(Hypertension. 2000;35:904.)
© 2000 American Heart Association, Inc.
Scientific Contributions |
Platelet-Derived Nitric Oxide and Coronary Risk Factors
From the Department of Internal Medicine III (H.I., Y.T., K.I., H.A., N.H., A.K., T.I.) and Cardiovascular Research Institute (T.M.), Kurume University School of Medicine, Kurume, Japan.
Correspondence to Hisao Ikeda, MD, PhD, Department of Internal Medicine III, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830-0011, Japan. E-mail ikeikeda{at}med.kurume-u.ac.jp
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Abstract |
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Abstract—Platelet aggregation is inhibited through a negative feedback mechanism by the L-arginine/nitric oxide (NO) pathway found in platelets themselves. We have shown that long-term smoking impairs the bioactivity of platelet-derived NO (PDNO), resulting in an increased platelet aggregability. However, little is known about the relation between other coronary risk factors and PDNO release. Accordingly, this study was undertaken to examine whether other coronary risk factors are related to the impairment of PDNO bioactivity. We measured collagen-induced PDNO release with an NO-selective electrode in 61 subjects (mean age 47 years, range 24 to 74 years) who underwent complete physical and laboratory examinations. There was a significant inverse correlation between PDNO release and the number of coronary risk factors (r=-0.61, P<0.001). Univariate analysis showed a significant inverse correlation between PDNO release and age (r=-0.33, P<0.01), mean arterial pressure (r=-0.40, P<0.002), total cholesterol level (r=-0.31, P<0.02), and LDL-cholesterol level (r=-0.33, P<0.02). PDNO release was significantly lower in long-term smokers than in nonsmokers (P<0.001). With multiple stepwise regression analysis, PDNO release correlated significantly and independently (r2=0.51), with smoking (F=37.8), age (F=7.1), and mean arterial pressure (F=5.1). Thus, we demonstrated that coronary risk factors are associated with an impairment of PDNO release by human platelets. Our findings may contribute to the understanding of the pathophysiological link between coronary risk factors and atherothrombotic disease.
Key Words: platelets • nitric oxide • risk factors • atherothrombosis
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Introduction |
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Platelets possess the functional L-arginine/nitric oxide (NO) pathway via constitutive NO synthase.1 2 Platelet-derived NO (PDNO) increases the intraplatelet level of cGMP and inhibits platelet aggregation.1 This modulation of platelet aggregation via the L-arginine/NO pathway is recognized as a negative-feedback mechanism to inhibit aggregation.3 4 Using an NO-selective electrode, we5 and others6 directly measured PDNO release during platelet aggregation, which was potentiated by L-arginine and attenuated by inhibitors of NO synthase, indicating the existence of an L-arginine/NO pathway in human platelets.1 7 PDNO has been shown to play a functional role in the inhibition of not only platelet activation but also platelet recruitment after aggregation.8 Recently, we have shown that long-term smoking impairs PDNO release, resulting in an increased platelet aggregability.5 However, little is known about the relation between other coronary risk factors and PDNO release. Accordingly, the present study was undertaken to investigate the hypothesis that other coronary risk factors impair platelet function and thereby affect PDNO release in subjects with major risk factors.
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Methods |
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Study Subjects
The study population consisted of 61 subjects who agreed to participate in the study. The subjects underwent complete routine physical and laboratory examinations, and their complete anamnesis was obtained. Of the 61 subjects, 42 were healthy volunteers, 12 had stable effort angina, and 7 had previous myocardial infarction. Medications, including long-acting nitrates, calcium channel blockers, ß-blockers, and ACE inhibitors, were withheld for
24 hours, and
aspirin was withheld for 1 week before the study. None of the
patients had received cholesterol-lowering agents. Patients
with acute coronary syndromes, valvular heart diseases,
or heart failure were excluded from the study. The present protocol
was approved by our institutional ethic committee, and informed consent
for the study was obtained from all patients.
Definition of Coronary Risk Factors
Coronary risk factors in this study were determined
according to the Sixth Report of the Joint National Committee on
Prevention, Detection, Evaluation, and Treatment of High Blood Pressure
report.9 Hypertension was defined as a systolic
blood pressure of 140 mm Hg, a diastolic blood
pressure of 90 mm Hg, or both.
Hypercholesterolemia was defined as a total
serum cholesterol level 220 mg/dL, which was measured
with an enzymatic method. None of the patients had documented familial
hyperlipidemia. Subjects who smoked 15 cigarettes per
day for 5 years were considered to be long-term smokers. Long -term
smokers were asked to abstain from smoking for 120 minutes before the
study to avoid the acute effects of smoking on platelet function.
