Aged platelets display diminished hemostatic and thrombotic potential in vitro. (A) Schematic outline showing pulse-labeled C57BL/6J mice (red arrow, X649; green arrow, X488). (B) MitoTracker and tetramethylrhodamine MFI of platelet age cohorts analyzed via flow cytometry (n = 4 per group; both P < .0001). (C) Representative micrographs of spread platelets and analysis of platelet size by area (n = 3 per group; P = .0060). (D) Platelet size measured by FSC-A (n = 4 per group; P = .0085). (E) Flow cytometric measurements of P-selectin expression (MFI) and GPIIbIIIa (αIIbβ3) integrin activation (MFI) in washed platelets after treatment with agonists relative to their expression after phosphate-buffered saline (PBS) treatment (n = 4 per group; all P < .0001). (F) Representative images of single-labeled platelet migration on labeled fibrinogen substrate and quantification of migration as cleared area (n = 4 per group; P = .0094); outlined area showing cleared substrate. (G) Representative micrographs showing single-cell clot retraction assay of pulse-labeled platelets with fibrinogen and platelet poor plasma (n = 3 per group; P = .0406); outlined area showing retracted substrate. (H) Representative micrographs of single-labeled platelets (using Image Calculator in ImageJ: subtracting X649 labeled from X488) showing in vitro thrombus formation with whole blood on collagen I (n = 3 per group); white dotted lines enclose area depicting thrombi; bar graph depicting percentage of single-labeled platelets in thrombus relative to the percentage of single-labeled platelets in blood (P = .0005). Statistical tests for all, ordinary 1-way ANOVA with the post hoc Dunnett multiple comparisons test compared with 0- to 12-hour group. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001; h, hour; ns, nonsignificant.