Functional evidence for NADPH oxidase activity. Gel-filtered human platelets were incubated with 25 μg/ml of control or patient Ab at 37°C, extracted, and immunoblotted. (A) Effect of Ab on AKT activation. C5, C15, C30, and P5, P15, and P30 refer to minutes of incubation with control or patient (P) Ab, respectively. LK-4 refers to incubation with irrelevant anti-GPIIIa mAb. Actin is internal control (representative of five experiments). AKT-P, phosphorylated AKT. (B) Effect of patient Ab on ERK-1/2 activation. Upper lanes refer to reactivity with Ab directed against phosphorylated ERK (ERK-P). Lower lanes refer to Ab directed against internal control ERK protein (representative of three experiments). (C) Effect of Ab on translocation of p67^phox, p47^phox, and PLA₂ from platelet cytosol to membrane. Platelets were incubated with Ab for 30 minutes at 37°C, extracted, separated into cytosol and membrane components, and then immunoblotted with specific Ab’s. M, marker for p67^phox and p47^phox Ab. Plt refers to platelets in buffer, prior to incubation. Cc, Cm and Pc, Pm refer to control and patient Ab directed against cytosol and membrane fractions, respectively. (D) Effect of 12(S)-HETE (200 nM) on translocation of p67^phox and p47^phox from platelet cytosol to membrane fractions. Hm, Hc, Cm, and Cc refer to cytoplasmic and membrane fractions from 12(S)-HETE and control incubations, respectively. PHm and PHc refer to incubation with 12(S)-HETE and protein kinase C inhibitor, bisindolylmaleimide at 20 nM. Representative of three experiments.