Superoxide Anion and Hydroxyl Radical Release by Collagen-induced Platelet Aggregation - Role of Arachidonic Acid Metabolism

Summary

Previous study demonstrated that platelets undergoing anoxia-reoxygenation generate superoxide anion (O₂ ⁻) and hydroxyl radical (OH°) which in turn contribute to activate arachidonic acid (AA) metabolism. However it has not been clarified if oxygen free radicals (OFRs) are also generated when platelets are aggregated by common agonists. We used two probes, i.e. lucigenin and salicylic acid (SA), to measure platelet release of O₂ ⁻ and OH°, respectively. Among the agonists used, such as ADP, thrombin and collagen, the release of O₂ ⁻ and OH° was observed mainly when platelets were stimulated with collagen. Such release was inhibited in platelets pre-treated by aspirin suggesting that AA metabolism was the main source of O₂ ⁻ and OH° formation. To further analyze this relationship, O₂ and OH° formation was measured if other oxidant species, namely O₂ ⁻ and OH°, contribute to the during AA-stimulated platelet aggregation (PA); we observed that O ⁻ and OH° release were dependent upon AA concentration. Furthermore, we found that the incubation of platelets with AACOCF3, a potent inhibitor of cytosolic phospholipase A₂, inhibited collagen-induced platelet O⁻ and OH° release. The incubation of platelets with salicylic acid or ascorbic acid, which blunt OH° and O₂ ⁻ respectively, inhibited both collagen-induced platelet aggregation and AA-release. This study demonstrated that collagen-induced platelet aggregation is associated with O₂ ⁻ and OH° formation, which is dependent upon AA release and analyzed if O₂ ⁻ and OH° are released during aggregation induced by metabolism.

Keywords

Platelet aggregation - free radicals - superoxide anion - arachidonic acid metabolism - collagen

• References

• 1 Iuliano L, Colavita AR, Leo R, Praticò D, Violi F. Oxygen free radicals and platelet activation. Free Rad Biol Med 1997; 22: 999-1006.
• 2 Marcus AJ, Silk ST, Safier LB, Ulmann HL. Superoxide production and reducing activity in human platelets. J Clin Invest 1977; 59: 149-58.
• 3 Del Principe D, Menichelli A, De Matteis W, Di Corpo M, Di Giulio S, Finazzi-Agrò A. Hydrogen peroxide has a role in the aggregation of human a platelets. FEBS Lett 1985; 185: 142-6.
• 4 Miller DM, Buettner GR, Aust SD. Transition metal as catalysts of autoxidation reactions. Free Radic Biol Med 1990; 08: 95-108.
• 5 Pignatelli P, Pulcinelli FM, Lenti L, Gazzaniga PP, Violi F. Hydrogen peroxide is involved in collagen-induced platelet activation. Blood 1997; 91 (02) 484-90.
• 6 Iuliano L, Pederson JZ, Praticò D, Rotilio G, Violi F. Role of hydroxyl radicals in the activation of human platelets. Eur J Biochem 1994; 221 (02) 695-704.
• 7 Leo R, Praticò D, Iuliano L, Pulcinelli FM, Ghiselli A, Pignatelli P, Colavita AR, FitzGerald GA, Violi F. Platelet activation by superoxide anion and hydroxyl radicals intrinsically generated by platelets that had undergone anoxia and then reoxygenated. Circulation 1997; 95: 885-891.
• 8 Riendau D, Guay J, Weech PK, Laliberté F, Yergey J, Li C, Desmarais S, Perrier H, Liu S, Nicoll-Griffith D, Street IP. Arachidonyl trifluorometyl ketone, a potent inhibitor of 85-kDa phospholipase A₂, blocks production of arachidonate and 12-hydroxyeicosatetraenoic acid by calcium ionophorechallenged platelets. J Biol Chem 1994; 269: 15619-24.
• 9 Gyllenhammar HJ. Lucigenin chemiluminescence in the assessment of neutrophil superoxide productin. J Immunol Methods 1987; 97: 209-13.
• 10 Ghiselli A, Laurenti O, De Mattia G, Maiani G, Ferro-Luzzi A. Salicylate hydroxylation as an early marker of in vivo oxidative stress in diabetic patients. Free Rad Biol Med 1992; 13: 621-6.
• 11 Rowe GT, Manson NM, Caplan M, Hess ML. Hydrogen peroxide and hydroxyl radical mediation of activated leukocyte depression of cardiac sarcoplasmic reticulum. Participation of the cyclooxygenase pathway. Circ Res 1983; 53: 584-91.
• 12 White CR, Brock TA, Chang LY, Crapo J, Briscoe P, Ku D, Bradley WA, Gianturco SH, Gore J, Freeman BA, Tarpey MM. Superoxide and peroxynitrite in atherosclerosis. Proc Natl Acad Sci 1994; 19: 1044-8.
• 13 Iuliano L, Praticò D, Ghiselli A, Bonavita MS, Violi F. Superoxide dismutase triggers activation of “primed” platelets. Arch Biochem Biophys 1991; 289: 180-3.