Author + information
- Received August 7, 2004
- Revision received September 23, 2004
- Accepted October 20, 2004
- Published online February 15, 2005.
- Hirohiko Morita, MD,
- Hisao Ikeda, MD, PhD* (, )
- Nobuya Haramaki, MD, PhD,
- Hiroyuki Eguchi, MD, PhD and
- Tsutomu Imaizumi, MD, PhD, FACC
- ↵*Reprint requests and correspondence:
Dr. Hisao Ikeda, Department of Internal Medicine III, Kurume University School of Medicine, 67 Asahi-machi, Kurume, 830-0011, Japan
Objectives We investigated whether and how soon smoking cessation ameliorates the smoking-induced intracellular oxidative stress and platelet aggregability in long-term smokers.
Background Smoking is a major risk factor of atherothrombosis. Smoking cessation reduces cardiac events. However, the underlying mechanisms of the beneficial effects remain to be elucidated.
Methods Twenty-seven male long-term smokers were divided into two groups. Group A (n = 14) quit smoking for four weeks whereas group B (n = 13) resumed smoking two weeks after quitting. Smoking status was monitored by measurement of urinary cotinine. Using gel-filtered platelets, agonist (adenosine diphosphate and collagen)-induced platelet aggregation, platelet-derived nitric oxide (PDNO), intraplatelet nitrotyrosine production, intraplatelet levels of the reduced form of glutathione (GSH) and its oxidized form (GSSG), and urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG) and urinary 8-iso-prostaglandin F2α(8-iso-PGF2α), as markers of systemic oxidative stress, were measured. The baseline measurements were similar between the two groups.
Results Smoking cessation quickly reduced agonist-induced platelet aggregations, intraplatelet nitrotyrosine level, and urinary productions of 8-OHdG and 8-iso-PGF2αby two weeks in both groups. In group A, they were maintained at the low levels until four weeks, whereas they were reversed by resmoking in group B; PDNO release and intraplatelet GSH/GSSG ratio were time-dependently increased by smoking cessation but reversed by resmoking.
Conclusions The present findings are the first demonstration that only two weeks of smoking cessation can ameliorate the enhanced platelet aggregability and intraplatelet redox imbalance in long-term smokers, possibly by decreasing oxidative stress. Our findings may strengthen the motivation for smokers to quit smoking.
Epidemiological studies have demonstrated that chronic smoking is a major risk factor for the development of atherosclerosis and thrombosis (1–3). These overwhelming epidemiological data closely link long-term smoking to adverse cardiovascular effects. Indeed, previous studies have shown the enhanced platelet aggregability (4–6) and the alterations in the clotting cascade in long-term smokers, suggesting the exaggerated risk of coronary artery thrombosis (7,8).
Platelets possess the L-arginine–nitric oxide (NO) pathway through constitutive NO synthase in humans (9,10). We have previously shown that long-term smoking impairs platelet-derived nitric oxide (PDNO) release (6), which acts as a negative feedback mechanism to inhibit not only platelet aggregation (11) but also recruitment after aggregation (12). Furthermore, we have recently shown that impaired PDNO bioactivity and augmented platelet aggregability in long-term smokers are related to the imbalance of the intraplatelet redox state, suggesting the smoking-induced oxidative stress for platelet activation (5,6). Thus, smoking-induced platelet-mediated thrombotic mechanisms may be involved in the pathophysiology of coronary artery disease of long-term smokers. It has been previously reported that smoking cessation is associated with a reduction in the risk of cardiovascular disease (13,14). However, the underlying mechanisms of the beneficial effects remain unclear. Accordingly, we investigated whether and how soon smoking cessation ameliorates impaired PDNO bioactivity and augmented platelet aggregability by improving the imbalance of the intraplatelet redox state. To assess this issue, we examined the effects of smoking cessation on platelet aggregation, PDNO bioactivity, intraplatelet nitrotyrosine production, the intraplatelet redox state, and systemic oxidative stress.
