This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Freedman, J. E. Articles by Vita, J. A. Search for Related Content PubMed PubMed Citation Articles by Freedman, J. E. Articles by Vita, J. A.

(Circulation. 1998;98:1481-1486.)
© 1998 American Heart Association, Inc.

Clinical Investigation and Reports

Impaired Platelet Production of Nitric Oxide Predicts Presence of Acute Coronary Syndromes

Jane E. Freedman, MD; Brian Ting, BS; Beth Hankin, BA; Joseph Loscalzo, MD, PhD; John F. Keaney, Jr, MD; ; Joseph A. Vita, MD

From the Departments of Pharmacology and Medicine, Georgetown University Medical Center, Washington, DC (J.E.F.), and the Whitaker Cardiovascular Institute and Evans Department of Medicine, Boston University School of Medicine, Boston, Mass (B.T., B.H., J.L., J.F.K., J.A.V.).

Correspondence to Dr Jane E. Freedman, Med-Dent Bldg Room NE 403, Georgetown University Medical Center, 3900 Reservoir Rd, NW, Washington, DC 20007. E-mail: Freedmaj{at}gunet.georgetown.edu

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background—Thrombus formation within a coronary vessel is the acute precipitating event in most acute coronary syndromes. Recently, constitutive nitric oxide synthase (cNOS) has been identified in human platelets, and platelet-derived nitric oxide has been shown to inhibit platelet recruitment after aggregation. However, its role in regulating platelet responses under normal or pathologic conditions has not yet been elucidated.

Methods and Results—We examined nitric oxide (NO) production by platelets isolated from 87 patients undergoing coronary angiography, 37 with stable angina and 50 with unstable angina or a myocardial infarction within 2 weeks. After stimulation with 5 µmol/L ADP, platelet aggregation and NO production were simultaneously measured with an NO-selective microelectrode adapted for use in a standard platelet aggregometer. Mean (±SEM) platelet-derived NO production was 1.78±0.36 pmol/10⁸ and 0.26±0.05 pmol/10⁸ platelets in coronary patients with stable angina and acute coronary syndromes, respectively (P=0.0001). By logistic regression analysis, heparin treatment (odds ratio 6.6, CI 1.9 to 22.8, P=0.003), lower platelet-NO production (odds ratio 4.0, CI 1.3 to 11.5, P=0.01), and extent of atherosclerosis (odds ratio 1.5, CI 1.1 to 2.0, P=0.02) were independent predictors of an acute coronary syndrome. In the subset of patients with angiographic evidence of atherosclerosis (n=83), logistic regression demonstrated that platelet NO production (odds ratio 3.9, CI 1.3 to 11.1, P=0.01) and heparin treatment (odds ratio 6.4, CI 1.9 to 22.0, P=0.004) were independent predictors of an acute coronary syndrome, whereas extent of atherosclerosis was not.

Conclusions—In summary, aggregating platelets from patients with acute coronary syndromes produce less NO. Since platelet aggregation and thrombus formation are implicated in unstable angina and myocardial infarction, impaired platelet-derived NO production may contribute to the development of acute coronary syndromes.

Key Words: platelets • nitric oxide • thrombosis • coronary disease

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Thrombus formation within a coronary vessel is the precipitating event in myocardial infarction and unstable angina, as documented by angiographic¹ and pathologic² studies. Rupture of atheromatous plaque in relatively mildly stenosed vessels and subsequent occlusive thrombus formation is believed to be responsible for most acute coronary syndromes.^{3 4 5} Both superficial and deep intimal injury lead to the adherence of platelets to the subendothelium and, subsequently, platelet activation. Once activated, platelets further stimulate thrombus formation and recruit additional platelets by releasing ADP and serotonin, producing thromboxane A₂, and promoting surface thrombin generation.⁶ Increased platelet-derived thromboxane and prostaglandin metabolites^{7 8} have been found in acute coronary syndromes, providing biochemical support for platelet activation. The importance of platelet activation in acute coronary syndromes is further supported by the clear clinical benefit of treatment with aspirin.^{9 10}

Platelet activation and recruitment are tightly regulated by products of the endothelium, including prostacyclin and NO (nitric oxide).^{11 12} NO inhibits platelet adhesion and aggregation,^{13 14} and prevents thrombosis in a model of endotoxin-induced glomerular damage.¹⁵ Endothelial production of bioactive NO is impaired in atherosclerosis¹⁶ and in the presence of coronary risk factors including hypercholesterolemia, diabetes mellitus, cigarette smoking, and hypertension.¹⁷ Furthermore, there is evidence that loss of endothelium-derived NO contributes to the pathogenesis of acute coronary syndromes.^{18 19}

Both constitutive and inducible nitric oxide synthase (NOS) have been identified in human platelets and megakaryocytoid cells,^{20 21 22 23} and studies report NO release from aggregating platelets.^{24 25 26} Platelet aggregation is enhanced by incubation with inhibitors of NOS and inhibited by incubation with the NOS substrate L-arginine.²⁷ Importantly, whereas platelet-derived NO appears to inhibit the primary aggregation response modestly, NO release from activated human platelets markedly inhibits platelet recruitment²⁶ and thus may limit progression of intra-arterial thrombosis. In vivo, systemic infusion of the NOS inhibitor L-N^G-monomethyl arginine citrate (L-NMMA) causes a reduction in bleeding time without change in vessel tone²⁸ and enhances platelet reactivity to various agonists,²⁹ supporting the clinical relevance of platelet-derived NO. Although the role of endothelium-derived NO has been extensively characterized, the relation between platelet-derived NO production and cardiovascular disease has not been previously investigated. Since coronary thrombosis is the precipitating event in most acute coronary syndromes and loss of platelet-derived NO promotes platelet recruitment, we sought to examine the relation between platelet NO production and clinical presentation in patients with coronary artery disease.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Patients
Consecutive patients referred to Boston Medical Center for cardiac catheterization were enrolled in accordance with the policies of the Institutional Review Board. Medications, including aspirin, were not discontinued. At the beginning of the catheterization, a 45-mL blood sample was collected for plasma and platelet analysis.

