Akut Hipergliseminin Trombositlerde Oksidatif Strese ve Nitrik Oksit Biyoyaralanımına Etkisi

Year 2015, Volume: 5 Issue: 4, 239 - 246, 29.12.2015

Azize Sener Nazli Gul Altındıs Ozge Dogan

https://doi.org/10.5455/musbed.20150715121543

Abstract

Amaç: Diyabette, trombositlerin aktivasyona ve trombus oluşumuna eğilimi artmaktadır. Nitrik oksit (NO), özellikle trombositten zengin trom¬buslara yeni trombositlerin katılmasını sınırlandırarak trombosit fonksi¬yonlarında önemli rol oynar. Ancak, NO hücrelerde oksidatif/nitrozatif stresi artırarak zararlı etki de gösterebilir. Bu çalışmada, istirahat halinde ve kollajenle uyarılmış trombositlerde akut yüksek glikozun trombosit agregasyonuna, oksidatif stres parametrelerine ve NO biyoyararlanımına etkileri araştırılmıştır. Ayrıca, kuersetin (süperoksit anyon süpürücüsü) kullanılarak yüksek glikozun NO biyoyararlanımı üzerindeki etkilerinde süperoksit üretiminin rolü incelenmiştir.
Yöntemler: Yıkanmış trombositler 5 mM D-glukoz (fizyolojik konsant¬rasyon, n:7) veya 25 mM D-glukoz (patofizyolojik konsantrasyon, n:7) veya kuersetin (10 μM) + 25 mM D-glukoz (n:7) ile 1 saat inkübe edildi. İnkübasyon sonrası, trombositlerde süperoksit üretimi, lipid peroksidas¬yonu (LPO), NO, nitrotirozin (NT) düzeyleri ve ilerlemiş glikasyon son ürünleri (AGE) ölçüldü. Ayrıca trombosit agregasyonu da incelendi.
Bulgular: Dinlenme halinde ve kollajenle aktive trombositlerin yüksek glukoz ile inkübasyonu LPO, NT ve AGE düzeylerinde 5mM glukoz konsantrasyonuna göre istatistiksel olarak anlamlı artışlara neden oldu (p<0.05). Yüksek glukoz kollajenle aktive trombositlerde trombosit agregasyonunu, süperoksit oluşumunu ve NO düzeylerini anlamlı olarak artırdı (p<0.05). Yüksek glukoz ile inkübe edilen trombositlerde, kuer¬setin anlamlı olarak NO üretiminin ve NO biyoyararlanımının artmasına neden olurken oksidatif stresi baskıladı (p<0.05).
Sonuç: Yüksek glukoz istirahat halinde ve özellikle kollajen ile aktive trombositlerde oksidatif stresi arttırır ve NO biyoyararlanımını azaltır. Yüksek glukoz aracılı süperoksit üretiminin bu etkiler üzerinde rolü olabi¬lir. Bulgularımız, diyabette trombosit-ilişkili komplikasyonların önlenme¬sinde kuersetinin yararlı etkisi olabileceğini göstermektedir.

Keywords

Hiperglisemi, kuersetin, kollajen, nitrik oksit, superoksit anyonu, trombosit

The effects of acute hyperglycemia on oxidative stress and nitric oxide bioavailability in platelets

Abstract

Objective: Platelet activation and thrombus formation tendency increase in diabetes mellitus. Nitric oxide (NO) synthesized by platelets plays important role in platelet functions and limits the recruitment of new platelets to the platelet-rich thrombus. However, NO may also be deleterious by elevating oxidative/nitrosative stress in cells. The aim of the present study was to examine effects of acute high glucose in both resting and collagen-stimulated platelets on platelet aggregation, oxidative stress and NO bioavailability. In addition, we investigated the role of superoxide production on NO bioavailability using quercetin as scavengers of superoxide anion.

Methods: Washed platelets were incubated with 5mM D-glucose (physiological concentration, n:7) or 25mM D-glucose (pathophysiological concentration, n:7) or quercetin (10 μM) plus 25 mM D-glucose (n:7) for 1 h. Superoxide production, lipid peroxidation (LPO), NO, nitrotyrosine (NT) levels and advanced glycation end products (AGE) in platelets were measured after incubation. Platelet aggregation was also investigated.

