Apoptotic markers in human blood platelets treated with peroxynitrite

Platelets are anucleated cells that upon activation by agonists or during storage may develop apoptotic events. The role of peroxynitrite and its reactive intermediates in apoptotic process in blood platelets is unknown. In order to study the appearance of biomarkers of apoptosis in platelets after treatment with peroxynitrite and with thrombin different markers were chosen: annexin V binding (phosphatidylserine exposure), platelet microparticle formation, mitochondrial membrane depolarization, caspase-3 activation and P-selectin expression. In gel-filtrated platelets treated with different concentrations of peroxynitrite (0.01, 0.1, 1.0 mM, 10 minute, 37 degrees C) a significant increase of phosphatidylserine exposure (about 36% at the highest concentration, p < 0.01) and the platelet microparticle formation were observed. Peroxynitrite caused a dose-dependent caspase-3 activation and depolarization of mitochondrial potential. The same apoptotic markers were appeared in thrombin-activated platelets. Dose-dependent tyrosine nitration in platelet proteins caused by peroxynitrite was reduced in the presence of (-)-epicatechin. Moreover, (-)-epicatechin distinctly reduced the level of apoptotic markers. The obtained results indicate that peroxynitrite responsible for oxidative/nitrative stress and changes in platelet function may promote in vitro apoptotic events in human gel-filtrated platelets via intrinsic pathway. Nitration of tyrosine seems to be partly associated with the appearance of apoptotic markers in platelets.