A research nurse identified the presence of the following risk factors: (1) age, (2) male sex, (3) clinical history of diabetes (fasting blood glucose >140 mg/dL or treatment with insulin or an oral hypoglycemic agent), (4) clinical history of hypertension (blood pressure >90 mm Hg diastolic or treatment for hypertension), (5) cigarette smoking (pack-years and most recent cigarette), (6) clinical history of hypercholesterolemias, and (7) family history of coronary disease (first-degree relative with myocardial infarction or cardiac death before age 55). Patients were also questioned about peripheral vascular disease, ethanol use, medications, and multivitamin use. Total cholesterol, HDL cholesterol, and triglycerides were measured in the Boston Medical Center clinical laboratory. LDL cholesterol was calculated with the Friedewald formula.³⁰

Assessment of Extent of Coronary Atherosclerosis
Coronary angiograms were analyzed off-line in a blinded fashion with the use of digital calipers to measure stenosis severity, and stenosis was defined as a dichotomous variable: if a stenotic lesion was >50%, that vessel was counted as stenosed. Patients were ranked as having 0- to 3-vessel disease. Extent of atherosclerosis was also quantified using the Hamsten extent score,³¹ which reflects the extent of early disease and is expressed on a scale of 0 (no disease) to 9 (extensive atherosclerosis in each of 15 coronary segments).

Classification of Clinical Disease Activity
We obtained a detailed angina history and reviewed the medical record for evidence of unstable angina as defined by Braunwald³² or myocardial infarction as indicated by the presence of typical symptoms, ischemic ECG changes, and a diagnostic elevation of CK_MB fraction. We categorized each subject as stable or having an acute coronary syndrome, depending on the presence or absence of unstable angina or a myocardial infarction within 2 weeks of study. Subjects were categorized by investigators blinded to platelet NO results.

Preparation of Samples
The blood was centrifuged (150g, 15 minutes, 22°C) and the supernatant, representing platelet-rich plasma (PRP), was separated. Gel-filtered platelets (GFP) were prepared by passing PRP over a Sepharose-2B column equilibrated with Tyrode's-HEPES buffered saline, as previously described.³³ Platelet counts were determined with the use of a Coulter Counter, model ZM (Coulter Electronics).

Measurement of Platelet NO Production and Aggregation
We adapted a NO-selective³⁴ micro-electrode (Inter Medical Co, Ltd) for use in a standard platelet aggregometer (Payton Associates) to monitor platelet NO production and aggregation simultaneously, as previously described.²⁶ Platelet NO production was quantitated as the integrated signal detected by the micro-electrode after platelet activation with 5 µmol/L ADP. Aggregation of GFP was monitored with a standard nephelometric technique as previously described.^{35 36}

Platelet NO production was initially studied in 41 normal controls (age 30±2 years) and ranged from 0.5 to 15.2 pmol/10⁸ platelets (mean 3.3±1.4 pmol/10⁸ platelets). To assess interassay reproducibility, GFP was prepared from 10 normal control subjects (age 31±2 years) on 2 different days. Platelet NO production in this population ranged from 0.5 to 14.7 pmol/10⁸ platelets (mean 3.6±1.0 pmol/10⁸ platelets). For each donor, the average difference in between-day determinations of NO production was 0.36±0.36 pmol/10⁸ platelets and the average within-day difference between determinations of NO production was 0.50±0.25 pmol/10⁸ platelets for each donor (P=NS). In each subject, the NOS inhibitor N-N^G nitroarginine methyl ester (L-NAME) inhibited at least 80% of the NO signal, confirming its dependence on NO production. The limit of detection for the assay is 0.05 pmol/10⁸ platelets.

Statistical Analysis
Clinical characteristics, coronary risk factors, angiographic findings, medication use, platelet function, and platelet NO production for the stable and acute coronary syndrome groups were compared with the use of the ² test for proportions, the 2-sample t tests for normally distributed continuous variables, and the Mann-Whitney U test for continuous variables with a nonnormal distribution. Normality was determined with the Kolmogorov-Smirnov algorithm. Platelet NO production had a nonnormal distribution. To determine which patient characteristics were associated with low platelet NO production, patients were categorized as having platelet NO production less than, greater than, or equal to the median value, and the 2 groups were compared with the use of the ² test or the 2-sample t test as appropriate. Logistic regression was used to identify independent predictors of an acute coronary syndrome. Statistical significance was accepted at the P<0.05 level. Analyses were completed using SPSS for Windows, Release 6.0 (SPSS, Inc). All data are expressed as mean±SEM.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Patient Characteristics
A total of 87 subjects were enrolled in the study. Fifty subjects had an acute coronary syndrome (unstable angina or myocardial infarction) and 37 subjects did not. The clinical characteristics, medications, and angiographic findings for the 2 groups are displayed in Table 1. As shown, the subjects with an acute coronary syndrome had more extensive coronary atherosclerosis and were more likely to be receiving nitrate or heparin treatment than were the stable subjects.

