Skip to main content
PMC home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1997 Jul 15;100(2):350–356. doi: 10.1172/JCI119540

Nitric oxide released from activated platelets inhibits platelet recruitment.

J E Freedman 1, J Loscalzo 1, M R Barnard 1, C Alpert 1, J F Keaney 1, A D Michelson 1
PMCID: PMC508197  PMID: 9218511

Abstract

Vessel injury and thrombus formation are the cause of most ischemic coronary syndromes and, in this setting, activated platelets stimulate platelet recruitment to the growing thrombus. Recently, a constitutive nitric oxide synthase (NOS) has been identified in human platelets. To further define the capacity of platelets to produce nitric oxide (NO), as well as to study the role of this NO in platelet recruitment, we adapted a NO-selective microelectrode for use in a standard platelet aggregometer, thereby permitting simultaneous measurement of platelet aggregation and NO production. Treatment of platelets with the NO synthase inhibitor -NG-nitroarginine methyl ester (L-NAME), reduced NO production by 92+/-8% in response to 5 microM ADP compared to control but increased aggregation by only 15+/-2%. In contrast, L-NAME had a more pronounced effect on platelet recruitment as evidenced by a 35+/-5% increase in the extent of aggregation, a 33+/-3% decrease in cyclic GMP content, and a 31+/-5% increase in serotonin release from a second recruitable population of platelets added to stimulated platelets at the peak of NO production. To study platelet recruitment accurately, we developed an assay that monitors two platelet populations simultaneously. Nonbiotinylated platelets were incubated with L-NAME or vehicle and activated with ADP. At peak NO production, biotinylated platelets were added. As measured by three-color flow cytometry, there was a 56+/-11% increase in the number of P selectin- positive platelets in the nonbiotinylated population treated with L-NAME as compared to control. When biotinylated platelets were added to the L-NAME-treated nonbiotinylated population, the number of P selectin positive biotinylated plate-lets increased by 180+/-32% as compared to biotinylated platelets added to the control. In summary, stimulated platelets produce NO that modestly inhibits platelet activation but markedly inhibits additional platelet recruitment. These data suggest that platelet-derived NO may regulate platelet recruitment to a growing thrombus.

