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. 1996 Feb 15;97(4):979–987. doi: 10.1172/JCI118522

Decreased platelet inhibition by nitric oxide in two brothers with a history of arterial thrombosis.

J E Freedman 1, J Loscalzo 1, S E Benoit 1, C R Valeri 1, M R Barnard 1, A D Michelson 1
PMCID: PMC507144  PMID: 8613552

Abstract

Highly reactive oxygen species rapidly inactivate nitric oxide (NO), and endothelial product which inhibits platelet activation. We studied platelet inhibition by NO in two brothers with a cerebral thrombotic disorder. Both children had hyperreactive platelets, as determined by whole blood platelet aggregometry and flow cytometric analysis of the platelet surface expression of P-selectin. Mixing experiments showed that the patients'platelets behaved normally in control plasma; however, control platelets suspended in patient plasma were not inhibited by NO. As determined by flow cytometry, in the presence of plasma from either patient there was normal inhibition of the thrombin-induced expression of platelet surface P-selectin by prostacyclin, but not NO. Using a scopoletin assay, we measured a 2.7-fold increase in plasma H2O2 generation in one patient and a 3.4-fold increase in the second patient, both compared woth control plasma. Glutathione peroxidase (GSH-Px) activity was decreased in the patients' plasmas compared with control plasma. The addition of exogenous GSH-Px led to restoration of platelet inhibition by NO. These data show that, in these patients' plasmas, impaired metabolism of reactive oxygen species reduces the bioavailability of NO and impairs normal platelet inhibitory mechanisms. These findings suggest that attenuated NO-mediated platelet inhibition produced by increased reactive oxygen species or impaired antioxidant defense may cause a thrombotic disorder in humans.