View this table: [in this window] [in a new window]   Table 1. Patient Characteristics

Platelet NO Production
For all patients, platelet NO production after activation by ADP ranged from 0.00 to 9.02, with a mean of 0.90±0.18 pmol/10⁸ platelets. The values for platelet NO production were not normally distributed and had a median value of 0.30 pmol/10⁸ platelets. As shown in representative tracings in Figure 1 and summarized in Figure 2, production of NO by platelets was lower in patients with an acute coronary syndrome than in stable patients. Extent of platelet aggregation and platelet count after gel filtration were similar in the 2 groups (Table 2). In addition, patients with significant coronary artery disease (stenosis 50%, n=73) and patients with no significant coronary artery disease (n=14) had platelet NO levels of 0.62±1.5 pmol/10⁸ platelets and 2.4±1.7 pmol/10⁸ platelets, respectively (P<0.0001). Platelet NO levels were not significantly different between patients with unstable angina and patients with myocardial infarction.

View larger version (7K): [in this window] [in a new window]   Figure 1. Effect of platelet activation on platelet NO production in patients with stable (left) and unstable (right) ischemic coronary syndromes. Shown are representative tracings of NO production by gel-filtered platelets stimulated with 5 µmol/L ADP as described in "Methods."

View larger version (19K): [in this window] [in a new window]   Figure 2. Platelet NO production in patients with stable and acute coronary syndromes. Platelet NO production after activation with ADP was significantly decreased (P=0.0001) in patients with acute coronary syndromes as compared with patients with stable disease.

View this table: [in this window] [in a new window]   Table 2. Platelet Parameters in Patients Referred for Cardiac Catheterization

To investigate whether low platelet NO production is an independent predictor of an acute coronary syndrome, we first identified potential confounders by comparing the clinical characteristics listed in Table 1 in the groups of patients with platelet NO production greater than and less than, or equal to the median value. Patients with low platelet NO production were more likely to be receiving heparin (49% versus 27%, P=0.04) and were more likely to be receiving nitrate therapy (72% versus 50%, P=0.03). There was a trend for a higher atherosclerosis extent score in the patients with low platelet NO production (3.6±0.3) compared with patients with high platelet NO production (2.8±0.2, P=0.06). No other clinical marker differed significantly according to level of NO production.

To identify the independent predictors of an acute coronary syndrome, a logistic regression model was constructed using these 4 variables: category of platelet NO production, heparin treatment, nitrate treatment, and atherosclerosis extent score. As shown in Table 3, the independent predictors of an unstable coronary syndrome were low platelet NO production, atherosclerosis extent score, and the presence of heparin treatment.

View this table: [in this window] [in a new window]   Table 3. Multivariate Analysis for Predictors of Acute Coronary Syndromes

No subject with angiographically normal arteries had an acute coronary syndrome, and we considered the possibility that the association between extent of atherosclerosis and an acute coronary syndrome was driven by this finding. Therefore, we investigated predictors of an acute coronary syndrome in the 83 patients with an atherosclerosis extent score >0. In this group of patients with proven coronary artery disease, logistic regression demonstrated that platelet NO production (odds ratio 3.9, CI 1.3 to 11.1, P=0.01) and heparin treatment (odds ratio 6.4, CI 1.9 to 22.0, P=0.004) were independent predictors of an acute coronary syndrome, whereas extent of atherosclerosis was not.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In this study, we evaluated stimulated platelet NO production in a group of 87 patients referred for cardiac catheterization. The patients with an acute coronary syndrome within 2 weeks of study had markedly lower platelet NO production than patients with stable or no angina. Acute coronary syndrome patients, not surprisingly, also were more likely to be receiving heparin and nitrate therapy and had more extensive coronary atherosclerosis. By logistic regression analysis, low platelet NO production, heparin therapy, and extent of coronary atherosclerosis were independent predictors of an acute coronary syndrome whereas nitrate treatment was not. Platelet NO production but not extent of atherosclerosis was an independent predictors of an acute coronary syndrome in the subset of patients with coronary artery disease, suggesting that once atherosclerosis is present, factors other than the extent of disease are important. Thus, low platelet NO production could be a contributing mechanism in the pathophysiology of acute coronary syndromes.

In the present study, heparin therapy was associated with both lower platelet NO production and acute coronary syndromes, raising the possibility that a confounding effect of heparin treatment might explain the strong relation between low platelet NO production and acute coronary syndromes. In high concentrations, heparin is known to decrease NO production in endothelial cells,³⁷ but there currently is no evidence for a direct effect of heparin on NO production in platelets. A direct heparin effect is unlikely to explain the present findings because platelet NO production remained an independent predictor of acute coronary syndromes after controlling for heparin treatment. Nitrate treatment also has the potential to affect platelet NO production. For example, it is known that NO can downregulate cNOS activity in cerebellum and human platelets,^{38 39} and it is possible that NO released from nitroglycerin had such an effect in platelets. In the present study, the relation between acute coronary syndromes and platelet NO production was independent of nitrate therapy. Nitrates are also known to inhibit platelet aggregability,⁴⁰ but we observed no difference in ex vivo platelet aggregation in patients with stable or acute coronary syndromes, further arguing against an important nitrate effect on platelet function in these patients. Aspirin also inhibits platelet aggregation and could influence platelet NO production; however, the present study had inadequate power to address this issue because only 6 subjects were not taking aspirin. Overall, the independent relation between platelet NO production and the presence of an acute coronary syndrome argues strongly against a confounding effect of concurrent medications.

