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Augmented Oxidative Stressof Platelets in Chronic SmokersMechanisms of Impaired Platelet-Derived NitricOxide Bioactivity and Augmented Platelet AggregabilityYoshinori Takajo, MD, Hisao Ikeda, MD, Nobuya Haramaki, MD, Toyoaki Murohara, MD,Tsutomu Imaizumi, MD, FACC

Kurume, Japan

OBJECTIVES We investigated whether impaired platelet-derived nitric oxide (PDNO) bioactivity andaugmented platelet aggregability in chronic smokers are related to the imbalance of theintraplatelet redox state through increased oxidative stress.

BACKGROUND Chronic smoking impairs PDNO release and augments platelet aggregability. However, theirmechanisms are unknown.

METHODS Collagen-induced PDNO release, platelet aggregation, plasma and intraplatelet vitamin Cand reduced glutathione (GSH), intraplatelet cyclic guanosine 3,5-monophosphate(cGMP) and intraplatelet nitrotyrosine production, which is a marker of the peroxynitriteformation, were measured in 11 chronic smokers and 10 age-matched nonsmokers.

RESULTS Release of PDNO and levels of intraplatelet cGMP were lower, and platelet aggregation wasgreater, in smokers than in nonsmokers. Intraplatelet vitamin C and GSH levels were lowerin smokers than in nonsmokers. Intraplatelet nitrotyrosine production was greater in smokersthan in nonsmokers. Next, we investigated the effects of oral vitamin C administration (2 g).After vitamin C administration, intraplatelet vitamin C levels were increased and not differentat 2 h between the two groups. Then, PDNO release, intraplatelet cGMP levels and plateletaggregation in smokers were restored to the levels of nonsmokers. In smokers, PDNO releaseand consumption of GSH during platelet aggregation were inversely correlated, andconsumption was much less after vitamin C administration. Vitamin C administrationdecreased intraplatelet nitrotyrosine production in smokers.

CONCLUSIONS Impaired PDNO bioactivity and augmented platelet aggregability may be caused by animbalance of the intraplatelet redox state through increased oxidative stress in smokers. (JAm Coll Cardiol 2001;38:13207) 2001 by the American College of Cardiology

Platelets possess the L-argininenitric oxide (NO) pathwaythrough constitutive NO synthase (NOS) in human plate-lets (1,2). Platelet aggregation is inhibited by L-arginine, aprecursor of NO, and potentiated by NG-monomethyl-L-arginine (L-NMMA), an inhibitor of NOS (1,3). Plateletaggregation is accompanied by an increase in the intracel-lular level of cyclic guanosine 3,5-monophosphate(cGMP) (1,3). These findings indicate evidence of thefunctional L-arginineNO pathway in platelets during ag-gregation, which acts as a negative feedback mechanism toinhibit not only platelet activation (4) but also recruitmentafter aggregation (5). Recently, it has been shown thatimpaired platelet-derived NO (PDNO) production maycontribute to the pathophysiology of acute coronary syn-dromes (6).

Chronic smoking is a powerful risk factor for the devel-opment of atherothrombosis (7). Recently, using an NO-selective electrode, we directly measured PDNO bioactivityduring platelet aggregation (3). The bioactivity of PDNO

was impaired in chronic smokers (3), although the mecha-nisms were not fully elucidated. Thus, in chronic smokers,production or release of PDNO may be impaired, orinactivation of PDNO may be augmented. There have beenseveral reports suggesting that smoking-related oxidativestress may inactivate NO and play a role in the impairedendothelium-dependent vasodilation in chronic smokers(8,9). Another possibility is that the disturbed intracellularredox state brought on by chronic smoking modulates theaction and metabolism of NO (10,11). However, it isimpossible to evaluate the intracellular redox state of theendothelium in vivo. In this study, to investigate thecontribution of the redox state to impaired PDNO bioac-tivity in chronic smokers, we investigated not only PDNOrelease, but also the intraplatelet redox state by measuringintraplatelet vitamin C, reduced glutathione (GSH) andintraplatelet nitrotyrosine production.

