Requirement of Ca²⁺ flux for oxidative platelet particle formation induced by Ca²⁺ ionophore through activation of the NADPH oxidase and 12-LO pathways. (A) Catalase and DPI inhibit Ca²⁺ ionophore–induced platelet particle formation. C1 and C2 indicate control buffer and control IgG (25 μg/mL), respectively; Pt, patient anti–GPIIIa49-66 Ab (25 μg/mL); Ct, catalase (100 μg/mL). Lane 0 is A23187 alone. Lanes 1, 2, and 3 refer to doubling concentration of either catalase starting at 50 μg/mL in the presence of 10 μM A23187 or DPI starting at 20 μM (n=4). (B) Ca²⁺ ionophore–induced platelet particle formation requires the NADPH oxidase pathway. Ctl and Pt refer to control and patient Ab. Ca²⁺ ionophore concentrations are given for wild-type and NADPH oxidase–deficient (gp91phox^−/−) platelets. (C) Ca²⁺ ionophore–induced platelet particle formation requires 12-LO (same designations as in panel B; Cig indicates control IgG). (D) Ab- or 12(S)-HETE–induced platelet particle formation is inhibited by Ca²⁺ chelator EGTA (extracellular) or BAPTA (intracellular). C1, C2, and C3 refer to control IgG without and with EGTA and BAPTA, respectively. Pt indicates patient Ab; PE, PB, and PEB, patient Ab in the presence of EGTA (100 μM), BAPTA (10 μM), and EGTA + BAPTA; H, 12(S)-HETE (0.1 μM); HE, HB, and HEB, 12(S)-HETE plus EGTA, BAPTA, and EGTA + BAPTA (n=4). SEM is given.