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Selected References

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  1. Avissar N., Ornt D. B., Yagil Y., Horowitz S., Watkins R. H., Kerl E. A., Takahashi K., Palmer I. S., Cohen H. J. Human kidney proximal tubules are the main source of plasma glutathione peroxidase. Am J Physiol. 1994 Feb;266(2 Pt 1):C367–C375. doi: 10.1152/ajpcell.1994.266.2.C367. [DOI] [PubMed] [Google Scholar]
  2. Avissar N., Whitin J. C., Allen P. Z., Wagner D. D., Liegey P., Cohen H. J. Plasma selenium-dependent glutathione peroxidase. Cell of origin and secretion. J Biol Chem. 1989 Sep 25;264(27):15850–15855. [PubMed] [Google Scholar]
  3. 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]
  4. Bevilacqua M., Butcher E., Furie B., Furie B., Gallatin M., Gimbrone M., Harlan J., Kishimoto K., Lasky L., McEver R. Selectins: a family of adhesion receptors. Cell. 1991 Oct 18;67(2):233–233. doi: 10.1016/0092-8674(91)90174-w. [DOI] [PubMed] [Google Scholar]
  5. Buczyński A., Wachowicz B., Kedziora-Kornatowska K., Tkaczewski W., Kedziora J. Changes in antioxidant enzymes activities, aggregability and malonyldialdehyde concentration in blood platelets from patients with coronary heart disease. Atherosclerosis. 1993 May;100(2):223–228. doi: 10.1016/0021-9150(93)90208-c. [DOI] [PubMed] [Google Scholar]
  6. Coller B. S., Peerschke E. I., Scudder L. E., Sullivan C. A. Studies with a murine monoclonal antibody that abolishes ristocetin-induced binding of von Willebrand factor to platelets: additional evidence in support of GPIb as a platelet receptor for von Willebrand factor. Blood. 1983 Jan;61(1):99–110. [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. Del Principe D., Menichelli A., De Matteis W., Di Giulio S., Giordani M., Savini I., Agro A. F. Hydrogen peroxide is an intermediate in the platelet activation cascade triggered by collagen, but not by thrombin. Thromb Res. 1991 Jun 1;62(5):365–375. doi: 10.1016/0049-3848(91)90010-t. [DOI] [PubMed] [Google Scholar]
  9. Dougherty J. H., Jr, Levy D. E., Weksler B. B. Platelet activation in acute cerebral ischaemia. Serial measurements of platelet function in cerebrovascular disease. Lancet. 1977 Apr 16;1(8016):821–824. doi: 10.1016/s0140-6736(77)92774-x. [DOI] [PubMed] [Google Scholar]
  10. Freedman J. E., Frei B., Welch G. N., Loscalzo J. Glutathione peroxidase potentiates the inhibition of platelet function by S-nitrosothiols. J Clin Invest. 1995 Jul;96(1):394–400. doi: 10.1172/JCI118047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Frei B., England L., Ames B. N. Ascorbate is an outstanding antioxidant in human blood plasma. Proc Natl Acad Sci U S A. 1989 Aug;86(16):6377–6381. doi: 10.1073/pnas.86.16.6377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gatter K. C., Cordell J. L., Turley H., Heryet A., Kieffer N., Anstee D. J., Mason D. Y. The immunohistological detection of platelets, megakaryocytes and thrombi in routinely processed specimens. Histopathology. 1988 Sep;13(3):257–267. doi: 10.1111/j.1365-2559.1988.tb02037.x. [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. Hsu-Lin S., Berman C. L., Furie B. C., August D., Furie B. A platelet membrane protein expressed during platelet activation and secretion. Studies using a monoclonal antibody specific for thrombin-activated platelets. J Biol Chem. 1984 Jul 25;259(14):9121–9126. [PubMed] [Google Scholar]
  15. Huie R. E., Padmaja S. The reaction of no with superoxide. Free Radic Res Commun. 1993;18(4):195–199. doi: 10.3109/10715769309145868. [DOI] [PubMed] [Google Scholar]
  16. Ignarro L. J., Byrns R. E., Buga G. M., Wood K. S. Endothelium-derived relaxing factor from pulmonary artery and vein possesses pharmacologic and chemical properties identical to those of nitric oxide radical. Circ Res. 1987 Dec;61(6):866–879. doi: 10.1161/01.res.61.6.866. [DOI] [PubMed] [Google Scholar]
  17. Ioannidis I., de Groot H. Cytotoxicity of nitric oxide in Fu5 rat hepatoma cells: evidence for co-operative action with hydrogen peroxide. Biochem J. 1993 Dec 1;296(Pt 2):341–345. doi: 10.1042/bj2960341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Keaney J. F., Jr, Puyana J. C., Francis S., Loscalzo J. F., Stamler J. S., Loscalzo J. Methylene blue reverses endotoxin-induced hypotension. Circ Res. 1994 Jun;74(6):1121–1125. doi: 10.1161/01.res.74.6.1121. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Leoncini G., Aprile B., Maresca M. p-Bromophenacyl bromide potentiates hydrogen peroxide formation in human platelets challenged by thrombin and inhibits aggregation. Biochem Mol Biol Int. 1993 Feb;29(2):317–325. [PubMed] [Google Scholar]