Vitamin C and GSH are the main water-soluble, low-molecular-weight antioxidants that directly scavenge oxygenfree radicals (1214). Vitamin C plays a central role in theregulation of the intracellular redox state by interacting withGSH (13,14), which modulates the action of NO (10,11).Moreover, vitamin C can replace GSH, and vice versa, asintracellular antioxidants (15). In this study, we found lower

levels of intraplatelet vitamin C and GSH in chronic

From the Department of Internal Medicine III and Cardiovascular Research

Institute, Kurume University School of Medicine, Kurume, Japan. This study wassupported in part by a grant-in-aid for scientific research (no. 10770333) from theMinistry of Education, Science, Sports and Culture of Japan, Tokyo, Japan.

Manuscript received October 18, 2000; revised manuscript received June 18, 2001,accepted July 12, 2001.

Journal of the American College of Cardiology Vol. 38, No. 5, 2001 2001 by the American College of Cardiology ISSN 0735-1097/01/$20.00Published by Elsevier Science Inc. PII S0735-1097(01)01583-2

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smokers, suggesting that the imbalance of the intraplateletredox state causes impaired bioactivity of PDNO. Accord-ingly, we examined the effects of oral administration of 2 gof vitamin C on PDNO bioactivity, platelet aggregation, theintraplatelet redox state and intraplatelet nitrotyrosine pro-duction.

METHODSStudy groups. The study groups consisted of 11 malesmokers who smoked at least 15 cigarettes/day for morethan five years and 10 healthy, age-matched male nonsmok-ers who never smoked (Table 1). They had no evidence ofother major risk factors, including hypercholesterolemia,hypertension and diabetes mellitus. Smokers had abstainedfrom smoking for at least 120 min before initiating theprotocol to avoid the acute effects of smoking on plateletfunction. The protocols were approved by the institutionalEthic Committees, and written, informed consent wasobtained from all subjects.

Preparation of platelet-rich plasma and washed platelets.Platelet suspensions were prepared as described previously(3,16). The platelet counts were adjusted to 12 105

platelets/l in Tyrodes solution, the composition of whichwas described previously (3).

Measurement of the electrical current with an NO-specific electrode. Measurements of NO were performedusing an NO meter (Model N0-501, Inter Medical Co.,

Tokyo, Japan), as described previously (3). After the base-line electric current was stabilized, a collagen-induced (3g/ml) electrical current was recorded at the rate of 20

mm/min, and a change in the peak electrical current wasconsidered as an index of the NO release.Measurement of intraplatelet cGMP levels. We mea-sured intraplatelet cGMP levels after collagen-induced (3g/ml) platelet aggregation by using a radioimmunoassaykit (Yamasa Shoyu Co., Chiba, Japan), as described previ-ously (3). The changes in the intraplatelet cGMP levels

were expressed by subtracting the levels in the absence ofcollagen from the levels in the presence of collagen.Measurement of platelet aggregation. We measuredcollagen-induced platelet aggregation, as described previ-ously (3). In brief, collagen (1, 3 or 5 g/ml) was added tothe washed platelet suspensions, and light transmission wasmonitored by using a platelet aggregometer (MDM He-matracer, MC Medical Co., Tokyo, Japan).Measurement of plasma and intraplatelet antioxidants.

We measured plasma and intraplatelet antioxidants, includ-ing vitamin C and GSH, by high-performance liquidchromatography with an electrochemical detection system(ECD-300, Eicom Co., Kyoto, Japan), as described previ-ously (17). Deproteinized samples were used for injection.Intraplatelet levels of antioxidants were determined bysubtracting the levels of platelet-poor plasma from those ofplatelet-rich plasma and were measured before and after thecollagen-induced (3 g/ml) aggregation. Then, consump-

tion of the intraplatelet antioxidants by aggregation wasdetermined by subtracting the levels of antioxidants afteraggregation from those before aggregation. Furthermore,

we estimated the mean platelet volume by using an elec-tronic particle volume analyzer (SE9000 Sysmex Co., Kobe,

