 18.4+4.7 23.0+5.8 23.4+6.0
20.7+6.7
19.0+5.5
Men and women, n = 77
16.1+8.0 ( n = 10) 16.8 ±6.7 (n = 26) 20.2+4.7 ( n = 10) 19.16+4.8 (n = 23) 25.4+5.3 ( n = 10) 24.2 ±5.8 (n = 28)
p >0.05 p values [ I and II: p> 0.05 between II and III: p> 0.05 groups ( l a n d III: 0.01 < p 0.05 p > 0.05 p > 0.05
p > 0.05 0.01 0.05 p< 0.01 p