Only one prior study has examined platelet NO production in human subjects, and it demonstrated lower values in chronic cigarette smokers compared with healthy, nonsmoking control subjects.⁴¹ Although we found that platelet NO production was similar in smokers and nonsmokers, differences in the study populations likely explain these seemingly disparate findings. Unlike the subjects studied by Ichiki and colleagues,⁴¹ the present study examined older patients, and the majority of these patients had symptomatic coronary artery disease. Both smokers and nonsmokers had relatively low platelet NO production compared with historical normal controls from our laboratory. As in the present study, Ichiki and colleagues demonstrated no relation between platelet NO production and extent of ex vivo platelet aggregation.⁴¹

The mechanism for decreased NO release from platelets in patients with acute coronary symptoms has not yet been established. A prominent feature of both abnormal platelet function and dysfunctional endothelium-dependent vasodilation in the setting of cardiovascular disease is increased oxidative stress. There is ample clinical evidence to suggest that oxidative stress and antioxidant status is important in normal platelet function. For example, alteration of the platelet redox status causes increased production of reactive oxygen species, including superoxide.⁴² Metabolism of reactive oxygen species by antioxidants may also alter platelet function. In patients with coronary artery disease, decreased plasma and platelet antioxidant activity is associated with increased platelet aggregability.⁴³ In a recent clinical study, vitamin E supplementation was associated with increased hemorrhagic stroke.⁴⁴ In addition, we have shown that the antioxidant enzyme glutathione peroxidase potentiates the inhibition of platelet function by NO through metabolism of reactive oxygen species⁴⁵ and that impairment of this process can lead to a clinical thrombotic disorder.⁴⁶

Impairment of endothelium-derived nitric oxide has been extensively characterized in patients with atherosclerosis,⁴⁷ risk factors for coronary artery disease,¹⁷ and acute coronary syndromes^{18 19} ; however, platelet NO production has not been previously studied in patients with coronary disease. By direct measurement of NO production, the present study indicates that impairment of platelet-derived NO may also be linked to the pathogenesis of acute coronary syndromes. Although platelet NO release does not have a major effect on the primary aggregation response, platelet-derived NO appears to play an important counterregulatory role after platelet activation by inhibiting recruitment of platelets to the growing thrombus.²⁶ Since platelet recruitment is a primary means of thrombus propagation, it is reasonable to speculate that loss of platelet NO could increase the risk for development of unstable angina or acute myocardial infarction where coronary thrombosis is a primary event. The findings of the present study support the clinical relevance of this mechanism.

In summary, low platelet NO production was independently associated with the presence of an acute coronary syndrome, a finding that could not be attributed to concurrent medical therapy or other clinical and coronary risk factors. It should be acknowledged that the present study is cross-sectional in nature, and we cannot exclude the possibility that some unmeasured confounding factor associated with acute myocardial infarction or unstable angina accounts for our findings. However, our results may support the hypothesis that impaired platelet-derived NO production contributes to the development of acute coronary syndromes in patients with coronary artery disease and may be a suitable target for therapy.

	Acknowledgments

 
Dr Freedman is the recipient of a Grant-in-Aid from the Massachusetts Affiliate of the American Heart Association and CIDA HL-03556. John F. Keaney is the recipient of CIDA HL-03195. Dr Vita is supported by NIH grants HL-53398 and HL-55993 and an Established Investigator Award from the American Heart Association. Dr Loscalzo is supported by NIH grants HL-8743, HL-5993, and HL-8976, as well as a Merit Award from the Veterans Administration.

Received March 24, 1998; revision received June 9, 1998; accepted June 13, 1998.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. DeWood MA, Spores J, Notske R, Mouser LT, Burroughs R, Golden MS, Lang HT. Prevalence of total coronary occlusion during the early hours of transmural myocardial infarction. N Engl J Med. 1980;303:897–902.[Abstract]

2. Falk E. Unstable angina with fatal outcome: dynamic coronary thrombosis leading to infarction and/or sudden death. Circulation. 1985;71:699–708.[Abstract/Free Full Text]

3. Falk E. Plaque rupture with severe pre-existing stenosis precipitating coronary thrombosis. Br Heart J. 1983;1983:127–134.

4. Davies MJ, Thomas AC. Thrombosis and acute coronary-artery lesions in sudden cardiac ischemic death. N Engl J Med. 1984;310:1137–1140.[Abstract]

5. Willerson J, Golino P, Eidt J, Campbell W, Buja L. Specific platelet mediators and unstable coronary artery lesions: experimental evidence and potential clinical implications. Circulation. 1989;80:198–205.[Abstract/Free Full Text]

6. Santol MT, Valles J, Marcus AJ, Safier M, Brodkman J, Islan N, Ullman HL, Eiroa AM, Aznar J. Enhancement of platelet reactivity and modulation of eicosanoid production by intact erythrocytes. J Clin Invest. 1991;87:571–580.