Results: Incubation of resting and collagen-activated platelets with high glucose resulted in significant elevations in LPO, NT and AGE levels when compared to 5mM glucose concentration (p<0.05). High glucose significantly increased platelet aggregation, superoxide formation and NO levels in collagen-activated platelets (p<0.05). Quercetin markedly increased production and bioavailability of NO and suppressed oxidative stress in high glucose incubated platelets (p<0.05).

Conclusion: High glucose increases oxidative stress and reduces bioavailability of NO in resting and especially collagen activated platelets. Hyperglycemia mediated superoxide production could be taken part on these effects. These findings would be suggested that there might be beneficial effects of quercetin in the prevention of platelet-related complications in diabetes mellitus.

Keywords

Hyperglycemia, quercetin, collagen, nitric oxide, superoxide anion

References

• 1. Coccheri S. Approaches to prevention of cardiovascular complications and events in diabetes mellitus. Drugs. 2007; 67(7): 997-1026. 2. Krotz F, Sohn HY and Pohl U. Reactive oxygen species: players in the platelet game. Arterioscler Thromb Vasc Biol. 2004; 24(11): 1988-1996.
• 3. Nathan C. Nitric oxide as a secretory product of mammalian cells. FASEB J. 1992; 6(12): 3051-3064.
• 4. 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; 25(3): 370-377.
• 5. Gkaliagkousi E, Ritter J and Ferro A. Platelet-derived nitric oxide signaling and regulation. Circ Res. 2007; 101(7): 654-662.
• 6. Zou MH, Shi C, Cohen RA. High glucose via peroxynitrite causes tyrosine nitration and inactivation of prostacyclin synthase that is associated with thromboxane/prostaglandin H2 receptor–mediated apoptosis and adhesion molecule expression in cultured human aortic endothelial cells. Diabetes. 2002; 51(1): 198-203. 7. Pacher P, Beckman JS, Liaudet L. Nitric oxide and peroxynitrite in health and disease. Physiol Rev. 2007; 87(1): 315-424.
• 8. Yamagishi SI, Edelstein D, Du, XL Brownlee M. Hyperglycemia potentiates collagen-induced platelet activation through mitochondrial superoxide overproduction. Diabetes. 2001; 50(6): 1491-1494.
• 9. Sener A, Egemen G, Cevik O, Yanikkaya-Demirel G, Apikoglu-Rabus S, Ozsavci D. In vitro effects of nitric oxide donors on apoptosis and oxidative/nitrative protein modifications in ADP-activated platelets. Hum Exp Toxicol. 2013; 32(3): 225-235.
• 10. Mosawy S, Effect of the flavonol quercetin on human platelet function: A review. Food and Public Health 2015; 5(1): 1-9.
• 11. Giardino I, Edelstein D, Brownlee M. Bcl-2 expression or antioxidants prevent hyperglycemia-induced formation of intracellular advanced glycation end products in bovine endothelial cells. J Clin Invest. 1996; 97(6): 1422-1428. 12. Amorini AM, Tuttobene M, Tomasello FM, Biazzo F, Gullotta S, De Pinto V, Lazzarino G, Tavazzi B. Glucose ameliorates the metabolic profile and mitochondrial function of platelet concentrates during storage in autologous plasma. Blood Transfus. 2013; 11(1): 61-70.
• 13. Fratantoni JC, Poindexter BJ, Measuring platelet aggregation with microplate reader. A new technical approach to platelet aggregation studies. Am J Clin Pathol. 1990; 94(5): 613-617.
• 14. Gyllenhammar H. Lucigenin chemiluminescence in the assessment of neutrophil superoxide production. J Immunol Methods. 1987; 97: 209-213.
• 15. Beuge JA, Aust SD. Microsomal lipid peroxidation. Methods Enzymol. 1978; 52: 302-311.
