Platelets possess the L-arginine–nitric oxide (NO) pathway through constitutive NO synthase (NOS) in human platelets (1- 2). Platelet aggregation is inhibited by L-arginine, a precursor of NO, and potentiated by N^G-monomethyl-L-arginine (L-NMMA), an inhibitor of NOS (1,3). Platelet aggregation is accompanied by an increase in the intracellular level of cyclic guanosine 3′,5′-monophosphate (cGMP) (1,3). These findings indicate evidence of the functional L-arginine–NO pathway in platelets during aggregation, which acts as a negative feedback mechanism to inhibit not only platelet activation (4) but also recruitment after aggregation (5). Recently, it has been shown that impaired platelet-derived NO (PDNO) production may contribute to the pathophysiology of acute coronary syndromes (6).

Chronic smoking is a powerful risk factor for the development of atherothrombosis (7). Recently, using an NO-selective electrode, we directly measured PDNO bioactivity during platelet aggregation (3). The bioactivity of PDNO was impaired in chronic smokers (3), although the mechanisms were not fully elucidated. Thus, in chronic smokers, production or release of PDNO may be impaired, or inactivation of PDNO may be augmented. There have been several reports suggesting that smoking-related oxidative stress may inactivate NO and play a role in the impaired endothelium-dependent vasodilation in chronic smokers (8- 9). Another possibility is that the disturbed intracellular redox state brought on by chronic smoking modulates the action and metabolism of NO (10- 11). However, it is impossible to evaluate the intracellular redox state of the endothelium in vivo. In this study, to investigate the contribution of the redox state to impaired PDNO bioactivity in chronic smokers, we investigated not only PDNO release, but also the intraplatelet redox state by measuring intraplatelet vitamin C, reduced glutathione (GSH) and intraplatelet nitrotyrosine production.

Vitamin C and GSH are the main water-soluble, low-molecular-weight antioxidants that directly scavenge oxygen free radicals (12- 14). Vitamin C plays a central role in the regulation of the intracellular redox state by interacting with GSH (13- 14), which modulates the action of NO (10- 11). Moreover, vitamin C can replace GSH, and vice versa, as intracellular antioxidants (15). In this study, we found lower levels of intraplatelet vitamin C and GSH in chronic smokers, suggesting that the imbalance of the intraplatelet redox state causes impaired bioactivity of PDNO. Accordingly, we examined the effects of oral administration of 2 g of vitamin C on PDNO bioactivity, platelet aggregation, the intraplatelet redox state and intraplatelet nitrotyrosine production.

The study groups consisted of 11 male smokers who smoked at least 15 cigarettes/day for more than five years and 10 healthy, age-matched male nonsmokers who never smoked (Table le1). They had no evidence of other major risk factors, including hypercholesterolemia, hypertension and diabetes mellitus. Smokers had abstained from smoking for at least 120 min before initiating the protocol to avoid the acute effects of smoking on platelet function. The protocols were approved by the institutional Ethic Committees, and written, informed consent was obtained from all subjects.

Platelet suspensions were prepared as described previously (3,16). The platelet counts were adjusted to 1–2 × 10⁵ platelets/μl in Tyrode’s solution, the composition of which was described previously (3).

Measurements of NO were performed using an NO meter (Model N0-501, Inter Medical Co., Tokyo, Japan), as described previously (3). After the baseline electric current was stabilized, a collagen-induced (3 μg/ml) 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.

We measured intraplatelet cGMP levels after collagen-induced (3 μg/ml) platelet aggregation by using a radioimmunoassay kit (Yamasa Shoyu Co., Chiba, Japan), as described previously (3). The changes in the intraplatelet cGMP levels were expressed by subtracting the levels in the absence of collagen from the levels in the presence of collagen.

We measured collagen-induced platelet aggregation, as described previously (3). In brief, collagen (1, 3 or 5 μg/ml) was added to the washed platelet suspensions, and light transmission was monitored by using a platelet aggregometer (MDM Hematracer, MC Medical Co., Tokyo, Japan).