Japan) and then calculated intraplatelet concentrations(mmol/l) of vitamin C.Detection of intraplatelet nitrotyrosine. We measuredintraplatelet nitrotyrosine production by using a modifiedmethod of a previous study (18). Immuno-labeling was per-formed by using a polyclonal antibody to nitrotyrosine as aprimary antibody and fluorescein isothiocyanate-conjugatedgoat anti-rabbit immunoglobulin G (IgG) as a secondaryantibody, and then analyzed with the FACScan (Becton-Deckinson, San Diego, California). The results were expressedas the percent changes in nitrotyrosine-specific staining ofplatelets by collagen-induced platelet aggregation in eachgroup. Platelets from smokers (n 5) were stimulated bycollagen in the presence of either NG-nitro-L-arginine meth-

ylester (L-NAME; 300 mol/l), an inhibitor of NOS, or4,5-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 intraplateletnitrotyrosine, indicating that nitrotyrosine is really a footprintof peroxynitrite, resulting from the interaction between NO

and superoxide anion.

Abbreviations and Acronyms

cGMP cyclic guanosine 3,5-monophosphateGSH reduced glutathioneIgG immunoglobulin GL-NAME NG-nitro-L-arginine methylesterL-NMMA NG-monomethyl-L-arginineNO nitric oxideNOS nitric oxide synthasePDNO platelet-derived nitric oxide

Table 1. Baseline Characteristics

Nonsmokers Smokersp

Value

Age (years) 32 1 34 1 NSMale/female 10/0 11/0 NSCigaretts per day 0 27 2

Smoking period (years) 0 15.2 1 Systolic/diastolic blood

pressure (mm Hg)120 4/73 3 123 4/76 2 NS

Heart rate (beats/min) 68 3 66 2 NSTotal cholesterol (mg/dl) 182 3 189 5 NSHDL cholesterol (mg/dl) 53 4 55 6 NSLDL cholesterol (mg/dl) 102 3 99 3 NSFasting blood sugar (mg/dl) 85 2 87 3 NSPlatelet count (104/l) 23 1 22 1 NSFibrinogen (mg/dl) 262 23 288 25 NSProthrombin time (%) 102 3 108 3 NSPlasma epinephrine (pg/ml) 38 3 47 3 NSPlasma norepinephrine (pg/ml) 409 45 442 54 NSNicotine (ng/ml) Not detectable 7.2 0.6

Data are expressed as the mean value

SE.HDL and LDL high and low density lipoprotein, respectively.

1321JACC Vol. 38, No. 5, 2001 Takajo et al.November 1, 2001:13207 Platelet-Derived Nitric Oxide and Oxidative Stress



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Effects of vitamin C administration. In all subjects, we

administered 2 g of vitamin C orally, and plasma andintraplatelet vitamin C levels were measured every 30 minfor 3 h. Measurements of the platelet-derived electricalcurrent, intraplatelet cGMP, platelet aggregation and GSH

were repeated 2 h after vitamin C administration.Statistical analysis. Data are presented as the mean

value SEM. Statistical comparisons between the groupswere performed by using the paired or unpaired Student ttest.Two-way repeated measures analysis of variance was used todetermine the effects of smoking and the time to effect after

vitamin C administration on plasma and intraplatelet vitaminC concentrations. The relationship between two variables was

analyzed by use of linear regression analysis. Differences wereconsidered statistically significant at p 0.05.

RESULTS

Platelet-derived electrical currents, intraplatelet cGMPlevels and platelet aggregation. Electrical currents (Fig.1A) and intraplatelet cGMP (Fig. 1B) levels were signifi-cantly lower, and platelet aggregation (Fig. 1C) was signif-icantly greater in smokers.Effects of vitamin C administration on platelet-derivedelectrical currents, intraplatelet cGMP levels and plate-

let aggregation. 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 (Fig. 1A and 1B).Vitamin C administration inhibited platelet aggregation insmokers, and platelet aggregation was no longer differentbetween the two groups (Fig. 1D).Plasma and intraplatelet antioxidants. Before vitamin Cadministration, plasma (Fig. 2A) and intraplatelet (Fig. 2B)

vitamin C levels and intraplatelet GSH levels (Fig. 2C) weresignificantly lower in smokers than in nonsmokers. After

