NAD(P)H oxidase inhibitors and superoxide scavengers reduce platelet aggregation, αIIbβ3 activation, and thrombus formation under high shear flow conditions. (A) Platelet-rich plasma was preincubated with DPI (50 μM) and apocynin (1.2 mM) and then stimulated with Trap6 (30 μM) and allowed to aggregate for 5 minutes, with stirring at 1000 rpm. Platelet aggregation was demonstrated by the change in light transmission. A representative aggregation tracing from 3 independent experiments is shown. (B) Washed human platelets (300 × 10⁹/L) were preincubated with DPI (10 μM), apocynin (600 μM), tiron (3 mM), MnTMPyP (100 μM), or ASA (100 μM) and stimulated with thrombin (0.2 U/mL) for 1 minute. Integrin αIIbβ3 activation was measured with FITC–PAC-1 monoclonal antibody (mAb). DPI, apocynin, tiron, and MnTMPyP, but not ASA, inhibited αIIbβ3 activation. Data are shown as arbitrary units, mean ± SD, thrombin-induced PAC-1 binding taken as 1 (*significantly different at P < .05 compared with thrombin; n = 5). In the same experimental conditions, platelets were analyzed for VASP phosphorylation²³  by Western blot and for cGMP levels. NAD(P)H oxidase inhibitors and superoxide scavengers did not significantly change either VASP phosphorylation or cGMP levels in platelets. Sodium nitroprusside (SNP) (1 μM) was used as a positive control for phosphorylated VASP (P-VASP) and cGMP. Data for cGMP are shown as fold increase, mean ± SD, thrombin taken as 1 (*significantly different at P < .05 compared with thrombin; n = 3). (C) ROS production and αIIbβ3 activation measured by FITC–PAC-1 mAb after stimulation with different platelet agonists, thrombin (0.2 U/mL), Trap6 (30 μM), U46619 (1 μM), convulxin (100 ng/mL), and ADP (50 μM). (D) Inhibition of thrombus formation after treatment of platelets with DPI and apocynin. Whole blood from different individuals was treated with DMSO (control), DPI ([ii] 100 μM), or apocynin ([iii] 1.2 mM) and was perfused over a collagen-coated surface for 4 minutes under high shear conditions (1000 s^–1). The chamber was rinsed, and phase-contrast images were taken with a Zeiss Axiovert 200 inverted microscope, using a 63 ×/0.75 numeric aperture objective (both from Carl Zeiss, Göttingen, Germany), and a CV-M300 CCD camera (Visitron, Munich, Germany) connected to an S-VHS video recorder (AG-7355; Panasonic, Matsushita Electric, Japan). Videotaped images were evaluated using Meta Morph (Visitron), a computer-assisted image analysis program. The results of the experiments are summarized as bar graphs showing percentage surface area coverage (iv). DPI and apocynin caused 18% ± 5% and 20% ± 6% inhibition of platelet adhesion, respectively (*significantly different at P < .05 compared with control; n = 3). The images shown are representative of 3 individual experiments.