Platelet activation responses are modulated by H₂O₂ in a CypD-dependent manner. (A) Platelets were stimulated with thrombin (0.5 U/mL) or thrombin plus H₂O₂ (200 μM) for 5 minutes. Platelets Δψ_m was assessed by flow cytometry using the cationic dye TMRM (n = 3). (B-D) Flow cytometry was performed to evaluate the percentage of (B) annexin V^high, (C) fibrinogen^high, and (D) P-selectin^high platelets (n = 4). #P < .05 compared with thrombin-stimulated CypD^+/+ platelets; *P < .05 for comparison between CypD^+/+ and CypD^−/− platelets. (B) Platelets were left unstimulated or stimulated for 5 minutes with thrombin (0.5 U/mL), thrombin plus convulxin (Cvx) (250 ng/mL), or thrombin plus H₂O₂. Error bars represent SE. (E) Two-color flow cytometry was performed using FITC-labeled antifibrinogen and PE-labeled JON/A. Gates are drawn to highlight activated platelets with high levels of JON/A binding and moderate fibrinogen binding (A1), and activated platelets with low levels of JON/A binding and high-level fibrinogen binding (A2) (n = 5).