Family history was considered positive if a first-degree relative had
clinical evidence of coronary artery disease (angina pectoris
or myocardial infarction) at the age of 
60 years. None of the
patients had a history of insulin-dependent diabetes mellitus or
elevated blood glucose. A composite risk factor score was calculated
for each subject, with 1 point given for each of the following: age of
>60 years, current smoker, hypertension,
hypercholesterolemia, and family history of
coronary artery disease.
Preparation of Washed Platelets
Platelet suspensions were prepared according to a previously
described method.5 10 Briefly, 20 mL of blood from all
subjects was collected through venipuncture into a plastic
tube containing 3.15% trisodium citrate (1:9 v/v) and prostacyclin (2
µg/mL; Sigma Chemical Co) and then centrifuged at
250g for 20 minutes at 22°C. The obtained supernatant,
platelet-rich plasma, was centrifuged again at
900g for 10 minutes at 22°C after the addition of
prostacyclin (300 ng/mL). The platelet pellet was resuspended in 5
mL of Ca2+ and Mg2+–free
Tyrode’s solution (pH 7.4) containing prostacyclin (300 ng/mL) and
centrifuged at 800g for 10 minutes at 37°C. The
platelet pellet was resuspended in Tyrode’s solution containing
Ca2+ and Mg2+. The
platelet counts were then adjusted to the range of 1 to
2x105 platelets/µL in Tyrode’s solution.
Tyrode’s solution consisted of the following components (in
mmol/L): NaCl 136, KCl 2.7, NaHCO3 12,
NaH2PO4 ·
2H2O 0.42, CaCl2 1.8,
MgCl2 · 6H2O 1,
glucose 5.5, and HEPES 5, pH 7.4.
Measurements of PDNO With an NO-Specific Electrode
We measured PDNO with an NO meter (model N0-501; Inter Medical
Co) that measures the picoampere-order redox current between the
working electrode and the counterelectrode, as previously
described.5 Briefly, the working electrode consisted of a
platinum/iridium alloy wire (OD 0.2 mm) coated with a 3-layer
membrane that included the KCl membrane, NO-selective resin, and normal
silicone membrane. The counterelectrode was made of carbon fiber (OD
0.5 mm).11 The NO meter and electrodes were placed
into an electromagnetic shield box to avoid the electrical perturbation
of the electrodes. The two electrodes were placed in a chamber
containing washed platelet suspensions. After the addition of
collagen (3 µg/mL), the working electrode was supplied with +0.6 V
for the electrochemical oxidation of NO diffusing through the membrane
to avoid currents from possible contaminants of oxygen because
electrode responses to oxygen changes were observed at voltages of
<+0.4 V.11 12 The obtained electrical current was
considered to be an index of NO release.
Statistical Analysis
Data are expressed as mean±SD. The number of risk factors was
considered a continuous variable and compared with PDNO release
with linear regression analysis. Repeated measures ANOVA was
applied for multiple comparisons. Univariate
analysis of the effects of each risk on PDNO release was
performed with linear regression for continuous variables (age;
systolic, diastolic, and mean blood pressures; and
total, HDL-, and LDL-cholesterol levels) and 1-way ANOVA
for categorical variables (gender, smoking, family history of
coronary artery disease, evidence of coronary artery
disease). Then, the interaction between various risk characteristics
and PDNO release was examined with multiple stepwise regression
analysis. Statistical significance was considered at the level
of P<0.05.
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Results |
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Clinical Characteristics of the Subjects
The subjects consisted of 52 men and 9 women (30 nonsmokers and 31 smokers, mean age 47 years, range 24 to 74 years). The averaged systolic, diastolic, and mean arterial pressures were 127±15, 76±10, and 93±10 mm Hg, respectively; 15 subjects had hypertension. Five subjects had a positive family history of coronary artery disease. The average levels of total, HDL-, and LDL-cholesterol and triglycerides were 192±27, 118±27, 48±13, and 128±56 mg/dL, respectively; 12 subjects had hypercholesterolemia.
PDNO and Coronary Risk Factors
Mean PDNO was 9.6±4 pA (range 3.5 to 22 pA). The mean number of
risk factors was 1.4±1.1, and the composite risk factor was 0 in 17, 1
in 17, 2 in 17, 3 in 8, and 4 in 2 subjects. PDNO release was
significantly lower in subjects with risk factors (8.3±2.4, 8.9±2.7,
6.4±3.1, and 4.5±0.7 pA for subjects with 1, 2, 3, and 4 risk
factors, respectively) than in subjects without risk factors (13.6±3.6
pA) (P<0.05). PDNO release exhibited a significant inverse
correlation with the number of risk factors (r=-0.61,
P<0.001; Figure 1).