The study groups consisted of 27 healthy male medical students in our university who smoked at least 15 cigarettes per day for more than 5 years (mean age, 27.4 years old). They were randomly divided into the two groups after baseline measurements (Table 1).Group A (n = 14) quit smoking for 28 days, whereas group B (n = 13) resumed smoking at day 14 after quitting. We told the volunteers at the beginning of the study not to change their lifestyle. Smokers had abstained from smoking for at least 120 min before initiating the protocol to avoid the acute effects of smoking on platelet function. Thereafter, blood sampling was performed at 7, 14, 21, and 28 days, and urinary sampling was performed at 14 and 28 days after the start of the protocol. Smoking status was monitored by weekly measurements of urinary cotinine in all subjects. The protocol was approved by the institutional ethics committee. Written informed consent was obtained from all subjects.
Preparation of washed platelets
Platelet suspensions were prepared as described previously (4). Briefly, blood (20 ml) was collected by venipuncture into a plastic tube containing 3.15% trisodium citrate. Platelet-rich plasma (PRP) and platelet-poor plasma (PPP) were prepared according to the previously described method (15). The platelets counts were adjusted to 2 × 105platelets/μl in Tyrode's solution, the composition of which was described previously (4).
Measurements of platelet aggregation
We measured adenosine diphosphate (ADP)- and collagen-induced platelet aggregations as described previously (5,6). In brief, ADP (5 and 10 μmol/l) and collagen (0.2 and 0.5 μg/ml) were added to the washed platelet suspensions, and light transmission was monitored by using a platelet aggregometer (MDM Hematracer, MC Medical Co., Tokyo, Japan).
Measurements of PDNO
We measured NO by using an NO meter (Model N0-501, Inter Medical Co., Tokyo, Japan) as described previously (4). After the baseline electric was stabilized, an ADP-induced (50 μmol/l) electrical current was recorded at the rate of 20 mm/min, and a change in the peak electrical current was considered as an index of the NO release.
Measurements of intraplatelet redox status
We measured intraplatelet GSH (the reduced form of glutathione) and GSSG (the oxidized form of glutathione) by high-performance liquid chromatography (HPLC) with an electrochemical detection system (ECD-300, Eicom Co., Kyoto, Japan) as previously described (16). The analytical column was a 150 × 4.6 mm, SC-5 ODS (Eicom Co). For HPLC measurements, PRP and PPP were mixed with HClO4(final concentration, 6%), sonicated for 5 s with a tip sonicator (Model MS-50, Heat System-Ultrasonic Inc., Farmingdale, New York), and centrifuged at 12,000 gfor 2 min. The supernatant was then stored at −80°C until injection into the HPLC column. Intraplatelet GSH/GSSG ratio was calculated as an index of intraplatelet oxidative stress.
Detection of intraplatelet nitrotyrosine
We measured intraplatelet nitrotyrosine production by using a modified method of a previous study. Immunolabeling was performed by using a polyclonal antibody to nitrotyrosine as a primary antibody and fluoresein isothiocyanate-conjugated goat anti-rabbit immunoglobulin G (IgG) as a secondary antibody, and then analyzed with the FACScan (Becton-Deckinson, San Diego, California). The results were expressed as the percent changes in nitrotyrosine-specific staining of platelets by collagen-induced platelet aggregation in each group.
Measurements of urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG) and 8-iso-prostaglandin pgf2α(8-iso-PGF2α)
We measured urinary 8-OHdG, an indicator of DNA oxidation (ELISA kit, Japan Institute for the Control of Aging, Tokyo, Japan), and urinary 8-iso-PGF2α, a product of lipid peroxidation (Cayman Chemical Company, Ann Arbor, Michigan), using 24-h urine samples, according to previously described procedures (17–19).
Values are presented as means ± SD. Statistical comparisons between groups were performed by an unpaired Student ttest. Multiple comparisons were analyzed by two-way repeated-measures analysis of variance with a post-hoc Scheffé's test. Differences were considered statistically significant at p < 0.05.
Baseline characteristics of the study subjects
The body mass index distribution of the subjects was similar between the two groups (Table 1). All subjects had no other major risk factors including hypercholesterolemia, hypertension, and diabetes mellitus. Furthermore, the two groups did not differ in levels of fibrinogen, plasma epinephrine, and norepinephrine. Urinary cotinine levels were elevated in both groups, indicating that they were smokers. The urinary cotinine levels in group A were not detectable after smoking cessation, but those in group B were reversed after resmoking (0 ng/ml at day 14 and 1,310 ± 720 ng/ml at day 28).