vitamin C administration, both plasma and intraplateletvitamin C levels were significantly increased, peaking at 2 hin both nonsmokers and smokers (Fig. 2A and 2B). Thepeak value of intraplatelet vitamin C was no longer different

between the two groups (Fig. 2B). Vitamin C administra-tion did not change intraplatelet GSH levels in either group(Fig. 2C).Consumption of intraplatelet vitamin C and GSH beforeand after vitamin C administration. Before vitamin Cadministration, consumption of intraplatelet vitamin C andGSH during platelet aggregation did not differ betweensmokers and nonsmokers (Fig. 3A and 3B). After vitamin Cadministration, consumption of intraplatelet vitamin C wassignificantly increased in both groups and were not differentbetween the two groups (Fig. 3A). After vitamin C admin-istration, although the consumption of intraplatelet GSH

was not changed in nonsmokers, it was significantly de-

Figure 1. Collagen-induced platelet-derived electrical currents (A) and intraplatelet cyclic guanosine 3,5-monophosphate (cGMP) levels (B) before andafter vitamin C administration in nonsmokers (n 10) and smokers (n 11). Collagen-induced platelet aggregation before (C) and after (D) vitamin Cadministration in nonsmokers (n 10) and smokers (n 11). Open bars nonsmokers; solid bars smokers; NS nonsignificant.

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Platelet-Derived Nitric Oxide and Oxidative Stress November 1, 2001:1320 7



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creased in smokers. Consequently, consumption of GSHduring platelet aggregation in smokers was much less afterthan before vitamin C administration (Fig. 3B).Relationship between consumption of intraplatelet GSHand platelet-derived electrical currents. In smokers, thecollagen-induced, platelet-derived electrical current exhib-ited a significant inverse correlation with the consumptionof intraplatelet GSH by platelet aggregation before (r 0.64, p 0.05) and after (r 0.67, p 0.03) vitaminC administration (Fig. 3D). No such relationship was foundin nonsmokers (Fig. 3C). Importantly, the line of in-traplatelet GSH consumption and platelet-derived electricalcurrent was shifted upward after vitamin C administrationin smokers.Intraplatelet nitrotyrosine production before and after

vitamin C administration. Figure 4 shows the representa-tive histograms of intraplatelet nitrotyrosine production in anonsmoker 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 insmokers was not observed when nonspecific IgG wasapplied instead of the nitrotyrosine antibody, or when thenitrotyrosine antibody was applied in the presence of excesssoluble nitrotyrosine (10 mmol/l). Vitamin C administra-

tion significantly decreased intraplatelet nitrotyrosine pro-

duction in smokers, and their levels were decreased to thelevels of nonsmokers (Fig. 4D).

DISCUSSION

The NO-selective electrode is specifically capable of mea-suring NO release (3,19,20). We previously showed thatthe electrode used in this study was sensitive enough todetect NO release from aggregated platelets, on the basis ofthe following findings (3). First, S-nitroso-N-acetyl-dl-penicillamine, a direct NO donor, dose dependently in-creased the electrical current. Second, the electrical currentshowed 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 electricalcurrent reflect the amount of NO released through theL-arginineNO pathway in aggregated platelets.

In the present study, the baseline characteristics did notdiffer between the two groups, except for plasma nicotinelevels (Table 1). Accordingly, it is likely that the presentobservations of impaired PDNO bioactivity and augmented

platelet aggregability are exclusively related to chronic

Figure 2. Time courses of plasma (A) and intraplatelet (B) levels of vitamin C in nonsmokers (n 10; open circles) and smokers (n 11; solid circles).*p 0.05 indicates the difference between nonsmokers and smokers before vitamin C administration. (C) Intraplatelet reduced glutathione (GSH) levelsbefore and after vitamin C administration in nonsmokers (n 10) and smokers (n 11). NS nonsignificant.