Full Text

The Full Text of this article is available as a PDF (241.6 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adams M. R., Forsyth C. J., Jessup W., Robinson J., Celermajer D. S. Oral L-arginine inhibits platelet aggregation but does not enhance endothelium-dependent dilation in healthy young men. J Am Coll Cardiol. 1995 Oct;26(4):1054–1061. doi: 10.1016/0735-1097(95)00257-9. [DOI] [PubMed] [Google Scholar]
  2. BORN G. V., CROSS M. J. THE AGGREGATION OF BLOOD PLATELETS. J Physiol. 1963 Aug;168:178–195. doi: 10.1113/jphysiol.1963.sp007185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bodzenta-Lukaszyk A., Gabryelewicz A., Lukaszyk A., Bielawiec M., Konturek J. W., Domschke W. Nitric oxide synthase inhibition and platelet function. Thromb Res. 1994 Sep 15;75(6):667–672. doi: 10.1016/0049-3848(94)90180-5. [DOI] [PubMed] [Google Scholar]
  4. Broekman M. J., Eiroa A. M., Marcus A. J. Inhibition of human platelet reactivity by endothelium-derived relaxing factor from human umbilical vein endothelial cells in suspension: blockade of aggregation and secretion by an aspirin-insensitive mechanism. Blood. 1991 Aug 15;78(4):1033–1040. [PubMed] [Google Scholar]
  5. Celi A., Pellegrini G., Lorenzet R., De Blasi A., Ready N., Furie B. C., Furie B. P-selectin induces the expression of tissue factor on monocytes. Proc Natl Acad Sci U S A. 1994 Sep 13;91(19):8767–8771. doi: 10.1073/pnas.91.19.8767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chen L. Y., Mehta J. L. 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 Jan;276(1):253–257. [PubMed] [Google Scholar]
  7. Cooke J. P., Stamler J., Andon N., Davies P. F., McKinley G., Loscalzo J. Flow stimulates endothelial cells to release a nitrovasodilator that is potentiated by reduced thiol. Am J Physiol. 1990 Sep;259(3 Pt 2):H804–H812. doi: 10.1152/ajpheart.1990.259.3.H804. [DOI] [PubMed] [Google Scholar]
  8. Davies M. J., Thomas A. Thrombosis and acute coronary-artery lesions in sudden cardiac ischemic death. N Engl J Med. 1984 May 3;310(18):1137–1140. doi: 10.1056/NEJM198405033101801. [DOI] [PubMed] [Google Scholar]
  9. DeWood M. A., Spores J., Notske R., Mouser L. T., Burroughs R., Golden M. S., Lang H. T. Prevalence of total coronary occlusion during the early hours of transmural myocardial infarction. N Engl J Med. 1980 Oct 16;303(16):897–902. doi: 10.1056/NEJM198010163031601. [DOI] [PubMed] [Google Scholar]
  10. Falk E. Plaque rupture with severe pre-existing stenosis precipitating coronary thrombosis. Characteristics of coronary atherosclerotic plaques underlying fatal occlusive thrombi. Br Heart J. 1983 Aug;50(2):127–134. doi: 10.1136/hrt.50.2.127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Falk E. Unstable angina with fatal outcome: dynamic coronary thrombosis leading to infarction and/or sudden death. Autopsy evidence of recurrent mural thrombosis with peripheral embolization culminating in total vascular occlusion. Circulation. 1985 Apr;71(4):699–708. doi: 10.1161/01.cir.71.4.699. [DOI] [PubMed] [Google Scholar]
  12. Fitzgerald D. J., Roy L., Catella F., FitzGerald G. A. Platelet activation in unstable coronary disease. N Engl J Med. 1986 Oct 16;315(16):983–989. doi: 10.1056/NEJM198610163151602. [DOI] [PubMed] [Google Scholar]
  13. Hawiger J., Parkinson S., Timmons S. Prostacyclin inhibits mobilisation of fibrinogen-binding sites on human ADP- and thrombin-treated platelets. Nature. 1980 Jan 10;283(5743):195–197. doi: 10.1038/283195a0. [DOI] [PubMed] [Google Scholar]
  14. Holmsen H., Dangelmaier C. A. Measurement of secretion of serotonin. Methods Enzymol. 1989;169:205–210. doi: 10.1016/0076-6879(89)69061-1. [DOI] [PubMed] [Google Scholar]
  15. Kestin A. S., Valeri C. R., Khuri S. F., Loscalzo J., Ellis P. A., MacGregor H., Birjiniuk V., Ouimet H., Pasche B., Nelson M. J. The platelet function defect of cardiopulmonary bypass. Blood. 1993 Jul 1;82(1):107–117. [PubMed] [Google Scholar]
  16. Little W. C., Constantinescu M., Applegate R. J., Kutcher M. A., Burrows M. T., Kahl F. R., Santamore W. P. Can coronary angiography predict the site of a subsequent myocardial infarction in patients with mild-to-moderate coronary artery disease? Circulation. 1988 Nov;78(5 Pt 1):1157–1166. doi: 10.1161/01.cir.78.5.1157. [DOI] [PubMed] [Google Scholar]
  17. Malinski T., Radomski M. W., Taha Z., Moncada S. Direct electrochemical measurement of nitric oxide released from human platelets. Biochem Biophys Res Commun. 1993 Jul 30;194(2):960–965. doi: 10.1006/bbrc.1993.1914. [DOI] [PubMed] [Google Scholar]