7. Hirsh P, Hillis L, Campbell W, Firth B, Willerson J. Release of prostaglandins and thromboxane into the coronary circulation in patients with ischemic heart disease. N Engl J Med. 1981;304:685–691.[Abstract]

8. Fitzgerald DJ, Roy L, Catella F, Fitzgerald A. Platelet activation in unstable coronary disease. N Engl J Med. 1986;315:983–989.[Abstract]

9. The RISC Group. Risk of myocardial infarction and death during treatment with low dose aspirin and intravenous heparin in men with unstable coronary artery disease. Lancet. 1990;336:827–830.[Medline] [Order article via Infotrieve]

10. Antiplatelet Trialists Collaboration. Collaborative overview of randomized trials of antiplatelet therapy, I: prevention of death, myocardial infarction, and stroke by prolonged antiplatelet therapy in various categories of patients. BMJ. 1994;308:81–106.[Abstract/Free Full Text]

11. de Graaf JC, Banga JD, Moncada S, Palmer RMJ, de Groot PG, Sixma JJ. Nitric oxide functions as an inhibitor of platelet adhesion under flow conditions. Circulation. 1992;85:2284–2290.[Abstract/Free Full Text]

12. Radomski MW, Palmer MJ, Moncada S. The role of nitric oxide and cGMP in platelet adhesion to vascular endothelium. Biochem Biophys Res Commun. 1987;148:1482–1489.[Medline] [Order article via Infotrieve]

13. Stamler J, Mendelsohn ME, Amarante P, Smick D, Andon N, Davies PF, Cooke JP, Loscalzo J. N-Acetylcysteine potentiates platelet inhibition by endothelium-derived relaxing factor. Circ Res. 1989;65:789–795.[Abstract/Free Full Text]

14. Cooke JP, Stamler J, Andon N, Davies PF, McKinley G, Loscalzo J. Flow stimulates endothelial cells to release a nitrovasodilator that is potentiated by reduced thiol. Am J Physiol. 1990;259:H804–H812.[Abstract/Free Full Text]

15. Shultz PJ, Raij L. Endogenously synthesized nitric oxide prevents endotoxin-induced glomerular thrombosis. J Clin Invest. 1992;90:1718–1725.

16. Bossaller C, Habib GB, Yamamoto H, Williams C, Wells S, Henry PD. Impaired muscarinic endothelium-dependent relaxation and cyclic guanosine 5-monophosphate formation in atherosclerotic human coronary artery and rabbit aorta. J Clin Invest. 1986;170–174.

17. Vita JA, Keaney JF Jr, Loscalzo J. Endothelial dysfunction in vascular disease. In: Loscalzo J, Creager MA, Dzau V, eds. Vascular Medicine. 2nd ed. Boston, Mass: Little Brown and Company; 1996:245–263.

18. Okumura K, Yasue H, Matsuyama K, Ogawa H, Morikami Y, Obata K, Sakaino N. Effect of acetylcholine on the highly stenotic coronary artery: difference between the constrictor response of the infarct-related coronary artery and that of the noninfarct-related artery. J Am Coll Cardiol. 1992;19:753–758.

19. Bogaty P, Hackett D, Davies G, Maseri A. Vasoreactivity of the culprit lesion in unstable angina. Circulation. 1994;90:5–11.[Abstract/Free Full Text]

20. 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]

21. Mehta JL, Chen LY, Kone BC, Mehta P, Turner P. Identification of constitutive and inducible forms of nitric oxide synthase in human platelets. J Lab Clin Med. 1995;125:370–377.[Medline] [Order article via Infotrieve]

22. Chen LY, Mehta JL. Further evidence of the presence of constitutive and inducible nitric oxide synthase isoforms in human platelets. J Cardiovasc Pharmacol. 1996;27:154–158.[Medline] [Order article via Infotrieve]

23. Pigazzi A, Fabian A, Johnson J, Upchuch GR, Loscalzo J. Identification of nitric oxide synthases in human megakaryocytes and platelets. Circulation. 1995;92:I-365. Abstract.

24. 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]

25. 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.[Abstract/Free Full Text]

26. Freedman JE, Michelson AM, Barnard MR, Alpert C, Keaney JF Jr, Loscalzo J. Nitric oxide release from activated platelets inhibits platelet recruitment. J Clin Invest. 1997;100:350–356.[Medline] [Order article via Infotrieve]

27. Chen LY, Mehta JL. Variable effects of L-arginine analogs on L-arginine-nitric oxide pathway in human neutrophils and platelets may relate to different nitric oxide synthase isoforms. J Pharmacol Exp Ther. 1996;276:253–257.[Abstract/Free Full Text]

28. Simon DI, Stamler JS, Loh E, Loscalzo J, Frances SA, Creager MA. Effect of nitric oxide synthase inhibition on bleeding time in humans. J Cardiovasc Pharmacol. 1995;26:339–342.[Medline] [Order article via Infotrieve]

29. Bodzenta-Lukaszyke A, Gabryelewicz A, Lukaszyk A, Bielawiec M, Konturek JW, Domschke W. Nitric oxide synthase inhibition and platelet function. Thromb Res. 1994;75:667–672.[Medline] [Order article via Infotrieve]

30. Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem. 1972;18:499–502.[Abstract]

31. Hamsten AG, Walldius G, Szamosi A, Dahlen G, deFaire U. Relationship of angiographically defined coronary artery disease to serum lipoprotein and apolipoproteins in young survivors of myocardial infarction. Circulation. 1986;73:1097–1100.[Abstract/Free Full Text]

32. Braunwald E. Unstable angina: a classification. Circulation. 1989;80:410–413.[Free Full Text]

33. Hawiger JS, Parkinson S, Timmons S. Prostacyclin inhibits mobilization of fibrinogen-binding sites on human ADP- and thrombin-treated platelets. Nature (London). 1980;283:195–198.[Medline] [Order article via Infotrieve]

34. Ichimori K, Ishida H, Fudahori M, Nakazawa H, Murakami E. Practical nitric oxide measurement employing a nitric oxide-selective electrode. Rev Sci Instruments. 1994;65:1–5.

