Nitric oxide (NO), a small gas molecule, has long been known to be a potent inhibitor of platelet function but the physiological and pathological implications of platelet inhibition by NO have not been well clarified. We recently showed that the addition of nitrite to platelet-rich plasma in the presence of erythrocytes could inhibit platelet aggregation and this inhibitory effect of nitrite + erythrocytes was enhanced by deoxygenation of erythrocytes as measured by P-selectin expression and cGMP production. In order to study the nitrite effect on platelets at different oxygen levels, we used the flow cytometric assays to detect platelet membrane surface markers upon activation. The P-selectin and activated gpIIb/IIIa expression on platelet membranes in response to ADP, collagen and thrombin stimulation was measured at various hematocrit and oxygen levels. Nitrite (0.1 to 1.0 μM) significantly decreased the percentage of these surface markers on the platelet membrane at the hematocrit values above 23% and oxygen levels lower than 49 mmHg. The inhibitory effect of nitrite was augmented by increasing hematocrit values and decreasing oxygen saturation. C-PTIO (an NO scavenger) prevented the platelet inhibition by nitrite + erythrocytes whereas the inhibitors of NO synthase and xanthine oxidoreductase had no effect. These results support the proposal that circulating nitrite decreases platelet reactivity in the presence of partially deoxygenated erythrocytes through its reduction to NO, which may also explain certain differences between arterial and venous thrombosis and support directly the role of deoxyhemoglobin in this process. We believe that our flow cytometric assays offer a possibility to identify the individual molecular process involved in these effects.
References
[1]
Mackman N (2008) Triggers, targets and treatments for thrombosis. Nature 451: 914–918. doi: 10.1038/nature06797
[2]
Offermanns S (2006) Activation of platelet function through G protein-coupled receptors. Circ Res 99: 1293–1304. doi: 10.1161/01.res.0000251742.71301.16
[3]
Broos K, Feys HB, De Meyer SF, Vanhoorelbeke K, Deckmyn H (2011) Platelets at work in primary hemostasis. Blood Rev 25: 155–167. doi: 10.1016/j.blre.2011.03.002
[4]
Naseem KM, Roberts W (2011) Nitric oxide at a glance. Platelets 22: 148–152. doi: 10.3109/09537104.2010.522629
[5]
Azuma H, Ishikawa M, Sekizaki S (1986) Endothelium-dependent inhibition of platelet aggregation. Br J Pharmacol 88: 411–415. doi: 10.1111/j.1476-5381.1986.tb10218.x
[6]
Radomski MW, Palmer RM, Moncada S (1987) Endogenous nitric oxide inhibits human platelet adhesion to vascular endothelium. Lancet 2: 1057–1058. doi: 10.1016/s0140-6736(87)91481-4
[7]
Govoni M, Jansson EA, Weitzberg E, Lundberg JO (2008) The increase in plasma nitrite after a dietary nitrate load is markedly attenuated by an antibacterial mouthwash. Nitric Oxide 19: 333–337. doi: 10.1016/j.niox.2008.08.003
[8]
Cosby K, Partovi KS, Crawford JH, Patel RP, Reiter CD, et al. (2003) Nitrite reduction to nitric oxide by deoxyhemoglobin vasodilates the human circulation. Nat Med 9: 1498–1505. doi: 10.1038/nm954
[9]
Nagababu E, Ramasamy S, Abernethy DR, Rifkind JM (2003) Active nitric oxide produced in the red cell under hypoxic conditions by deoxyhemoglobin-mediated nitrite reduction. J Biol Chem 278: 46349–46356. doi: 10.1074/jbc.m307572200
[10]
Rassaf T, Flogel U, Drexhage C, Hendgen-Cotta U, Kelm M, et al. (2007) Nitrite reductase function of deoxymyoglobin: oxygen sensor and regulator of cardiac energetics and function. Circ Res 100: 1749–1754. doi: 10.1161/circresaha.107.152488
[11]
Zhang Z, Naughton DP, Blake DR, Benjamin N, Stevens CR, et al. (1997) Human xanthine oxidase converts nitrite ions into nitric oxide (NO). Biochem Soc Trans 25: 524S.
