Nix-mediated-platelet mitophagy regulates platelet mitochondrial quality and platelet activity. Platelets were isolated from the blood of Nix^−/− mice and WT mice. The prepared platelets (3 × 10⁸/mL) were treated with FCCP (5 μM) for 2 hours at room temperature, and then analyzed for mitochondrial quality and platelet activation. (A-B) OCR of platelets from Nix^−/− mice and WT controls (WT) was measured by Seahorse analysis (A); statistical analysis of the data are presented in panel B. (C) Statistical analysis of the ATP production capacity of platelets from Nix^−/− mice and WT controls. (D-E) Platelets were treated with the selective mitochondrial dye nonyl acridine orange (NAO) and analyzed by flow cytometry to determine the mitochondrial mass (D). Statistical analysis of the data are presented in panel E. (F-G) Mitochondrial membrane potential was detected by flow cytometry using TMRE staining (F); statistical analysis of the data are shown in panel G. (H-I) Platelet aggregation induced by thrombin, collagen, ADP, and U46619 were detected (H) and the aggregation amplitudes were analyzed (I). Baseline of platelet aggregation trace was indicated with arrowhead. (J-K) P-selectin surface expression and α_IIbβ₃ activation were analyzed on platelets stimulated with 0.05 U/mL α-thrombin by flow cytometry using antibodies against P-selectin (J) and the active α_IIbβ₃, respectively. Statistical analysis of the data are shown (K). (L-M) Platelet mitophagy was analyzed by western blotting using the indicated antibodies (L). The grayscale values of all the bands were measured with ImageJ software and the statistical data are illustrated (M). ANOVA was used. *P < .05; **P < .01; N = 6 for each group.