Univariate analysis showed a significant inverse
correlation between PDNO release and age (r=-0.33,
P<0.01; Figure 2A),
systolic blood pressure (r=-0.38,
P<0.005), diastolic blood pressure
(r=-0.34, P<0.01), mean arterial
pressure (r=-0.40, P<0.01; Figure 2B),
total cholesterol level (r=-0.31,
P<0.02; Figure 2C), and LDL-cholesterol
level (r=-0.33, P<0.02). PDNO release did not
correlate with triglycerides or HDL-cholesterol
levels. PDNO release was significantly lower in smokers than in
nonsmokers (P<0.001; Figure 2D) and significantly
lower in subjects with coronary artery disease than in healthy
volunteers (10.3±4.1 versus 8.0±3.1 pA, P<0.05). However,
neither gender nor family history of coronary artery disease
seemed to affect PDNO release. Multiple stepwise regression
analysis (Table) revealed that PDNO
release significantly and independently
(r2=0.51) correlated with smoking,
age, and mean arterial pressure.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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In the present study, the most important findings were that (1) the significant inverse correlation was found between PDNO release and the number of risk factors and that (2) PDNO release independently correlated with smoking, aging, and hypertension. Thus, our findings suggest that impairment of PDNO release by platelets is associated with coronary risk factors in humans.
It has been shown that an NO-selective electrode is specifically capable of measuring real-time release of NO from endothelial cells11 13 14 and platelets.6 15 We also previously showed that the electrode used in this study was sufficiently sensitive to detect the release of NO from aggregated platelets on the basis of the following findings.5 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 from 1 to 5 µg/mL (r=0.94). Third, the collagen-induced electrical current and intraplatelet levels of cGMP were increased by L-arginine and attenuated by NG-monomethyl-L-arginine, an inhibitor of NO synthesis. 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.
We previously reported that long-term smoking impairs PDNO release.5 The same findings were obtained in this study. We further examined whether other major risk factors are associated with impaired PDNO release during platelet aggregation. A significant inverse correlation was observed between PDNO release and the number of risk factors. Univariate analysis showed that PDNO release was inversely related to risk factors such as aging, hypertension, and hypercholesterolemia. Thus, our findings suggest not only that individual risk factors impair PDNO release but also that PDNO release is further impaired when the number of risk factors increases. When the multiple stepwise regression model was used to predict PDNO release with age, gender, cholesterol level, blood pressure, smoking, and family history as independent variables, PDNO release was found to be independently related to smoking, aging, and hypertension. Most importantly, PDNO release showed the strongest negative correlation with smoking. Accordingly, among major risk factors, smoking is the most powerful factors affecting PDNO release. Taken together, our findings may suggest that the L-arginine/NO pathway, as a negative feedback mechanism to inhibit platelet aggregation, is impaired in subjects with major risk factors, leading to an increased platelet aggregability and the development of atherothrombotic disease.
In the present study, the presence of coronary risk factors was associated with an impairment of PDNO release in human platelets. These risk factors have been shown to impair endothelium-dependent vasodilation16 17 18 and to reduce bioactivity of NO19 in the human coronary arteries. On the bases of the present and other studies, coronary risk factors may impair the L-arginine/NO pathway in both endothelial cells and platelets. Epidemiological studies have demonstrated that exposure to multiple risk factors increases the occurrence of atherosclerosis and thrombosis.20 21 22 Because endogenous NO has antiatherothrombotic actions,8 23 our findings may contribute to an understanding of the pathophysiological link between risk factors and atherothrombotic disease.
The correlations between each risk factor and PDNO release in this study were relatively weak. A possible reason is that the present study included a relatively small number of subjects compared with epidemiological trials. Hence, it is possible that a significant independent correlation between PDNO release and other risk factors, such as hypercholesterolemia, diabetes mellitus, or family history, would be shown in a larger sample size. In this study, the r2 value for the multiple stepwise regression model of PDNO release with all independent variables was 0.51, implying that 51% of the variability of PDNO release could be explained by the risk factors. Finally, we were unable to determine with our method whether coronary risk factors impair the release of PDNO or increase the inactivation of PDNO released from platelets.
In conclusion, the present study demonstrates that coronary risk factors are associated with an impairment of PDNO release in human platelets.
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Acknowledgments |
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This work was supported in part by a Grant-in-Aid for scientific research from the Ministry of Education, Science, Sports and Culture of Japan, Tokyo, and by a research grant from the Kimura Memorial Heart Foundation, Kurume, Japan.
Received September 17, 1999; first decision October 14, 1999; accepted November 30, 1999.
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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1. Radomski MW, Palmer RMJ, Moncada S. An L-arginine/nitric oxide pathway present in human platelets regulates aggregation. Proc Natl Acad Sci U S A. 1990;87:5193–5197.
