OxLDL/CD36–mediated H₂O₂generation promotes protein sulfenylation in platelets. OxLDL promotes protein sulfenylation over time (A) and in a concentration-dependent manner (B). (A-B) Human platelets (3 × 10⁸/mL) were pretreated with 1 mM alkyne-containing benzothiazine-based probe (BTD-alkyne) for 15 minutes, followed by stimulation with 50 µg/mL oxLDL or treatment with 0.55 mM glucose and 1 U/mL glucose oxidase up to 60 minutes for a continuous low flux of H₂O₂ (A) or with increasing concentrations of LDL or oxLDL for 15 minutes (B). A control without BTD-alkyne was incorporated in panel B. Cysteine sulfenylation by oxLDL (C) and nonoxidized CD36 ligand MRP-14 (D) were decreased by the CD36-blocking antibody. Platelets were pretreated with 1 µg/mL nonimmunizing control antibody or the CD36-blocking monoclonal antibody FA6-152 (FA6) for 15 minutes in the presence of 0.1 mM BTD-alkyne followed by platelet activation with 50 µg/mL oxLDL (C) or 4 µg/mL MRP-14 (D) for 15 minutes. (E) Cysteine sulfenylation by oxLDL/CD36 signaling is through an H₂O₂-dependent mechanism. Platelets were pretreated with 1000 U/mL denatured PEG-catalase or functional PEG-catalase for 15 minutes in the presence of 0.1 mM BTD-alkyne followed by platelet activation with 50 µg/mL LDL or oxLDL for 15 minutes. In all panels, platelets were lysed with radioimmunoprecipitation assay buffer followed by modification of the alkyne of BTD-alkyne with biotin-PEG3-azide via azide-alkyne cycloaddition (click chemistry). P values were determined by 1-way analysis of variance with Dunnett’s post hoc analysis. *P < .05 compared with buffer treatment (A-B,E). *P < .05 with their respective comparisons (C-D). N = 5 separate donors (A), N = 4 separate donors (B), N = 4 separate donors (C-D), and N = 3 separate donors (E). Data are expressed as mean ± SEM. ns, not significant.