Regulation of platelet α_IIbβ₃ activation and Ca²⁺ mobilization by apelin-13. (A) Flow cytometry analysis of the integrin α_IIbβ₃ activation detected by the recognition by the antibody JON/A of the activated conformation of mouse α_IIbβ₃ integrin after stimulation by thrombin (Thr), ADP, or U-46619 of washed mouse platelets treated with 10 μM apelin-13 (+) or not (−). Results are expressed as relative mean fluorescence intensity (MFI) ± SEM, in arbitrary units, from at least 3 experiments. Statistical significance was determined by unpaired Student t test (***P < .001). (B) Platelet adhesion of washed human platelets (10⁷) in presence of apyrase (2 U/mL) and preincubated with or without apelin-13 (10 μM) to immobilized collagen (50 μg/mL), von Willebrand factor (VWF; 50 μg/mL), or fibrinogen (100 μg/mL). Platelet adhesion to VWF was performed in the presence of botrocetin (5 μg/mL) and to fibrinogen with and without the activation by thrombin (100 mU/mL). Data are expressed as mean relative adhesion ± SEM of at least 3 independent experiments, and statistical significance was determined by unpaired Student t test (*P < .05; **P < .01). (C) Effect of apelin-13 (10 μM) on intracellular Ca²⁺ mobilization in platelets activated by thrombin (100 mU/mL) (left). Bar graph denotes the corresponding percentages of Ca²⁺ mobilization (right). (D) Effect of apelin-13 on intracellular Ca²⁺ mobilization in ADP (10 μM)-activated platelets. (E) Effect of apelin-13 (10 μM) on intracellular Ca²⁺ mobilization in convulxin (500 pM)-activated platelets. Platelet aggregation induced by convulxin (200 pM) is also indicated. (F) Effect of apelin-13 on intracellular Ca²⁺ mobilization in U-46619 (200 nM)-activated platelets. (G) Effect of apelin-13 (10 μM) on intracellular Ca²⁺ mobilization in the absence or presence of wortmannin (10 nM) in platelets activated by thrombin (100 mU/mL). Results are representative of 4 experiments and expressed as the relative MFI, in arbitrary units (left). Bar graph denotes the corresponding percentages of Ca²⁺ mobilization (right). Values are mean ± SEM (n = 4 per group). Statistical significance was determined by unpaired Student t test (***P < .001). Ca²⁺, calcium ion; ns, not significant.