Platelets shift toward inflammatory function during in vitro storage. (A) Schematic outline of platelet concentrate sampling scheme. (B) Flow cytometric analysis of baseline surface marker expression PS exposure (0.0170), CD36 (0.0028), and CD47 (0.0045) relative to time point 1 [TP1]; percentage of reticulated platelets depicted by thiazole orange positive cells (n = 3; P < .0001; RM 1-way ANOVA with the post hoc Dunnett multiple comparisons test compared with TP1). (C) Representative micrographs and analysis of spreading of unactivated or stimulated (4-μM adenosine 5′-diphosphate [ADP] + 2-μM U46619) platelets (n = 3); 2-way RM ANOVA (comparison between PBS and ADP, P = .0064) with post hoc Holm-Šídák multiple comparisons test compared with TP1. (D) Representative micrographs showing in vitro thrombus formation (n = 5); bar graph showing the percentage of thrombus area per field of view (P = .0058). (E) Representative micrographs of procoagulant activation of platelets seeded on collagen I/fibrinogen matrix; graphs showing percentage of procoagulant platelets (P = .0107) and percentage of ballooned procoagulant platelets relative to TP1 (P = .001). (F) Flow cytometric analysis showing percentage of P-selectin–positive PS⁺ platelets (n = 4; P = .0105). (G) Schematic outline of platelet-neutrophil coincubation (n = 3); flow cytometric analysis showing percentage of platelets aggregating with neutrophils (0.0274); statistical tests for panels D-G, RM 1-way ANOVA with the post hoc Dunnett multiple comparisons test compared with TP7. (H) In vitro aged donor C57BL/6J platelets, freshly isolated (day 0 [D0], n = 4) or stored for 2 days in DSD (day 2 [D2], n = 7) were transfused into thrombocytopenic C57BL/6J recipient mice (n = 4 per group); LPS was given intranasally to induce acute lung injury (ALI) in recipients; blood sampled at 0 and 6 hours after ALI and BALF was collected. (I) Bar graph depicting decline of transfused platelets, mixed-effects model (REML), and comparison between transfused groups (P = .0015 with the post hoc Šídák multiple comparisons test). (J) Bar graphs showing surface marker expression: CD11b (P = .0344) and CD66a (P = .0316) of neutrophils in circulation after ALI (unpaired t test, 2-tailed). (K) Bar graphs depicting the percentage of transfused in vitro aged platelets aggregating with CD45⁺ cells (P = .0203) and the percentage of platelets aggregating with Ly-6G⁺ cells (P = .0890) in BALF (unpaired t test, 2-tailed). (L) Assessment of cytokine measurements in BALF (2-way ANOVA, comparison between transfused groups; P = .0014, with the post hoc Šídák multiple comparisons test; additional cytokines shown in supplemental Figure 11H. (M) Clinical progression of ALI in recipients that received transfusion with D0 or D2 in vitro–aged platelets (2-way RM ANOVA, comparison between transfused groups; P < .0001, with the post hoc Šídák multiple comparisons test). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001; h, hour; IL, interleukin; LPS I.N., lipopolysaccharide intranasally; ns, nonsignificant; TNF-α, tumor necrosis factor α.