We measured plasma and intraplatelet antioxidants, including vitamin C and GSH, by high-performance liquid chromatography with an electrochemical detection system (ECD-300, Eicom Co., Kyoto, Japan), as described previously (17). Deproteinized samples were used for injection. Intraplatelet levels of antioxidants were determined by subtracting the levels of platelet-poor plasma from those of platelet-rich plasma and were measured before and after the collagen-induced (3 μg/ml) aggregation. Then, consumption of the intraplatelet antioxidants by aggregation was determined by subtracting the levels of antioxidants after aggregation from those before aggregation. Furthermore, we estimated the mean platelet volume by using an electronic particle volume analyzer (SE9000 Sysmex Co., Kobe, Japan) and then calculated intraplatelet concentrations (mmol/l) of vitamin C.

We measured intraplatelet nitrotyrosine production by using a modified method of a previous study (18). Immuno-labeling was performed by using a polyclonal antibody to nitrotyrosine as a primary antibody and fluorescein 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. Platelets from smokers (n = 5) were stimulated by collagen in the presence of either N^G-nitro-L-arginine methylester (L-NAME; 300 μmol/l), an inhibitor of NOS, or 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron; 1 mmol/l), an intracellular scavenger of superoxide anion. L-NAME and Tiron completely inhibited the production of intraplatelet nitrotyrosine, indicating that nitrotyrosine is really a “footprint” of peroxynitrite, resulting from the interaction between NO and superoxide anion.

In all subjects, we administered 2 g of vitamin C orally, and plasma and intraplatelet vitamin C levels were measured every 30 min for 3 h. Measurements of the platelet-derived electrical current, intraplatelet cGMP, platelet aggregation and GSH were repeated 2 h after vitamin C administration.

Data are presented as the mean value ± SEM. Statistical comparisons between the groups were performed by using the paired or unpaired Student t test. Two-way repeated measures analysis of variance was used to determine the effects of smoking and the time to effect after vitamin C administration on plasma and intraplatelet vitamin C concentrations. The relationship between two variables was analyzed by use of linear regression analysis. Differences were considered statistically significant at p < 0.05.

Electrical currents (Figure 1) and intraplatelet cGMP (Figure 1) levels were significantly lower, and platelet aggregation (Figure 1) was significantly greater in smokers.

After vitamin C administration, electrical currents and intraplatelet cGMP levels were significantly increased in nonsmokers and smokers. These values were no longer different between the two groups (Figure 1). Vitamin C administration inhibited platelet aggregation in smokers, and platelet aggregation was no longer different between the two groups (Figure 1).

Before vitamin C administration, plasma (Figure 2) and intraplatelet (Figure 2) vitamin C levels and intraplatelet GSH levels (Figure 2) were significantly lower in smokers than in nonsmokers. After vitamin C administration, both plasma and intraplatelet vitamin C levels were significantly increased, peaking at 2 h in both nonsmokers and smokers (Figure 2). The peak value of intraplatelet vitamin C was no longer different between the two groups (Figure 2). Vitamin C administration did not change intraplatelet GSH levels in either group (Figure 2).

Before vitamin C administration, consumption of intraplatelet vitamin C and GSH during platelet aggregation did not differ between smokers and nonsmokers (Figure 3). After vitamin C administration, consumption of intraplatelet vitamin C was significantly increased in both groups and were not different between the two groups (Figure 3). After vitamin C administration, although the consumption of intraplatelet GSH was not changed in nonsmokers, it was significantly decreased in smokers. Consequently, consumption of GSH during platelet aggregation in smokers was much less after than before vitamin C administration (Figure 3).

In smokers, the collagen-induced, platelet-derived electrical current exhibited a significant inverse correlation with the consumption of intraplatelet GSH by platelet aggregation before (r = −0.64, p < 0.05) and after (r = −0.67, p < 0.03) vitamin C administration (Figure 3). No such relationship was found in nonsmokers (Figure 3). Importantly, the line of intraplatelet GSH consumption and platelet-derived electrical current was shifted upward after vitamin C administration in smokers.

(Figure 4) shows the representative histograms of intraplatelet nitrotyrosine production in a nonsmoker and a smoker. Before vitamin C administration, the percent change in intraplatelet nitrotyrosine production was significantly higher in smokers than in nonsmokers. This increased intraplatelet nitrotyrosine formation in smokers was not observed when nonspecific IgG was applied instead of the nitrotyrosine antibody, or when the nitrotyrosine antibody was applied in the presence of excess soluble nitrotyrosine (10 mmol/l). Vitamin C administration significantly decreased intraplatelet nitrotyrosine production in smokers, and their levels were decreased to the levels of nonsmokers (Figure 4).