1323JACC Vol. 38, No. 5, 2001 Takajo et al.November 1, 2001:1320 7 Platelet-Derived Nitric Oxide and Oxidative Stress



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smoking. Thus, our previous and present findings indicatethat the L-arginineNO pathway, as a negative feedbackmechanism of platelet aggregation, is impaired in chronicsmokers, resulting in increased platelet aggregability. How-ever, the mechanisms of impaired PDNO bioactivity andaugmented platelet aggregability in chronic smokers wereunknown previously.Oxidative stress in chronic smokers. A previous study(21) and our studies indicate that smoking induces oxidativestress, which is associated with decreased plasma vitamin Clevels. Moreover, we demonstrated for the first time, to thebest 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 considerthat vitamin C protects against free radical-mediated inac-tivation of PDNO. Thus, our results may suggest thatoxidative stress associated with decreased intraplatelet vita-min C is related to impaired bioactivity of PDNO inchronic smokers.

In addition to vitamin C, intracellular GSH also serves asa free radical scavenger (12,13). Furthermore, GSH notonly regulates the intracellular redox state, but also modu-lates 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 thiolenhances NO activity (23). These findings indicate thatintracellular GSH plays an important role in modulating theaction and metabolism of NO. Accordingly, we examinedthe intraplatelet GSH level. It was confirmed, for the firsttime, that the intraplatelet GSH level is significantly lowerin smokers. Taken together, our findings indicate thatsmoking-related oxidative stress decreases not only plasma

vitamin C levels but also intraplatelet vitamin C and GSHlevels, resulting in an imbalance of the intraplatelet redoxstate. To further address this issue, we measured intraplate-let peroxynitrite production, a reaction product of superox-ide anion and NO (24), which was significantly higher insmokers. Based on these findings, we assumed that theimbalance of the intraplatelet redox state is responsible forthe impaired PDNO release in chronic smokers. To furthertest this hypothesis, we investigated whether modulation ofthe intraplatelet redox state by vitamin C administrationaffects PDNO release and platelet aggregation.Effects of vitamin C administration. We determined thetime course of plasma and intraplatelet vitamin C levelsafter oral administration of vitamin C (2 g). A similar doseof vitamin C has been reported to improve endothelial

function in patients with coronary artery disease (25). Both

Figure 3. Consumption of intraplatelet vitamin C (A) and reduced glutathione (GSH) (B) by platelet aggregation before and after vitamin C administrationin nonsmokers (n 10) and smokers (n 11). Correlation between the collagen-induced platelet-derived electrical current and consumption ofintraplatelet GSH by platelet aggregation before and after vitamin C administration in (C) nonsmokers (n 10) and (D) smokers (n 11). NS nonsignificant.





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plasma and intraplatelet vitamin C levels peaked 2 h afteroral vitamin C administration in nonsmokers and smokers.

The peak value of intraplatelet vitamin C was no longerdifferent between the two groups. Therefore, we chose tostudy 2 h after 2 g of oral vitamin C administration. VitaminC increased PDNO release and intracellular cGMP insmokers and nonsmokers. These values were no longerdifferent between smokers and nonsmokers after vitamin Cadministration, and enhanced platelet aggregability insmokers was normalized. Thus, our results provide moredirect evidence that the imbalance of the intraplatelet redoxstate is responsible for the impairment of PDNO releaseand augmented platelet aggregability in chronic smokers.

There are several possible mechanisms of the antioxidanteffects of vitamin C on platelets. First, one may considerthat increased plasma vitamin C protected against freeradicalmediated inactivation of PDNO. In this study, theplasma vitamin C level was increased from 28 (baseline

value) to 107 mol/l at 2 h. However, a recent in vitro studyby Jackson et al. (26) showed that impaired endothelium-derived NO release by superoxide was not prevented by

vitamin C at concentrations 150 mol/l. Accordingly,this possibility is unlikely. Second, it is possible that theprotective 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 thatvitamin C was effective in competing with NO for super-oxide at concentrations 1 mmol/l. Thus, they speculatedthat the vasorelaxing effects of exogenously administered

vitamin C on the constricted vascular strips by superoxidewere due to the intracellular increases in vitamin C, al-though they did not measure intracellular vitamin C con-centrations. In this study, we measured intraplatelet andplasma vitamin C concentrations in smokers at 2 h after