2. Sase K, Michel T. Expression of constitutive endothelial nitric oxide synthase in human blood platelets. Life Sci. 1995;57:2049–2055.[Medline] [Order article via Infotrieve]
3. Moncada S, Palmer RMJ, Higgs EA. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev. 1991;43:109–142.[Medline] [Order article via Infotrieve]
4. Radomski MW, Palmer RM, Moncada S. Modulation of platelet aggregation by an L-arginine-nitric oxide pathway. Trends Pharmacol Sci. 1991;12:87–88.[Medline] [Order article via Infotrieve]
5.
Ichiki K, Ikeda H, Haramaki N, Ueno T, Imaizumi T.
Long-term smoking impaired platelet-derived nitric oxide release.
Circulation. 1996;94:3109–3114.
6. Malinski T, Radomski MW, Taha Z, Moncada S. Direct electrochemical measurement of nitric oxide released from human platelets. Biochem Biophys Res Commun. 1993;194:960–965.[Medline] [Order article via Infotrieve]
7. Muruganandam A, Mutus B. Isolation of nitric oxide synthase from human platelets. Biochim Biophys Acta. 1994;1200:1–6.[Medline] [Order article via Infotrieve]
8. Freedman JE, Michelson AM, Barnard MR, Alpert C, Keaney JFJ, Loscalzo J. Nitric oxide release from activated platelets inhibits platelet recruitment. J Clin Invest. 1997;100:350–356.[Medline] [Order article via Infotrieve]
9.
The Sixth Report of the Joint National Committee on
Prevention, Detection, Evaluation, and Treatment of High Blood
Pressure. Arch Intern Med. 1997;157:2413–2446.
10. Radomski M, Moncada S. An improved method for washing of human platelets with prostacyclin. Thromb Res. 1983;30:383–389.[Medline] [Order article via Infotrieve]
11. Ichimori K, Ishida H, Fukahori M, Nakazawa H, Murakami E. Practical nitric oxide measurement employing a nitric oxide-selective electrode. Rev Sci Instrum. 1994;65:1–5.
12. Shibuki K. An electrochemical microprobe for detecting nitric oxide release in brain tissue. Neurosci Res. 1990;9:69–76.[Medline] [Order article via Infotrieve]
13. Malinski T, Taha Z. Nitric oxide release from a single cell measured in situ by a porphyrinic-based microsensor. Nature. 1992;358:676–678.[Medline] [Order article via Infotrieve]
14. Tsukahara H, Gordienko DV, Goligorsky MS. Continuous monitoring of nitric oxide release from human umbilical vein endothelial cells. Biochem Biophys Res Commun. 1993;193:722–729.[Medline] [Order article via Infotrieve]
15. Lantoine F, Brunet A, Bedioui F, Devynck J, Devynck MA. Direct measurement of nitric oxide production in platelets: relationship with cytosolic Ca2+ concentration. Biochem Biophys Res Commun. 1995;215:842–848.[Medline] [Order article via Infotrieve]
16.
Vita JA, Treasure CB, Nabel EG, David MMJ, Fish R,
Yeung AC, Vekshtein VI, Selwyn AP, Ganz P. Coronary vasomotor
response to acetylcholine relates to risk factors for coronary
artery disease. Circulation. 1990;81:491–497.
17. Egashira K, Inou T, Hirooka Y, Yamada A, Maruoka Y, Kai H, Sugimachi M, Suzuki S, Takeshita A. Impaired coronary blood flow response to acetylcholine in patients with coronary risk factors and proximal atherosclerotic lesions. J Clin Invest. 1993;91:29–37.
18. Celermajer DS, Sorensen KE, Bull C, Robinson J, Deanfield JE. Endothelium-dependent dilation in the systemic arteries of asymptomatic subjects relates to coronary risk factors and their interaction. J Am Coll Cardiol. 1994;24:1468–1474.[Abstract]
19. Quyyumi AA, Dakak N, Andrews NP, Husain S, Arosa S, Gilligan DM, Panza JA, Cannon RR. Nitric oxide activity in the human coronary circulation. impact of risk factors for coronary atherosclerosis. J Clin Invest. 1995;95:1747–1755.
20. The Pooling Project Research Group. Relationship of blood pressure, serum cholesterol, smoking habit, relative weight and ECG abnormalities to incidence of major coronary events: final report of the Pooling Project. J Chron Dis. 1978;31:201–306.[Medline] [Order article via Infotrieve]
21. Castelli WP. Epidemiology of coronary heart disease: the Framingham Study. Am J Med. 1984;76(suppl 2A):4–12.
22.
Lipid Research Clinics Program. The Lipid Research
Clinics Primary Prevention Trial results. JAMA. 1984;251:351–364.
23. Cooke JP, Singer AH, Tsao P, Zera P, Rowan RA, Billingham ME. Antiatherogenic effects of L-arginine in the hypercholesterolemic rabbit. J Clin Invest. 1992;90:1168–1172.
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