[12]
Millar TM, Stevens CR, Benjamin N, Eisenthal R, Harrison R, et al. (1998) Xanthine oxidoreductase catalyses the reduction of nitrates and nitrite to nitric oxide under hypoxic conditions. FEBS Lett 427: 225–228. doi: 10.1016/s0014-5793(98)00430-x
[13]
Benjamin N, O'Driscoll F, Dougall H, Duncan C, Smith L, et al. (1994) Stomach NO synthesis. Nature 368: 502. doi: 10.1038/368502a0
[14]
Lundberg JO, Weitzberg E, Lundberg JM, Alving K (1994) Intragastric nitric oxide production in humans: measurements in expelled air. Gut 35: 1543–1546. doi: 10.1136/gut.35.11.1543
[15]
Carlsson S, Wiklund NP, Engstrand L, Weitzberg E, Lundberg JO (2001) Effects of pH, nitrite, and ascorbic acid on nonenzymatic nitric oxide generation and bacterial growth in urine. Nitric Oxide 5: 580–586. doi: 10.1006/niox.2001.0371
[16]
Bryan NS, Calvert JW, Elrod JW, Gundewar S, Ji SY, et al. (2007) Dietary nitrite supplementation protects against myocardial ischemia-reperfusion injury. Proc Natl Acad Sci U S A 104: 19144–19149. doi: 10.1073/pnas.0706579104
[17]
Machha A, Schechter AN (2011) Dietary nitrite and nitrate: a review of potential mechanisms of cardiovascular benefits. Eur J Nutr 50: 293–303. doi: 10.1007/s00394-011-0192-5
[18]
Srihirun S, Sriwantana T, Unchern S, Kittikool D, Noulsri E, et al. (2012) Platelet inhibition by nitrite is dependent on erythrocytes and deoxygenation. PLoS One 7: e30380. doi: 10.1371/journal.pone.0030380
[19]
Velmurugan S, Kapil V, Ghosh SM, Davies S, McKnight A, et al. (2013) Antiplatelet effects of dietary nitrate in healthy volunteers: Involvement of cGMP and influence of sex. Free Radic Biol Med. Jun 24. doi:10.1016/j.freeradbiomed.2013.06.031. [Epub ahead of print].
Shattil SJ, Hoxie JA, Cunningham M, Brass LF (1985) Changes in the platelet membrane glycoprotein IIb.IIIa complex during platelet activation. J Biol Chem 260: 11107–11114.
[22]
Alheid U, Frolich JC, Forstermann U (1987) Endothelium-derived relaxing factor from cultured human endothelial cells inhibits aggregation of human platelets. Thromb Res 47: 561–571. doi: 10.1016/0049-3848(87)90361-6
[23]
Bult H, Fret HR, Van den Bossche RM, Herman AG (1988) Platelet inhibition by endothelium-derived relaxing factor from the rabbit perfused aorta. Br J Pharmacol 95: 1308–1314. doi: 10.1111/j.1476-5381.1988.tb11769.x
[24]
Lundberg JO, Weitzberg E, Gladwin MT (2008) The nitrate-nitrite-nitric oxide pathway in physiology and therapeutics. Nat Rev Drug Discov 7: 156–167. doi: 10.1038/nrd2466
[25]
Laustiola KE, Vuorinen P, Porsti I, Metsa-Ketela T, Manninen V, et al. (1991) Exogenous GTP enhances the effects of sodium nitrite on cyclic GMP accumulation, vascular smooth muscle relaxation and platelet aggregation. Pharmacol Toxicol 68: 60–63. doi: 10.1111/j.1600-0773.1991.tb01209.x
[26]
Keszler A, Piknova B, Schechter AN, Hogg N (2008) The reaction between nitrite and oxyhemoglobin: a mechanistic study. J Biol Chem 283: 9615–9622. doi: 10.1074/jbc.m705630200
[27]
Sibmooh N, Piknova B, Rizzatti F, Schechter AN (2008) Oxidation of iron-nitrosyl-hemoglobin by dehydroascorbic acid releases nitric oxide to form nitrite in human erythrocytes. Biochemistry 47: 2989–2996. doi: 10.1021/bi702158d
[28]
Richardson G, Hicks SL, O'Byrne S, Frost MT, Moore K, et al. (2002) The ingestion of inorganic nitrate increases gastric S-nitrosothiol levels and inhibits platelet function in humans. Nitric Oxide 7: 24–29. doi: 10.1016/s1089-8603(02)00010-1
[29]
Webb AJ, Patel N, Loukogeorgakis S, Okorie M, Aboud Z, et al. (2008) Acute blood pressure lowering, vasoprotective, and antiplatelet properties of dietary nitrate via bioconversion to nitrite. Hypertension 51: 784–790. doi: 10.1161/hypertensionaha.107.103523
[30]
Park JW, Piknova B, Huang PL, Noguchi CT, Schechter AN (2013) Effect of blood nitrite and nitrate levels on murine platelet function. PLoS One 8: e55699. doi: 10.1371/journal.pone.0055699
[31]
Becattini C, Agnelli G, Schenone A, Eichinger S, Bucherini E, et al. (2012) Aspirin for preventing the recurrence of venous thromboembolism. N Engl J Med 366: 1959–1967. doi: 10.1056/nejmoa1114238
[32]
Becker RC (2012) Aspirin and the prevention of venous thromboembolism. N Engl J Med 366: 2028–2030. doi: 10.1056/nejme1201911