  21. Levine P. H., Weinger R. S., Simon J., Scoon K. L., Krinsky N. I. Leukocyte-platelet interaction. Release of hydrogen peroxide by granulocytes as a modulator of platelet reactions. J Clin Invest. 1976 Apr;57(4):955–963. doi: 10.1172/JCI108372. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Loscalzo J. N-Acetylcysteine potentiates inhibition of platelet aggregation by nitroglycerin. J Clin Invest. 1985 Aug;76(2):703–708. doi: 10.1172/JCI112024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Maddipati K. R., Marnett L. J. Characterization of the major hydroperoxide-reducing activity of human plasma. Purification and properties of a selenium-dependent glutathione peroxidase. J Biol Chem. 1987 Dec 25;262(36):17398–17403. [PubMed] [Google Scholar]
  24. Marcus A. J., Safier L. B. Thromboregulation: multicellular modulation of platelet reactivity in hemostasis and thrombosis. FASEB J. 1993 Apr 1;7(6):516–522. doi: 10.1096/fasebj.7.6.8472890. [DOI] [PubMed] [Google Scholar]
  25. Marcus A. J., Silk S. T., Safier L. B., Ullman H. L. Superoxide production and reducing activity in human platelets. J Clin Invest. 1977 Jan;59(1):149–158. doi: 10.1172/JCI108613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. McEver R. P. Properties of GMP-140, an inducible granule membrane protein of platelets and endothelium. Blood Cells. 1990;16(1):73–83. [PubMed] [Google Scholar]
  27. 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]
  28. Mendelsohn M. E., O'Neill S., George D., Loscalzo J. Inhibition of fibrinogen binding to human platelets by S-nitroso-N-acetylcysteine. J Biol Chem. 1990 Nov 5;265(31):19028–19034. [PubMed] [Google Scholar]
  29. Michelson A. D., Barnard M. R. Plasmin-induced redistribution of platelet glycoprotein Ib. Blood. 1990 Nov 15;76(10):2005–2010. [PubMed] [Google Scholar]
  30. Michelson A. D., Ellis P. A., Barnard M. R., Matic G. B., Viles A. F., Kestin A. S. Downregulation of the platelet surface glycoprotein Ib-IX complex in whole blood stimulated by thrombin, adenosine diphosphate, or an in vivo wound. Blood. 1991 Feb 15;77(4):770–779. [PubMed] [Google Scholar]
  31. Michelson A. D., Loscalzo J., Melnick B., Coller B. S., Handin R. I. Partial characterization of a binding site for von Willebrand factor on glycocalicin. Blood. 1986 Jan;67(1):19–26. [PubMed] [Google Scholar]
  32. Michelson A. D. Platelet activation by thrombin can be directly measured in whole blood through the use of the peptide GPRP and flow cytometry: methods and clinical applications. Blood Coagul Fibrinolysis. 1994 Feb;5(1):121–131. doi: 10.1097/00001721-199402000-00014. [DOI] [PubMed] [Google Scholar]
  33. Noronha-Dutra A. A., Epperlein M. M., Woolf N. Reaction of nitric oxide with hydrogen peroxide to produce potentially cytotoxic singlet oxygen as a model for nitric oxide-mediated killing. FEBS Lett. 1993 Apr 19;321(1):59–62. doi: 10.1016/0014-5793(93)80621-z. [DOI] [PubMed] [Google Scholar]
  34. Pai T. G., Payne W. J., LeGall J. Use of a chemiluminescence detector for quantitation of nitric oxide produced in assays of denitrifying enzymes. Anal Biochem. 1987 Oct;166(1):150–157. doi: 10.1016/0003-2697(87)90557-4. [DOI] [PubMed] [Google Scholar]
  35. Pascual P., Martinez-Lara E., Bárcena J. A., López-Barea J., Toribio F. Direct assay of glutathione peroxidase activity using high-performance capillary electrophoresis. J Chromatogr. 1992 Oct 2;581(1):49–56. doi: 10.1016/0378-4347(92)80446-w. [DOI] [PubMed] [Google Scholar]
  36. Portal B. C., Richard M. J., Faure H. S., Hadjian A. J., Favier A. E. Altered antioxidant status and increased lipid peroxidation in children with cystic fibrosis. Am J Clin Nutr. 1995 Apr;61(4):843–847. doi: 10.1093/ajcn/61.4.843. [DOI] [PubMed] [Google Scholar]
  37. Radi R., Beckman J. S., Bush K. M., Freeman B. A. Peroxynitrite oxidation of sulfhydryls. The cytotoxic potential of superoxide and nitric oxide. J Biol Chem. 1991 Mar 5;266(7):4244–4250. [PubMed] [Google Scholar]
  38. Radomski M. W., Palmer R. M., Moncada S. Comparative pharmacology of endothelium-derived relaxing factor, nitric oxide and prostacyclin in platelets. Br J Pharmacol. 1987 Sep;92(1):181–187. doi: 10.1111/j.1476-5381.1987.tb11310.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Radomski M. W., Palmer R. M., Moncada S. The role of nitric oxide and cGMP in platelet adhesion to vascular endothelium. Biochem Biophys Res Commun. 1987 Nov 13;148(3):1482–1489. doi: 10.1016/s0006-291x(87)80299-1. [DOI] [PubMed] [Google Scholar]
  40. Riela A. R., Roach E. S. Etiology of stroke in children. J Child Neurol. 1993 Jul;8(3):201–220. doi: 10.1177/088307389300800302. [DOI] [PubMed] [Google Scholar]