35. Born GV, Cross MJ. The aggregation of blood platelets. J Physiol (London). 1963;168:178–195.

36. Freedman JE, Farhat J, Loscalzo J, Keaney JF Jr. Alpha-tocopherol inhibits aggregation of human platelets by a protein kinase C-dependent mechanism. Circulation. 1996;94:2434–2440.[Abstract/Free Full Text]

37. Upchurch GR, Welch GN, Freedman JF, Fabian AJ, Pigazzi A, Scribner AM, Alpert CS, Keaney JFJ, Loscalzo J. High-dose heparin decreases nitric oxide production by cultured bovine endothelial cells. Circulation. 1997;95:2115–2121.[Abstract/Free Full Text]

38. Rogers NE, Ignarro LJ. Constitutive nitric oxide synthase from cerebellum is reversibly inhibited by nitric oxide from L-arginine. Biochem Biophys Res Commun. 1992;189:242–249.[Medline] [Order article via Infotrieve]

39. Chen LY, Mehta JL. Downregulation of nitric oxide synthase activity in human platelets by nitroglycerin and authentic nitric oxide. J Invest Med. 1997;45:69–74.[Medline] [Order article via Infotrieve]

40. Loscalzo J. N-Acetylcysteine potentiates inhibition of platelet aggregation by nitroglycerine. J Clin Invest. 1985;76:703–708.

41. Ichiki K, Ikeda H, Haramaki N, Ueno T, Imaizumi T. Long-term smoking impairs platelet-derived nitric oxide release. Circulation. 1996;94:3109–3114.[Abstract/Free Full Text]

42. Marcus AJ, Silk ST, Safier LB, Ullman HL. Superoxide production and reducing activity in human platelets. J Clin Invest. 1977;59:149–158.

43. Buczynski AB, Wachowicz B, Dedziora-Kornatowska K, Tkaczewski W, Kedziora J. Changes in antioxidant enzyme activities, aggregability, and malonyldialdehyde concentration in blood platelet from patients with coronary heart disease. Atherosclerosis. 1993;100:223–228.[Medline] [Order article via Infotrieve]

44. The Alpha-Tocopherol Beta-Carotene Cancer Prevention Study Group. The effect of vitamin E and beta carotene on the incidence of lung cancer and other cancers in male smokers. N Engl J Med. 1994;330:1029–1035.[Abstract/Free Full Text]

45. Freedman JE, Frei B, Welch GN, Loscalzo J. Glutathione peroxidase potentiates the inhibition of platelet function by S-nitrosothiols. J Clin Invest. 1995;96:394–400.

46. Freedman JE, Loscalzo J, Benoit SE, Valeri CR, Barnard MR, Michelson AD. Decreased platelet inhibition by nitric oxide in two brothers with a history of arterial thrombosis. J Clin Invest. 1996;97:979–987.[Medline] [Order article via Infotrieve]

47. Ludmer P, Selwyn A, Shook T, Wayne R, Mudge G, Alexander R, Ganz P. Paradoxical vasoconstriction induced by acetylcholine in atherosclerotic coronary arteries. N Engl J Med. 1986;315:1046–1051.Thrombus formation within a coronary vessel is the acute precipitating event in most acute coronary syndromes. Nitric oxide synthase has been identified in human platelets, and platelet-derived nitric oxide has been shown to inhibit platelet recruitment after aggregation. Platelet nitric oxide (NO) production was measured in patients with stable or unstable coronary syndromes. In patients with angiographic evidence of atherosclerosis (n=83), logistic regression demonstrated that platelet NO production (odds ratio 3.9, CI 1.3 to 11.1, P=0.01) was an independent predictor of an acute coronary syndrome. In summary, aggregating platelets from patients with acute coronary syndromes produce less NO.[Abstract]

This article has been cited by other articles:

				P. O'Kane, L. Xie, Z. Liu, L. Queen, G. Jackson, Y. Ji, and A. Ferro Aspirin acetylates nitric oxide synthase type 3 in platelets thereby increasing its activity Cardiovasc Res, July 1, 2009; 83(1): 123 - 130. [Abstract] [Full Text] [PDF]

				J. E. Freedman Oxidative Stress and Platelets Arterioscler Thromb Vasc Biol, March 1, 2008; 28(3): s11 - s16. [Abstract] [Full Text] [PDF]

				E. Gkaliagkousi, J. Ritter, and A. Ferro Platelet-Derived Nitric Oxide Signaling and Regulation Circ. Res., September 28, 2007; 101(7): 654 - 662. [Abstract] [Full Text] [PDF]

				T. M.C. Brunini, A. C. Mendes-Ribeiro, J. C. Ellory, and G. E. Mann Platelet nitric oxide synthesis in uremia and malnutrition: A role for L-arginine supplementation in vascular protection? Cardiovasc Res, January 15, 2007; 73(2): 359 - 367. [Abstract] [Full Text] [PDF]

				Y. Shimasaki, Y. Saito, M. Yoshimura, S. Kamitani, Y. Miyamoto, I. Masuda, M. Nakayama, Y. Mizuno, H. Ogawa, H. Yasue, et al. The Effects of Long-term Smoking on Endothelial Nitric Oxide Synthase mRNA Expression in Human Platelets as Detected With Real-time Quantitative RT-PCR Clinical and Applied Thrombosis/Hemostasis, January 1, 2007; 13(1): 43 - 51. [Abstract] [PDF]

