In Vitro Susceptibility of Wistar Rat Platelets to Hydrogen Peroxide and AAPH-Induced Oxidative Stress

Hydroxyl and peroxyl radicals are biologically active species because of their likelihood to damage cellular constituents. An in vitro study on Wistar rats was conducted to investigate the influence of hydrogen peroxide (H₂O₂) and 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) on platelets and compare the vulnerability of platelets to oxidative stress (OS) induced by these two free radical initiators. Isolated platelets were divided into controls (without free radical initiators; n = 5) and experimentals (with free radical initiators; n = 5). Different concentrations (0.5, 1.0 and 2.0) of free radical initiators H₂O₂ and AAPH were used to treat the platelets and incubated for 5, 15 and 30 min. Biomarkers such as platelet aggregation, superoxide generation, lipid peroxidation (thiobarbituric acid reactive substances, conjugate dienes), protein oxidation (protein carbonyls, sulfhydryls) and antioxidant enzymes were assessed. In H₂O₂ and AAPH treated platelets, though OS was observed at concentrations of 0.5 and 1.0 mM, platelets could tolerate the oxidative insult. Treatment of platelets with 2.0 mM H₂O₂ demonstrated the onset of irreversible changes in platelets as observed in the results of increased superoxide generation and lipid peroxidation products. In 2.0 mM AAPH platelets, the oxidative damage was evident as indicated through increased aggregation, superoxide generation and conjugate dienes and lower protein sulfhydryls. Platelets were more susceptible to AAPH than H₂O₂, as AAPH acted on both lipids and proteins whereas H₂O₂ acted only on lipids. This study gives insight on platelet survival under different OS situations.