Reduced bleeding times in apelin^−/− mice and aggregation of apelin^−/− platelets. (A) Tail-bleeding times (left) and platelet count (right) in WT and apelin^−/− mice. (B) Images of WT and apelin^−/− platelet ultrastructure obtained by electron microscopy. Bar represents 1 μm. (C) Aggregation of washed WT and apelin^−/− platelets induced by indicated concentration of collagen, thrombin, ADP, or U-46619. In several experiments, apelin^−/− platelets were challenged with apelin-13 (10 μM). Traces are representative of at least 3 independent experiments. Results are expressed as the percentage change in light transmission with respect to the blank (buffer without platelets), set at 100%. (D) Aggregation of WT and apelin^−/− platelets induced by convulxin (250 and 350 pM). Traces are representative of at least 3 independent experiments. Results are expressed as the percentage change in light transmission with respect to the blank (buffer without platelets), set at 100%. (E) Immunoblot of GPVI signaling by analysis of tyrosine phosphorylation of Syk (p-Syk) and LAT (p-LAT) in WT and apelin^−/− platelets after activation for 2 min by 250 pM of convulxin (Cvx) in the presence of Leo.H4 (20 μg/mL) to prevent outside-in signaling induced by αIIbβ3 engagement, and in the presence of apyrase (5 U/mL) plus indomethacin (5 μM) to prevent platelet secretion. The expression of 14.3.3ζ was used as loading control. Results are representative of 3 experiments.