				S. R. Willoughby, S. Stewart, A. S. Holmes, Y. Y. Chirkov, and J. D. Horowitz Platelet Nitric Oxide Responsiveness: A Novel Prognostic Marker in Acute Coronary Syndromes Arterioscler Thromb Vasc Biol, December 1, 2005; 25(12): 2661 - 2666. [Abstract] [Full Text] [PDF]

				J. E. Freedman Molecular Regulation of Platelet-Dependent Thrombosis Circulation, October 25, 2005; 112(17): 2725 - 2734. [Abstract] [Full Text] [PDF]

				V. Mollace, C. Muscoli, E. Masini, S. Cuzzocrea, and D. Salvemini Modulation of Prostaglandin Biosynthesis by Nitric Oxide and Nitric Oxide Donors Pharmacol. Rev., June 1, 2005; 57(2): 217 - 252. [Abstract] [Full Text] [PDF]

				H. Morita, H. Ikeda, N. Haramaki, H. Eguchi, and T. Imaizumi Only two-week smoking cessation improves platelet aggregability and intraplatelet redox imbalance of long-term smokers J. Am. Coll. Cardiol., February 15, 2005; 45(4): 589 - 594. [Abstract] [Full Text] [PDF]

				R. Stocker and J. F. Keaney Jr. Role of Oxidative Modifications in Atherosclerosis Physiol Rev, October 1, 2004; 84(4): 1381 - 1478. [Abstract] [Full Text] [PDF]

				G. Vilahur, E. Segales, E. Salas, and L. Badimon Effects of a Novel Platelet Nitric Oxide Donor (LA816), Aspirin, Clopidogrel, and Combined Therapy in Inhibiting Flow- and Lesion-Dependent Thrombosis in the Porcine Ex Vivo Model Circulation, September 21, 2004; 110(12): 1686 - 1693. [Abstract] [Full Text] [PDF]

				S. Fichtlscherer, S. Breuer, V. Schachinger, S. Dimmeler, and A. M. Zeiher C-reactive protein levels determine systemic nitric oxide bioavailability in patients with coronary artery disease Eur. Heart J., August 2, 2004; 25(16): 1412 - 1418. [Abstract] [Full Text] [PDF]

				U. Landmesser, B. Hornig, and H. Drexler Endothelial Function: A Critical Determinant in Atherosclerosis? Circulation, June 1, 2004; 109(21_suppl_1): II-27 - II-33. [Abstract] [Full Text] [PDF]

				S. Rajagopalan, E. C. Somers, R. D. Brook, C. Kehrer, D. Pfenninger, E. Lewis, A. Chakrabarti, B. C. Richardson, E. Shelden, W. J. McCune, et al. Endothelial cell apoptosis in systemic lupus erythematosus: a common pathway for abnormal vascular function and thrombosis propensity Blood, May 15, 2004; 103(10): 3677 - 3683. [Abstract] [Full Text] [PDF]

				G. Vilahur, M. I. Baldellou, E. Segales, E. Salas, and L. Badimon Inhibition of thrombosis by a novel platelet selective S-nitrosothiol compound without hemodynamic side effects Cardiovasc Res, March 1, 2004; 61(4): 806 - 816. [Abstract] [Full Text] [PDF]

				T. Heitzer, I. Ollmann, K. Koke, T. Meinertz, and T. Munzel Platelet Glycoprotein IIb/IIIa Receptor Blockade Improves Vascular Nitric Oxide Bioavailability in Patients With Coronary Artery Disease Circulation, August 5, 2003; 108(5): 536 - 541. [Abstract] [Full Text] [PDF]

				P. D. O'Kane, L. R. Queen, Y. Ji, V. Reebye, P. Stratton, G. Jackson, and A. Ferro Aspirin modifies nitric oxide synthase activity in platelets: effects of acute versus chronic aspirin treatment Cardiovasc Res, July 1, 2003; 59(1): 152 - 159. [Abstract] [Full Text] [PDF]

				O. Gorchakova, W. Koch, N. von Beckerath, J. Mehilli, A. Schomig, and A. Kastrati Association of a genetic variant of endothelial nitric oxide synthase with the 1 year clinical outcome after coronary stent placement Eur. Heart J., May 1, 2003; 24(9): 820 - 827. [Abstract] [Full Text] [PDF]

				M. H. Shishehbor, R. J. Aviles, M.-L. Brennan, X. Fu, M. Goormastic, G. L. Pearce, N. Gokce, J. F. Keaney Jr, M. S. Penn, D. L. Sprecher, et al. Association of Nitrotyrosine Levels With Cardiovascular Disease and Modulation by Statin Therapy JAMA, April 2, 2003; 289(13): 1675 - 1680. [Abstract] [Full Text] [PDF]

				M. Liu, A. Wallmon, C. Olsson-Mortlock, R. Wallin, and T. Saldeen Mixed tocopherols inhibit platelet aggregation in humans: potential mechanisms Am. J. Clinical Nutrition, March 1, 2003; 77(3): 700 - 706. [Abstract] [Full Text] [PDF]

				L. Cominacini, A. Fratta Pasini, U. Garbin, A. Pastorino, A. Rigoni, C. Nava, A. Davoli, V. Lo Cascio, and T. Sawamura The platelet-endothelium interaction mediated by lectin-like oxidized low-density lipoprotein receptor-1 reduces the intracellular concentration of nitric oxide in endothelial cells J. Am. Coll. Cardiol., February 5, 2003; 41(3): 499 - 507. [Abstract] [Full Text] [PDF]