• 16. Bradford MM. A rapid and sensitive method for quantititation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem. 1976; 7: 248-254.
• 17. Robak J, Gryglewski RJ. Flavonoids are scavengers of superoxide anions. Biochem Pharmacol. 1988; 37(5): 837-841.
• 18. Tang WH, Stitham J, Gleim S, Di Febbo C, Porreca E, Fava C, Tacconelli S, Capone M, Evangelista V, Levantesi G, Wen L, Martin K, Minuz P, Rade J, Patrignani P, Hwa J. Glucose and collagen regulate human platelet activity through aldose reductase induction of thromboxane. J Clin Invest. 2011; 121(11): 4462-4476. 19. Hubbard GP, Stevens JM, Cicmil M, Sage T, Jordan PA, Williams CM, Lovegrove JA, Gibbins JM. Quercetin inhibits collagen-stimulated platelet activation through inhibition of multiple components of the glycoprotein VI signaling pathway. J Thromb Haemost. 2003; 1(5): 1079-1088.
• 20. Sener A, Ozsavci D, Oba R, Demirel GY, Uras F, Yardimci KT. Do platelet apoptosis, activation, aggregation, lipid peroxidation and platelet-leukocyte aggregate formation occur simultaneously in hyperlipidemia? Clin Biochem. 2005; 38(12): 1081-1087.
• 21. Pignatelli P, Di Santo S, Buchetti B, Sanguigni V, Brunelli A, Violi F. Polyphenols enhance platelet nitric oxide by inhibiting protein kinase C-dependent NADPH oxidase activation: effect on platelet recruitment. FASEB J. 2006; 20(8): 1082-1089.
• 22. Mosawy S, Jackson DE, Woodman OL, Linden MD. The flavonols quercetin and 3’,4’-dihydroxyflavonol reduce platelet function and delay thrombus formation in a model of type 1 diabetes. Diab Vasc Dis Res. 2014; 11(3): 174-181.
• 23. Koya D, King GL. Protein kinase C activation and the development of diabetic complications. Diabetes. 1998; 47(6): 859-866.
• 24. Way KJ, Katai N, King GL. Protein kinase C and the development of diabetic vascular complications. Diabetic Medicine. 2001; 18(12): 945-959. 25. Chappey O, Dosquet C, Wautier MP, Wautier JL. Advanced glycation end products, oxidant stress and vascular lesions. Eur J Clin Invest. 1997; 27(2): 97-108.
• 26. Harris CS. Inhibition of advanced glycation end product formation by medicinal plant extracts correlates with phenolic metabolites and antioxidant activity. Planta Med. 2011; 77(2): 196-204.
• 27. Brownlee M. The pathobiology of diabetic complications aunifying mechanism. Diabetes. 2005; 54(6): 1615-1625. 28. Quijano C, Castro L, Peluffo G, Valez V, Radi R. Enhanced mitochondrial superoxide in hyperglycemic endothelial cells: direct measurements and formation of hydrogen peroxide and peroxynitrite. Am J Physiol Heart Circ Physiol. 2007; 293(6): H3404-3414. 29. Freedman J, Ting B, Hankin B, Loscalzo J, Keaney JF, Vita JA. Impaired platelet production of nitric oxide predicts presence of acute coronary syndromes. Circulation. 1998; 98(15): 1481-146.
• 30. Martina V, Bruno GA, Trucco F, Zumpano E, Tagliabue M, Di Bisceglie C, Pescarmona G. Platelet cNOS activity is reduced in patients with IDDM and NIDDM. Thromb Haemost. 1998; 79(3): 520-522.
• 31. Mutus B, Rabini RA, Staffolani R, Ricciotti R, Fumelli P. Homocysteine-induced inhibition of nitric oxide production in platelets: a study on healthy and diabetic subjects. Diabetologia. 2001; 44(8): 979-982.
• 32. Massucco P, Mattiello L, Russo I, Traversa M, Doronzo G, Anfossi G, Trovati M. High glucose rapidly activates the nitric oxide/cyclic nucleotide pathway in human platelets via an osmotic mechanism. Thromb Haemost. 2005; 93(3): 517-526.