  18. Mellion B. T., Ignarro L. J., Ohlstein E. H., Pontecorvo E. G., Hyman A. L., Kadowitz P. J. Evidence for the inhibitory role of guanosine 3', 5'-monophosphate in ADP-induced human platelet aggregation in the presence of nitric oxide and related vasodilators. Blood. 1981 May;57(5):946–955. [PubMed] [Google Scholar]
  19. Michelson A. D., Benoit S. E., Furman M. I., Breckwoldt W. L., Rohrer M. J., Barnard M. R., Loscalzo J. Effects of nitric oxide/EDRF on platelet surface glycoproteins. Am J Physiol. 1996 May;270(5 Pt 2):H1640–H1648. doi: 10.1152/ajpheart.1996.270.5.H1640. [DOI] [PubMed] [Google Scholar]
  20. Palabrica T., Lobb R., Furie B. C., Aronovitz M., Benjamin C., Hsu Y. M., Sajer S. A., Furie B. Leukocyte accumulation promoting fibrin deposition is mediated in vivo by P-selectin on adherent platelets. Nature. 1992 Oct 29;359(6398):848–851. doi: 10.1038/359848a0. [DOI] [PubMed] [Google Scholar]
  21. Radomski M. W., Palmer R. M., Moncada S. An L-arginine/nitric oxide pathway present in human platelets regulates aggregation. Proc Natl Acad Sci U S A. 1990 Jul;87(13):5193–5197. doi: 10.1073/pnas.87.13.5193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Santos M. T., Valles J., Marcus A. J., Safier L. B., Broekman M. J., Islam N., Ullman H. L., Eiroa A. M., Aznar J. Enhancement of platelet reactivity and modulation of eicosanoid production by intact erythrocytes. A new approach to platelet activation and recruitment. J Clin Invest. 1991 Feb;87(2):571–580. doi: 10.1172/JCI115032. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sase K., Michel T. Expression of constitutive endothelial nitric oxide synthase in human blood platelets. Life Sci. 1995;57(22):2049–2055. doi: 10.1016/0024-3205(95)02191-k. [DOI] [PubMed] [Google Scholar]
  24. Shultz P. J., Raij L. Endogenously synthesized nitric oxide prevents endotoxin-induced glomerular thrombosis. J Clin Invest. 1992 Nov;90(5):1718–1725. doi: 10.1172/JCI116045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Simon D. I., Stamler J. S., Loh E., Loscalzo J., Francis S. A., Creager M. A. Effect of nitric oxide synthase inhibition on bleeding time in humans. J Cardiovasc Pharmacol. 1995 Aug;26(2):339–342. doi: 10.1097/00005344-199508000-00022. [DOI] [PubMed] [Google Scholar]
  26. Stamler J., Mendelsohn M. E., Amarante P., Smick D., Andon N., Davies P. F., Cooke J. P., Loscalzo J. N-acetylcysteine potentiates platelet inhibition by endothelium-derived relaxing factor. Circ Res. 1989 Sep;65(3):789–795. doi: 10.1161/01.res.65.3.789. [DOI] [PubMed] [Google Scholar]
  27. Valles J., Santos M. T., Marcus A. J., Safier L. B., Broekman M. J., Islam N., Ullman H. L., Aznar J. Downregulation of human platelet reactivity by neutrophils. Participation of lipoxygenase derivatives and adhesive proteins. J Clin Invest. 1993 Sep;92(3):1357–1365. doi: 10.1172/JCI116709. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Vita J. A., Treasure C. B., Nabel E. G., McLenachan J. M., Fish R. D., Yeung A. C., Vekshtein V. I., Selwyn A. P., Ganz P. Coronary vasomotor response to acetylcholine relates to risk factors for coronary artery disease. Circulation. 1990 Feb;81(2):491–497. doi: 10.1161/01.cir.81.2.491. [DOI] [PubMed] [Google Scholar]
  29. Winn R. K., Liggitt D., Vedder N. B., Paulson J. C., Harlan J. M. Anti-P-selectin monoclonal antibody attenuates reperfusion injury to the rabbit ear. J Clin Invest. 1993 Oct;92(4):2042–2047. doi: 10.1172/JCI116799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Yao S. K., Ober J. C., Krishnaswami A., Ferguson J. J., Anderson H. V., Golino P., Buja L. M., Willerson J. T. Endogenous nitric oxide protects against platelet aggregation and cyclic flow variations in stenosed and endothelium-injured arteries. Circulation. 1992 Oct;86(4):1302–1309. doi: 10.1161/01.cir.86.4.1302. [DOI] [PubMed] [Google Scholar]
  31. Zhou Q., Hellermann G. R., Solomonson L. P. Nitric oxide release from resting human platelets. Thromb Res. 1995 Jan 1;77(1):87–96. doi: 10.1016/0049-3848(95)90868-g. [DOI] [PubMed] [Google Scholar]
  32. de Graaf J. C., Banga J. D., Moncada S., Palmer R. M., de Groot P. G., Sixma J. J. Nitric oxide functions as an inhibitor of platelet adhesion under flow conditions. Circulation. 1992 Jun;85(6):2284–2290. doi: 10.1161/01.cir.85.6.2284. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation

RESOURCES