At baseline, agonist (ADP or collagen)-induced platelet aggregations were similar between the two groups. In group A, agonist-induced platelet aggregations significantly and time-dependently decreased throughout the smoking cessation (Fig. 1).In group B, agonist-induced platelet aggregations significantly and time-dependently decreased until day 14, but those rapidly returned to the baseline level by resmoking.
At baseline, the PDNO level was similar between the two groups. In group A, PDNO significantly and time-dependently increased throughout the smoking cessation (Fig. 2).In group B, PDNO significantly and time-dependently increased by day 14, but it quickly returned to the baseline level by resmoking.
Intraplatelet GSH/GSSG ratio
At baseline, the intraplatelet GSH/GSSG ratio was similar between the two groups. In group A, the ratio significantly and time-dependently increased throughout the smoking cessation (Fig. 3).In group B, the ratio significantly and time-dependently increased by day 14 after smoking cessation, but it quickly returned to the baseline level by resmoking.
At baseline, the intraplatelet nitrotyrosine level was similar between the two groups. In group A, it significantly and time-dependently decreased throughout the smoking cessation (Fig. 4).In group B, it significantly and time-dependently decreased by day 14 after smoking cessation, but it quickly returned to the baseline level by resmoking.
Markers of systemic oxidative stress
At baseline, the urinary levels of 8-OHdG and 8-iso- PGF2αwere similar between the two groups. In group A, they significantly and time-dependently decreased throughout the smoking cessation (Fig. 5).In group B, they time-dependently decreased by day 14 after smoking cessation, but they quickly returned to the baseline levels by resmoking.
How soon is platelet aggregability reversible after smoking cessation? It is well known that platelet aggregability is more augmented in long-term smokers than in nonsmokers (4–6). Although the importance of primary prevention of smoking is well recognized, quitting smoking is not so appreciated; long-term smokers tend to underestimate the benefit of quitting smoking and are pessimistic to quit smoking. The reason for this underestimation and pessimism may derive from the fact that many long-term smokers have established atherosclerosis and from the fact that they cannot see the immediate benefits by themselves. As far as we know, there has been only one study that examined acute effects of smoking cessation on platelet aggregability (20). They reported that augmented platelet aggregability in long-term smokers was not reversible in four to six weeks. However, their study enrolled very few subjects, and, moreover, they did not monitor smoking status by measuring blood or urinary nicotine levels. As apparent from the study design, our study was carefully designed with a monitor of smoking status by measuring nicotine (cotinine) levels. Our results showed that only two-week smoking cessation reversed the augmented platelet aggregability in long-term smokers. In the present study, the baseline characteristics did not differ between the two groups. Furthermore, the urinary cotinine levels were not detectable after smoking cessation, and they returned to the baseline value after resmoking. Thus, the platelet aggregability was exclusively related to the smoking status. The results of our study may be in agreement with those that examined acute effects of smoking cessation on endothelial function (21) in which they showed that only one day of smoking cessation reversed endothelial dysfunction of human veins in long-term smokers. Although we did not examine platelet aggregability one day after smoking cessation, platelet aggregability was not fully reversed until day 14. It is not clear why smoking cessation reversed endothelial dysfunction within one day after smoking cessation whereas it took two weeks for platelet dysfunction to recover. Anyway, our results may provide some motivation for smokers to quit smoking.
Mechanisms of rapidly reversed platelet aggregability in long-term smokers
In order to examine the mechanisms by which platelet aggregability was rapidly reversed by smoking cessation, we measured PDNO and oxidative stress. Platelets possess the L-arginine–NO pathway through constitutive NO synthase in humans (10,22); PDNO acts as a negative feedback mechanism for platelet aggregation (11). In our previous studies (4,6), we have shown that PDNO was lower in long-term smokers than in nonsmokers and that impaired PDNO bioactivity is responsible for the augmented platelet aggregability in long-term smokers. Thus, in this study we measured PDNO and platelet aggregability at the same time after smoking cessation. Our results indicate that the changes in PDNO and platelet aggregability were parallel during smoking cessation and resmoking. Accordingly, the present observations suggest that the improved platelet aggregability is exclusively related to augmented PDNO. To further address the mechanism for augmented PDNO, we measured intraplatelet peroxynitrite production, the reaction product of superoxide anion and NO (23). Intraplatelet nitrotyrosine was significantly decreased after smoking cessation, whereas it was significantly increased after resmoking. Thus, our findings may indicate that the bioactivity of NO is impaired by oxidative stress in long-term smokers and is rapidly reversed after smoking cessation, resulting in improved platelet aggregability.
