Rejuvenation rescues the functional defects of BAK/BAX-deficient platelets. (A) Platelet counts and percentage reticulated thiazole orange (TO) positive platelets postplatelet depletion by APS in wild-type mice. n = 4 (untreated) and n = 8 (APS treated) mice. (B) TO-positive platelets 24 to 96 hours after APS treatment. n = 3 to 6 mice per group and point. Data are presented as mean ± SD. (C-D) Platelet age was synchronized by APS-induced platelet depletion 72 hours before platelet harvest. Agonist-induced platelet activation determined by (C) JON/A or (D) P-selectin positive platelets by flow cytometry. Platelets were washed and counts adjusted before 20 minutes incubation with ADP 12.5 µM (37°C) Convulxin 12.5-50 ng/mL (RT) and PAR4-AP 0.06-0.5 mM (RT). n = 3 biological replicates per genotype. Data are presented as mean ± SD. (E) Maximal platelet aggregation in response to PAR4-AP (50 µM). Mice were depleted of platelets (APS) 72 hours before platelet purification. Platelets were washed and counts adjusted. Bak^+/+Bax^+/+ (n = 5), Bak^−/−Bax^fl/fl (n = 6), Bak^+/+Bax^{Pf4∆/Pf4∆} (n = 2), and Bak^−/−Bax^{Pf4∆/Pf4∆} (n = 5) mice. Representative experiment included. (F) ATP release after platelet aggregation in response to PAR4-AP (50 µM). Mice were depleted of platelets (APS) 72 hours before platelet purification. Platelets were washed and counts adjusted before agonist stimulation. ATP levels were determined in platelet supernatants by cell titer glo assay. n = 3 mice per genotype. Data are presented as mean ratio/wild-type (wt) ± SD. (G) Tail bleeding time into 37°C saline (3-mm tail amputation). Maximal time was set as 600 seconds. The bleeding time was determined as the time from the tail amputation to the moment the blood flow stopped for more than 1 minute. Mice were untreated or depleted of platelets (APS) 72 hours before assay. Each symbol represents an individual mouse. Data are presented as mean. Unpaired Student t test with 2-tailed P values. *P < .05; **P < .005; ***P < .001.