Combination of AMPKi, Sirt3 activator, and antioxidants significantly ameliorate platelet survival in vitro and in vivo. (A) The aggregation, apoptosis, MMP, and mtROS levels were measured in human platelets stored for 6 days with 0.5 mM NMN, 5 mM NAC, 4 µM AMPKi, or 0.5 mM NMN + 5 mM NAC + 4 µM AMPKi. n = 3. (B) The survival of human platelets in immunodeficient NCG mice was detected; these platelets were stored for 6 days in vitro with 0.5 mM NMN, 5 mM NAC, and 4 µM AMPKi before transfusion. n = 3. (C) The survival of mouse platelets in WT mice was detected; these platelets were stored for 2 days in vitro with 0.5 mM NMN, 5 mM NAC, 4 µM AMPKi, or the combination of 0.5 mM NMN, 5 mM NAC, and 4 µM AMPKi. n = 3. (D) Schematic model of how CPT2 K79 acetylation induces short life span of platelets. On the one hand, AMPK activation leads to increased CPT1 activity and LCAC production; on the other hand, NAD⁺ exhaustion and Sirt3 dysfunction results in the over-acetylation of CPT2 K79 and the decrease in CPT2 activity. The incongruous changes in CPT1 and CPT2 activities lead to the accumulation of LCAC and mitochondrial damage. Blocking LCAC generation by inhibition of AMPK or CPT1, activation of Sirt3, and antioxidants tremendously retarded platelet storage lesion by single or combined use in vitro and in vivo. *P < .0332, ***P < .0002, ****P < .0001. LCFA, long-chain fatty acid; OAA, oxaloacetate; αKG，α-ketoglutarate.