As previously discussed, it is likely that oxidative stress impairs bioactivity of PDNO, which is rapidly improved by smoking cessation. In order to further address this issue, we measured urinary levels of 8-OHdG, an indicator of DNA oxidation and 8-iso-PGF2α, a product of lipid peroxidation, as markers of systemic oxidative stress. Although these two markers have been reported to be elevated in chronic smokers (24–26), we, for the first time, show that these markers were decreased after smoking cessation and were increased after resmoking. We also measured intraplatelet levels of GSH and GSSG and obtained the ratio of GSH/GSSG for a marker of intraplatelet oxidative stress. In our previous study, we reported that GSH/GSSG ratio was lower in long-term smokers than in nonsmokers (5). Intracellular GSH serves as a free radical scavenger (27,28). Moreover, GSH not only regulates the intracellular redox state but also modulates the action and metabolism of NO (29,30). Indeed, it has been shown in cultured endothelial cells that depletion of GSH decreases synthesis of NO (31) and that reduced thiol enhances NO activity (32). These findings indicate that intracellular GSH plays an important role in modulating the action and metabolism of NO. In our recent study, we reported that smoking-induced oxidative stress is associated with decreased intraplatelet GSH levels, resulting in an imbalance of the intraplatelet redox state in long-term smokers (5,6). However, the effects of smoking cessation on the intraplatelet redox state in long-term smokers were unknown previously. In this study, we measured not only GSH but also its oxidized form, GSSG. The behavior of intraplatelet GSH/GSSG ratio was similar to that of peroxynitrite and PDNO during smoking cessation and resmoking. In our previous studies using NG-nitro-L-arginine methylester, an inhibitor of NO synthase, or 4,5-dihydroxy-1,3-benzene disulfonic acid, an intracellular scavenger of superoxide anion, we confirmed that intraplatelet nitrotyrosine was really a “footprint” of peroxynitrite, resulting from the interaction between NO and superoxide anion (6,33). These findings indicate that smoking cessation rapidly improves the imbalance of the intraplatelet redox state in long-term smokers. Taken together, present findings indicate that smoking cessation rapidly improves not only the systemic oxidative stress but also the imbalance of intracellular redox state, resulting in improvements of bioactivity of PDNO and platelet aggregability in long-term smokers. Finally, it is interesting to note that the changes in intraplatelet markers of redox state after smoking cessation were greater than those of systemic oxidative stress. Thus, intraplatelet GSH/GSSG ratio and nitrotyrosine may be more sensitive and useful markers of oxidative stress than 8-OHdG and 8-iso-PGF2αin the blood.
The present study has some study limitations. In this study, because we studied young, healthy individuals, caution is warranted when extrapolating present findings to patients with atherosclerosis or to those with multiple cardiovascular risk factors.
In conclusion, the present study, to the best of our knowledge, provides the first demonstration that only two weeks of smoking cessation ameliorates the enhanced platelet aggregability and intraplatelet redox imbalance through improvement of oxidative stress. Our findings may contribute to the understanding of the pathophysiological link of smoking cessation to beneficial cardiovascular effects. Furthermore, present findings may strengthen the motivation for smokers, especially patients with atherothrombosis, to quit smoking.
The authors are grateful to Kimiko Kimura for her excellent technical assistance.
- Abbreviations and acronyms
- adenosine diphosphate
- reduced form of glutathione
- oxidized form of glutathione
- nitric oxide
- platelet-derived nitric oxide
- platelet-rich plasma
- platelet-poor plasma
- 8-iso-prostaglandin F2α
- Received August 7, 2004.
- Revision received September 23, 2004.
- Accepted October 20, 2004.
- American College of Cardiology Foundation
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