vitamin C administration, which were 8.3 mmol/l and 107mol/l, respectively. Thus, our findings indicate that in-traplatelet vitamin C concentrations are higher in plateletsthan in plasma and are sufficient enough to scavengesuperoxide anion. Third, the antioxidative effects of vitaminC may have prevented oxidation of intraplatelet GSH,resulting in the preservation of GSH levels. However,

vitamin C administration did not change intraplatelet GSHlevels in nonsmokers and smokers. The process of plateletaggregation involves the generation of oxygen free radicals(27), and intracellular antioxidants such as vitamin C andGSH may be consumed during platelet aggregation. Ac-cordingly, we calculated consumption of intracellular vita-min C and GSH during platelet aggregation, and theconsumption rates were similar between the two groups

before vitamin C administration. After vitamin C adminis-

Figure 4. (AC) Representative histograms of intraplatelet nitrotyrosine production in a nonsmoker and a smoker. In the smoker before vitamin Cadministration, the fluorescent peak of nitrotyrosine in platelets stimulated by collagen (3 g/ml) (open area) shifted to the right from fluorescent peaksof unstimulated platelets (solid area). In the smoker after vitamin C administration, the histograms were identical to those of the nonsmoker. (D)Intraplatelet nitrotyrosine production by collagen-induced platelet aggregation in nonsmokers (n 8) and in smokers (n 8).




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tration, consumption of intraplatelet vitamin C was signif-icantly increased in both nonsmokers and smokers, and,again, no difference was found between the two groups.However, consumption of intraplatelet GSH after vitaminC administration was significantly lower in smokers than innonsmokers. These findings suggest that in smokers, vita-

min C may have preserved intraplatelet GSH during plate-let aggregation.

To test this hypothesis, the correlation between PDNOand the consumption of intraplatelet GSH by plateletaggregation was evaluated. A significant and inverse corre-lation was found in smokers but not in nonsmokers.Furthermore, the curve was shifted upward after vitamin Cadministration, indicating more PDNO release at the sameconsumption of intraplatelet GSH. These findings suggestthat impaired PDNO bioactivity in smokers was restored byintraplatelet GSH after vitamin C administration. Our datasupport the notion that vitamin C spares GSH from

oxidation and preserves intracellular GSH levels under thecondition of increased oxidative stress (15), resulting in animproved intracellular redox state (13). In this study, wefurther examined whether improved PDNO bioactivity by

vitamin C administration in smokers was caused by theantioxidant effects of vitamin C. This notion was supportedby evidence that vitamin C administration decreased in-traplatelet nitrotyrosine production in smokers to the levelsof nonsmokers. Taken together, our findings suggest that insmokers, vitamin C administration improves PDNO releasethrough the antioxidant effects of vitamin C.Clinical implications. An important corollary to the redox

hypothesis of smoking-induced hyperaggregability found inthis study is the possibility that antioxidant treatment willreduce atherothrombosis. However, in clinical trials (28,29),antioxidant therapy with vitamin E in cardiovascular diseasedid not consistently improve the outcome. A possible reasonfor these disappointing results may be due to the lipophilicproperty of vitamin E. In contrast to the hydrophilicantioxidants, such as vitamin C, the effects of lipophilicantioxidants may act on lipids, such as membranes andcholesterol. Accordingly, vitamin C might be a betterantioxidant in terms of cellular redox modulation. There-fore, the current results may encourage the endeavor of afuture, large, prospective study to test a long-term regimenof vitamin C on cardiovascular morbidity and mortality.Conclusions. Some previous studies have shown aug-mented platelet aggregation in chronic smokers (30) andbeneficial effects of antioxidant therapy on platelet functionin humans (31,32). However, there is no report examiningthe impact of antioxidant therapy on platelet function inchronic smokers. The present study, to the best of ourknowledge, provides the first evidence that the imbalance ofthe intraplatelet redox state caused by increased oxidativestress may be an important mechanism for the impairedPDNO bioactivity in chronic smokers. Accordingly, atherapeutic strategy focusing on improvement of the intra-

cellular redox state may be considered in chronic smokers.

Reprint requests and correspondence: Dr. Hisao Ikeda, Depart-ment of Internal Medicine III, Kurume University School ofMedicine, 67 Asahi-machi, Kurume 830-0011, Japan. E-mail:[email protected].

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