The NO-selective electrode is specifically capable of measuring NO release (3,19- 20). We previously showed that the electrode used in this study was sensitive enough to detect NO release from aggregated platelets, on the basis of the following findings (3). First, S-nitroso-N-acetyl-dl-penicillamine, a direct NO donor, dose dependently increased the electrical current. Second, the electrical current showed a high correlation with collagen concentrations (r = 0.94) used for platelet aggregation. Third, the collagen-induced electrical current and intraplatelet cGMP levels were increased by L-arginine and attenuated by L-NMMA. Fourth, a good correlation was found between collagen-induced intraplatelet cGMP and the electrical current (r = 0.73). Thus, we confirmed that the changes in the electrical current reflect the amount of NO released through the L-arginine–NO pathway in aggregated platelets.

In the present study, the baseline characteristics did not differ between the two groups, except for plasma nicotine levels (Table le1). Accordingly, it is likely that the present observations of impaired PDNO bioactivity and augmented platelet aggregability are exclusively related to chronic smoking. Thus, our previous and present findings indicate that the L-arginine–NO pathway, as a negative feedback mechanism of platelet aggregation, is impaired in chronic smokers, resulting in increased platelet aggregability. However, the mechanisms of impaired PDNO bioactivity and augmented platelet aggregability in chronic smokers were unknown previously.

A previous study (21) and our studies indicate that smoking induces oxidative stress, which is associated with decreased plasma vitamin C levels. Moreover, we demonstrated for the first time, to the best of our knowledge, that intraplatelet vitamin C levels were significantly lower in smokers than in nonsmokers. Because vitamin C is a potent antioxidant, one may consider that vitamin C protects against free radical-mediated inactivation of PDNO. Thus, our results may suggest that oxidative stress associated with decreased intraplatelet vitamin C is related to impaired bioactivity of PDNO in chronic smokers.

In addition to vitamin C, intracellular GSH also serves as a free radical scavenger (12- 13). Furthermore, GSH not only regulates the intracellular redox state, but also modulates the action and metabolism of NO (10- 11). It has also been shown in cultured endothelial cells that depletion of GSH decreases synthesis of NO (22) and that reduced thiol enhances NO activity (23). These findings indicate that intracellular GSH plays an important role in modulating the action and metabolism of NO. Accordingly, we examined the intraplatelet GSH level. It was confirmed, for the first time, that the intraplatelet GSH level is significantly lower in smokers. Taken together, our findings indicate that smoking-related oxidative stress decreases not only plasma vitamin C levels but also intraplatelet vitamin C and GSH levels, resulting in an imbalance of the intraplatelet redox state. To further address this issue, we measured intraplatelet peroxynitrite production, a reaction product of superoxide anion and NO (24), which was significantly higher in smokers. Based on these findings, we assumed that the imbalance of the intraplatelet redox state is responsible for the impaired PDNO release in chronic smokers. To further test this hypothesis, we investigated whether modulation of the intraplatelet redox state by vitamin C administration affects PDNO release and platelet aggregation.

We determined the time course of plasma and intraplatelet vitamin C levels after oral administration of vitamin C (2 g). A similar dose of vitamin C has been reported to improve endothelial function in patients with coronary artery disease (25). Both plasma and intraplatelet vitamin C levels peaked 2 h after oral vitamin C administration in nonsmokers and smokers. The peak value of intraplatelet vitamin C was no longer different between the two groups. Therefore, we chose to study 2 h after 2 g of oral vitamin C administration. Vitamin C increased PDNO release and intracellular cGMP in smokers and nonsmokers. These values were no longer different between smokers and nonsmokers after vitamin C administration, and enhanced platelet aggregability in smokers was normalized. Thus, our results provide more direct evidence that the imbalance of the intraplatelet redox state is responsible for the impairment of PDNO release and augmented platelet aggregability in chronic smokers.