  41. Rodvein R., Lindon J. N., Levine P. H. Physiology and ultrastructure of the blood platelet following exposure to hydrogen peroxide. Br J Haematol. 1976 May;33(1):19–26. doi: 10.1111/j.1365-2141.1976.tb00968.x. [DOI] [PubMed] [Google Scholar]
  42. Root R. K., Metcalf J., Oshino N., Chance B. H2O2 release from human granulocytes during phagocytosis. I. Documentation, quantitation, and some regulating factors. J Clin Invest. 1975 May;55(5):945–955. doi: 10.1172/JCI108024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Rubbo H., Radi R., Trujillo M., Telleri R., Kalyanaraman B., Barnes S., Kirk M., Freeman B. A. Nitric oxide regulation of superoxide and peroxynitrite-dependent lipid peroxidation. Formation of novel nitrogen-containing oxidized lipid derivatives. J Biol Chem. 1994 Oct 21;269(42):26066–26075. [PubMed] [Google Scholar]
  44. Schlossman S. F., Boumsell L., Gilks W., Harlan J. M., Kishimoto T., Morimoto C., Ritz J., Shaw S., Silverstein R. L., Springer T. A. CD antigens 1993. Blood. 1994 Feb 15;83(4):879–880. [PubMed] [Google Scholar]
  45. Shatos M. A., Doherty J. M., Hoak J. C. Alterations in human vascular endothelial cell function by oxygen free radicals. Platelet adherence and prostacyclin release. Arterioscler Thromb. 1991 May-Jun;11(3):594–601. doi: 10.1161/01.atv.11.3.594. [DOI] [PubMed] [Google Scholar]
  46. Shattil S. J., Cunningham M., Hoxie J. A. Detection of activated platelets in whole blood using activation-dependent monoclonal antibodies and flow cytometry. Blood. 1987 Jul;70(1):307–315. [PubMed] [Google Scholar]
  47. Shattil S. J., Hoxie J. A., Cunningham M., Brass L. F. Changes in the platelet membrane glycoprotein IIb.IIIa complex during platelet activation. J Biol Chem. 1985 Sep 15;260(20):11107–11114. [PubMed] [Google Scholar]
  48. 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]
  49. 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]
  50. Smith A. M., Chen L. W., Thomas M. R. Selenate fortification improves selenium status of term infants fed soy formula. Am J Clin Nutr. 1995 Jan;61(1):44–47. doi: 10.1093/ajcn/61.1.44. [DOI] [PubMed] [Google Scholar]
  51. Stamler J. S., Jaraki O., Osborne J., Simon D. I., Keaney J., Vita J., Singel D., Valeri C. R., Loscalzo J. Nitric oxide circulates in mammalian plasma primarily as an S-nitroso adduct of serum albumin. Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7674–7677. doi: 10.1073/pnas.89.16.7674. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Stamler J. S., Osborne J. A., Jaraki O., Rabbani L. E., Mullins M., Singel D., Loscalzo J. Adverse vascular effects of homocysteine are modulated by endothelium-derived relaxing factor and related oxides of nitrogen. J Clin Invest. 1993 Jan;91(1):308–318. doi: 10.1172/JCI116187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Stamler J. S., Vaughan D. E., Loscalzo J. Synergistic disaggregation of platelets by tissue-type plasminogen activator, prostaglandin E1, and nitroglycerin. Circ Res. 1989 Sep;65(3):796–804. doi: 10.1161/01.res.65.3.796. [DOI] [PubMed] [Google Scholar]
  54. 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]
  55. Starkebaum G., Harlan J. M. Endothelial cell injury due to copper-catalyzed hydrogen peroxide generation from homocysteine. J Clin Invest. 1986 Apr;77(4):1370–1376. doi: 10.1172/JCI112442. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Stenberg P. E., McEver R. P., Shuman M. A., Jacques Y. V., Bainton D. F. A platelet alpha-granule membrane protein (GMP-140) is expressed on the plasma membrane after activation. J Cell Biol. 1985 Sep;101(3):880–886. doi: 10.1083/jcb.101.3.880. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Stocker R., Bowry V. W., Frei B. Ubiquinol-10 protects human low density lipoprotein more efficiently against lipid peroxidation than does alpha-tocopherol. Proc Natl Acad Sci U S A. 1991 Mar 1;88(5):1646–1650. doi: 10.1073/pnas.88.5.1646. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Violi F., Ghiselli A., Iuliano L., Alessandri C., Cordova C., Balsano F. Influence of hydroxyl radical scavengers on platelet function. Haemostasis. 1988;18(2):91–98. doi: 10.1159/000215788. [DOI] [PubMed] [Google Scholar]
  59. Yamamoto N., Greco N. J., Barnard M. R., Tanoue K., Yamazaki H., Jamieson G. A., Michelson A. D. Glycoprotein Ib (GPIb)-dependent and GPIb-independent pathways of thrombin-induced platelet activation. Blood. 1991 Apr 15;77(8):1740–1748. [PubMed] [Google Scholar]
  60. 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]

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