Platelet aging proteomics. (A-E) Rosa26-DTRxPF4cre mice were administered with DT, intraperitoneally every 48 hours; mixed-aged platelet cohorts were isolated from Cre– mice 4 days after serial DT injections (n = 4); platelets aged >4 days were collected 4 days after serial DT injections in Cre+ mice (n = 4); platelets aged <1 day were collected 8 days after serial DT injections in Cre+ mice during platelet recovery phase (n = 4); schematic outline (A); principal component analysis of all 2062 proteins quantified (n = 4) (B); volcano plot (C) of a student t test (P < .05; |log2 fold change| > 2) comparing 3124 proteins in the young (left) and old (right) cohorts; heat map (D) of the 447 significant proteins from panel C; Fisher exact test enrichment analysis (false discovery rate <0.02; count ≥10, top 10) of gene ontology biological pathway terms (E) among differential proteins from panel C. (F) Proteomic findings were confirmed by analyzing surface marker expression of single-labeled platelets in pulse-labeled C57BL/6J mice (n = 4 per group) via whole blood flow cytometry; CD36 (P = .0009), C3 (P = .0013) and fibrinogen (P = .0035); statistical tests, ordinary 1-way ANOVA with the post hoc Dunnett multiple comparisons test compared with the 0- to 12-hour group. ∗∗P < .01; ∗∗∗P < .001; FC, fold change; h, hour; LFQ, label-free quantification; ns, nonsignificant; rRNA, ribosomal RNA.
