F1KO mice and wild-type (WT) mice (A–I), Fundc1fl/fl::Pf4Cre- and Fundc1fl/fl::Pf4Cre+ mice (J–M) and Atg5fl/fl::Pf4Cre- and Atg5fl/fl::Pf4Cre+ mice (N–Q) were treated for 72 hr under 8% hypoxia. (A). The oxygen consumption rate (OCR) of platelets was measured by Seahorse (A, left) and was normalized to the level in normoxic WT platelets (A, right). (B) ATP level was detected and normalized to the percentage in normoxic WT platelets. (C) Mitochondrial mass was measured by flow cytometry after staining with NAO (20 nM) and was normalized to the percentage in normoxic WT platelets. (D) Mitochondrial membrane potential was analyzed by flow cytometry and normalized to the percentage in normoxic WT platelets. (E) ROS levels were detected by flow cytometry. DCF fluorescence intensity was quantified using BD FACS Calibur and CellQuest software. ROS levels were normalized to the percentage in normoxic WT platelets. (F, J, N) Platelet aggregation was analyzed (n = 16) using a turbidometric aggregometer. (G, K, O) The correlation between mitochondrial OCR and aggregation was analyzed in platelets treated with different concentrations of thrombin (0.00625, 0.0125, 0.025, 0.05, 0.10, 0.20, 0.40 U/mL). (H, L, P) Platelet spreading was analyzed after treatment with 0.05 U/ml thrombin. At least 96 platelets were analyzed in each group. (I, M, Q) P-selectin expression on platelets after activation with 0.05 U/ml thrombin was analyzed by flow cytometry. Data from three separate experiments are presented as mean ± s.e.m. *p<0.05. **p<0.01. ***p<0.001.