There are several possible mechanisms of the antioxidant effects of vitamin C on platelets. First, one may consider that increased plasma vitamin C protected against free radical–mediated inactivation of PDNO. In this study, the plasma vitamin C level was increased from 28 (baseline value) to 107 μmol/l at 2 h. However, a recent in vitro study by Jackson et al. (26) showed that impaired endothelium-derived NO release by superoxide was not prevented by vitamin C at concentrations <150 μmol/l. Accordingly, this possibility is unlikely. Second, it is possible that the protective effects of vitamin C on PDNO release are mediated by the antioxidant property of vitamin C at the intraplatelet level. Jackson et al. (26) demonstrated that vitamin C was effective in competing with NO for superoxide at concentrations ≥1 mmol/l. Thus, they speculated that the vasorelaxing effects of exogenously administered vitamin C on the constricted vascular strips by superoxide were due to the intracellular increases in vitamin C, although they did not measure intracellular vitamin C concentrations. In this study, we measured intraplatelet and plasma vitamin C concentrations in smokers at 2 h after vitamin C administration, which were 8.3 mmol/l and 107 μmol/l, respectively. Thus, our findings indicate that intraplatelet vitamin C concentrations are higher in platelets than in plasma and are sufficient enough to scavenge superoxide anion. Third, the antioxidative effects of vitamin C may have prevented oxidation of intraplatelet GSH, resulting in the preservation of GSH levels. However, vitamin C administration did not change intraplatelet GSH levels in nonsmokers and smokers. The process of platelet aggregation involves the generation of oxygen free radicals (27), and intracellular antioxidants such as vitamin C and GSH may be consumed during platelet aggregation. Accordingly, we calculated consumption of intracellular vitamin C and GSH during platelet aggregation, and the consumption rates were similar between the two groups before vitamin C administration. After vitamin C administration, consumption of intraplatelet vitamin C was significantly increased in both nonsmokers and smokers, and, again, no difference was found between the two groups. However, consumption of intraplatelet GSH after vitamin C administration was significantly lower in smokers than in nonsmokers. These findings suggest that in smokers, vitamin C may have preserved intraplatelet GSH during platelet aggregation.

To test this hypothesis, the correlation between PDNO and the consumption of intraplatelet GSH by platelet aggregation was evaluated. A significant and inverse correlation was found in smokers but not in nonsmokers. Furthermore, the curve was shifted upward after vitamin C administration, indicating more PDNO release at the same consumption of intraplatelet GSH. These findings suggest that impaired PDNO bioactivity in smokers was restored by intraplatelet GSH after vitamin C administration. Our data support the notion that vitamin C spares GSH from oxidation and preserves intracellular GSH levels under the condition of increased oxidative stress (15), resulting in an improved intracellular redox state (13). In this study, we further examined whether improved PDNO bioactivity by vitamin C administration in smokers was caused by the antioxidant effects of vitamin C. This notion was supported by evidence that vitamin C administration decreased intraplatelet nitrotyrosine production in smokers to the levels of nonsmokers. Taken together, our findings suggest that in smokers, vitamin C administration improves PDNO release through the antioxidant effects of vitamin C.

An important corollary to the redox hypothesis of smoking-induced hyperaggregability found in this study is the possibility that antioxidant treatment will reduce atherothrombosis. However, in clinical trials (28- 29), antioxidant therapy with vitamin E in cardiovascular disease did not consistently improve the outcome. A possible reason for these disappointing results may be due to the lipophilic property of vitamin E. In contrast to the hydrophilic antioxidants, such as vitamin C, the effects of lipophilic antioxidants may act on lipids, such as membranes and cholesterol. Accordingly, vitamin C might be a better antioxidant in terms of cellular redox modulation. Therefore, the current results may encourage the endeavor of a future, large, prospective study to test a long-term regimen of vitamin C on cardiovascular morbidity and mortality.

Some previous studies have shown augmented platelet aggregation in chronic smokers (30) and beneficial effects of antioxidant therapy on platelet function in humans (31- 32). However, there is no report examining the impact of antioxidant therapy on platelet function in chronic smokers. The present study, to the best of our knowledge, provides the first evidence that the imbalance of the intraplatelet redox state caused by increased oxidative stress may be an important mechanism for the impaired PDNO bioactivity in chronic smokers. Accordingly, a therapeutic strategy focusing on improvement of the intracellular redox state may be considered in chronic smokers.