Knockout of Nix increases the life span of platelets by inhibiting the autophagic degradation of the mitochondrial protein Bcl-xL in vivo. (A-B) Nix^−/− and WT mice were injected IV with NHS-biotin (A). Whole blood was collected at various time points and platelets were isolated and prepared. Flow cytometry was used to analyze the platelet life span. (B) The data were analyzed using CellQuest software (BD Biosciences). (C) Platelets were collected by cell sorting as described in panel A, and western blotting was used to analyze platelet mitophagy. (D-F) The adoptive platelet transfer assay was conducted as shown in panel D, and Nix^−/− and WT mice were IV injected with NHS-biotin, 30 minutes after the injection, platelets were isolated and resuspended in saline solution. The prepared platelets were then IV injected into recipient mice as indicated. Biotinylated platelets were analyzed by flow cytometry. (E) Data from 3 separate experiments are shown as mean ± standard error of the mean. (F) Western blotting was used to analyze mitophagy of the biotin-labeled platelets from the recipient mice. (G-H) Nix^−/− and WT mice were injected IV with NHS-biotin, and the pretreated mice were further injected intraperitoneally with chloroquine (CQ; 1 mg/kg). Whole blood was collected at various time points and platelets were isolated and prepared. Flow cytometry was used to analyze the platelet life span. (G) The data were analyzed using CellQuest software (BD Biosciences). (H) Platelets were collected as described in panel A, and western blotting was used to analyze platelet mitophagy. (C,F,H) 6 mice of WT and 6 mice of Nix^−/− were used in 1 experiment, and 3 independent experiments with total of 18 mice were used. The grayscale values of all the western blot bands were measured with ImageJ software and the statistical data are illustrated. Student t test. *P < .05; **P < .01; ***P < .001.