Priming of platelet α(IIb)β₃ by oxidants is associated with tyrosine phosphorylation of β₃

Reactive oxygen species play an important role at the site of vascular injuries and arterial thromboses. We studied the mechanism mediating platelet aggregation induced by H₂O₂, a major cellular oxidant. Exposure to H₂O₂ triggered platelet aggregation, but only when the platelets were stirred. Strong platelet aggregation induced by H₂O₂ required the presence of the tyrosine phosphatase inhibitor sodium orthovanadate (NaVO₄) and was dependent on the participation of integrin α(IIb)β₃ (glycoprotein IIb- IIIa). A specific inhibitor of α(IIb)β₃ blocked platelet aggregation induced by H₂O₂ and NaVO₄, thus confirming that aggregation requires this receptor. In the presence of H₂O₂ and NaVO₄, multiple platelet substrates were phosphorylated on tyrosine. Such tyrosine kinase response was necessary but not sufficient to activate α(IIb)β₃, as detected by binding of soluble fibrinogen to platelets. Stirring of the platelets exposed to H₂O₂ and NaVO₄ was also needed to allow for binding of fibrinogen to α(IIb)β₃. The tyrosine kinase inhibitor genistein was able to block platelet aggregation induced by H₂O₂ and NaVO₄, thus confirming that tyrosine kinase activity was needed to trigger α(IIb)β3 activation on stirring. N-Acetyl-L-cysteine, a cell-permeant antioxidant, blocked the tyrosine phosphorylation of platelet substrates and also the platelet aggregation induced by H₂O₂ and NaVO₄. We found that β₃ was phosphorylated on tyrosine in platelets exposed to H₂O₂ and NaVO₄, even in the absence of aggregation. Hence, tyrosine phosphorylation of β₃ might contribute to the 'priming' of α(IIb)β₃ induced by H₂O₂ and NaVO₄, whereby the receptor can become activated on stirring of the platelets.