				S. Kanaya, H. Ikeda, N. Haramaki, T. Murohara, and T. Imaizumi Intraplatelet Tetrahydrobiopterin Plays an Important Role in Regulating Canine Coronary Arterial Thrombosis by Modulating Intraplatelet Nitric Oxide and Superoxide Generation Circulation, November 13, 2001; 104(20): 2478 - 2484. [Abstract] [Full Text] [PDF]

				Y. Takajo, H. Ikeda, N. Haramaki, T. Murohara, and T. Imaizumi Augmented oxidative stress of platelets in chronic smokers: Mechanisms of impaired platelet-derived nitric oxide bioactivity and augmented platelet aggregability J. Am. Coll. Cardiol., November 1, 2001; 38(5): 1320 - 1327. [Abstract] [Full Text] [PDF]

				A. Huang, H. Xiao, J. M. Samii, J. A. Vita, and J. F. Keaney Jr. Contrasting effects of thiol-modulating agents on endothelial NO bioactivity Am J Physiol Cell Physiol, August 1, 2001; 281(2): C719 - C725. [Abstract] [Full Text] [PDF]

				J. E. Freedman, C. Parker III, L. Li, J. A. Perlman, B. Frei, V. Ivanov, L. R. Deak, M. D. Iafrati, and J. D. Folts Select Flavonoids and Whole Juice From Purple Grapes Inhibit Platelet Function and Enhance Nitric Oxide Release Circulation, June 12, 2001; 103(23): 2792 - 2798. [Abstract] [Full Text] [PDF]

				D. Li, T. Saldeen, F. Romeo, and J. L. Mehta Different Isoforms of Tocopherols Enhance Nitric Oxide Synthase Phosphorylation and Inhibit Human Platelet Aggregation and Lipid Peroxidation: Implications in Therapy with Vitamin E Journal of Cardiovascular Pharmacology and Therapeutics, June 1, 2001; 6(2): 155 - 161. [Abstract] [PDF]

				S. J. Duffy, J. A. Vita, M. Holbrook, P. L. Swerdloff, and J. F. Keaney Jr Effect of Acute and Chronic Tea Consumption on Platelet Aggregation in Patients With Coronary Artery Disease Arterioscler Thromb Vasc Biol, June 1, 2001; 21(6): 1084 - 1089. [Abstract] [Full Text] [PDF]

				J. Loscalzo Nitric Oxide Insufficiency, Platelet Activation, and Arterial Thrombosis Circ. Res., April 27, 2001; 88(8): 756 - 762. [Abstract] [Full Text] [PDF]

				A.C. Mendes Ribeiro, T.M.C. Brunini, J.C. Ellory, and G.E. Mann Abnormalities in L-arginine transport and nitric oxide biosynthesis in chronic renal and heart failure Cardiovasc Res, March 1, 2001; 49(4): 697 - 712. [Abstract] [Full Text] [PDF]

				D Tousoulis, C Tentolouris, T Crake, G Goumas, C Stefanadis, P Toutouzas, and G Davies Complex stenosis morphology and vasomotor responses to inhibition of nitric oxide synthesis Heart, November 1, 2000; 84(5): 529 - 534. [Abstract] [Full Text]

				U. Laufs, K. Gertz, P. Huang, G. Nickenig, M. Bohm, U. Dirnagl, M. Endres, and C. J. Vaughan Atorvastatin Upregulates Type III Nitric Oxide Synthase in Thrombocytes, Decreases Platelet Activation, and Protects From Cerebral Ischemia in Normocholesterolemic Mice Editorial Comment Stroke, October 1, 2000; 31(10): 2442 - 2449. [Abstract] [Full Text] [PDF]

				L. R. Queen, B. Xu, K. Horinouchi, I. Fisher, and A. Ferro {beta}2-Adrenoceptors Activate Nitric Oxide Synthase in Human Platelets Circ. Res., July 7, 2000; 87(1): 39 - 44. [Abstract] [Full Text] [PDF]

				Y. Yang and J. Loscalzo Regulation of Tissue Factor Expression in Human Microvascular Endothelial Cells by Nitric Oxide Circulation, May 9, 2000; 101(18): 2144 - 2148. [Abstract] [Full Text] [PDF]

				T. J. Anderson Assessment and treatment of endothelial dysfunction in humans J. Am. Coll. Cardiol., September 1, 1999; 34(3): 631 - 638. [Full Text] [PDF]

				C. S Hayward, R. P Kelly, and P. S Macdonald Inhaled nitric oxide in cardiology practice Cardiovasc Res, August 15, 1999; 43(3): 628 - 638. [Abstract] [Full Text] [PDF]

				J. E. Freedman, R. Sauter, E. M. Battinelli, K. Ault, C. Knowles, P. L. Huang, and J. Loscalzo Deficient Platelet-Derived Nitric Oxide and Enhanced Hemostasis in Mice Lacking the NOSIII Gene Circ. Res., June 25, 1999; 84(12): 1416 - 1421. [Abstract] [Full Text] [PDF]

				A. Huang, J. A. Vita, R. C. Venema, and J. F. Keaney Jr. Ascorbic Acid Enhances Endothelial Nitric-oxide Synthase Activity by Increasing Intracellular Tetrahydrobiopterin J. Biol. Chem., June 2, 2000; 275(23): 17399 - 17406. [Abstract] [Full Text] [PDF]

				B. Yan and J. W. Smith A Redox Site Involved in Integrin Activation J. Biol. Chem., December 15, 2000; 275(51): 39964 - 39972. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Freedman, J. E. Articles by Vita, J. A. Search for Related Content PubMed PubMed Citation Articles by Freedman, J. E. Articles by Vita, J. A.