ROS produced from platelet NOX2 regulate P-selectin exposure and GPIbα function. (A) Mouse or (B) human platelets were pretreated with vehicle, catalase (1000 U/mL), dimethylsulfoxide (0.1%), or DPI (10 µM), and incubated with or without thrombin (0.025 U/mL in [B]). The surface expression of P-selectin (CD62P) was measured by flow cytometry. (C) Human platelets were treated with 1 μg/mL mouse immunoglobulin G1 (IgG1) or an anti-GPIbα antibody (VM16d), vehicle, or catalase followed by incubation with 10 μg/mL αMβ2 in the presence or absence of 0.5 mM MnCl₂. The bound αMβ2 was determined by flow cytometry using goat anti-human β2 antibodies. (D) αMβ2 binding was measured in WT and NOX2 KO platelets as described in (C). (E-F) Mouse platelets were pretreated with or without catalase or 1 μM H₂O₂ and then incubated with 10 µg/mL vWF and 10 mM EDTA in the presence or absence of 10 µg/mL botrocetin. vWF binding was analyzed by flow cytometry using anti-vWF antibodies. Flow cytometric data are shown as the geometric mean fluorescence intensity (MFI) value or fold increase obtained by normalization of the MFI of antibodies to that of control IgG. (G) Mouse platelets were incubated with 10 µg/mL vWF and 10 µg/mL botrocetin. In some experiments, NOX2 KO platelets were pretreated with 0.1 to 1 μM H₂O₂. Platelet agglutination was measured in an aggregometer. All data represent the mean ± standard deviation (SD) (n = 4-5). *P < .05, **P < .01, or ***P < .001 between two groups after Student t test.