Old platelets show significantly reduced hemostatic capacity. (A and B) Flow cytometric analysis of platelets from mice 5 d after Kupffer cell depletion shows increased P-selectin exposure in resting conditions, but decreased P-selectin exposure after stimulation with thrombin (Thr) compared with control platelets (A) and significantly reduced αIIbβ3 integrin activation (JON/A-PE binding; B) after stimulation with thrombin. Data represent means ± SEM (n = 5 mice per group). (C) Representative aggregation traces of platelets isolated from control mice or mice 5 d after Kupffer cell depletion stimulated with 0.25 U/ml thrombin. (D and E) Maximum aggregation (A_max; D) and aggregation at the end time point (A_end; E) of platelets isolated from control mice or mice 5 d after Kupffer cell depletion. Data represent means ± SEM (n = 3 mice per group). Control mice and mice 5 d after Kupffer cell depletion were subjected to the tail bleeding time assay. (F) Time until bleeding cessation; values >20 min indicate that bleeding did not stop within the 20-min observation period of the experiment. (G) Quantification of the amount of blood loss. Data represent means ± SEM (n = 10 mice per group). (H) SD-IVM was performed on the liver of control mice after injection of cold-stored platelets. Kupffer cells and platelets were labeled using anti-F4/80 and CellTracker Red dye, respectively. Representative image taken at 30 min after injection. Scale bar is 70 µm. (I) Quantification of the accumulation of cold-stored platelets in wild-type and Asgr1^−/− mice as well as Asgr1^−/− mice treated with MGL-blocking antibody. n ≥ 4 mice per group, normalized to control mice values. Unpaired two-tailed t test (A, B, D, E, and G) was used to determine statistical significance. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Results from all experiments shown are representative of at least three independent experiments.