Effect of apelin-13 on cAMP, cGMP, and thromboxane B₂ synthesis and platelet secretion. Accumulation of cAMP (A) and cGMP (B) was determined in resting and thrombin-activated platelets (5 minutes) in the absence or presence of apelin-13 (10 μM). Results represent the mean ± SEM of at least 4 separate experiments, each performed in triplicate. Statistical significance was determined by unpaired Student t test (*P < .05; **P < .001). (C) cGMP content in platelets in the presence or absence of the nitric oxide synthase inhibitor N^G-methyl-L-arginine (L-NAME) (1 mM) for 5 minutes was measured in untreated or apelin (10 μM)-treated platelets. Results are the mean ± SEM of at least 3 separate experiments, each performed in triplicate (Student t test; **P < .001). (D) Accumulation of cGMP was determined in untreated or apelin-treated platelets (5 minutes) in the absence or presence of sodium nitroprusside (SNP; 10 μM). Results represent the mean ± SEM of 3 separate experiments, each performed in triplicate. (E) Thromboxane B₂ (TXB₂) content was determined in resting and thrombin-activated platelets after their incubation in the absence or presence of apelin-13 (10 μM) for 5 minutes. Results represent the mean ± SEM of 3 separate experiments, each performed in triplicate (Student t test, **P < .01; ***P < .001). (F) Aggregation of washed mouse platelets induced by arachidonic acid (39 μM) with or without incubation with apelin-13 (10 μM). Traces are representative of at least 3 independent experiments (supplemental Figure 3). Results are expressed as the percentage change in light transmission with respect to the blank (buffer without platelets; Ctl), set at 100%. (G) Dense granule secretion was evaluated by measuring the ATP release after the aggregation induced by the indicated concentration of thrombin, U-46619, and ADP of human platelets preincubated with or without apelin-13 (10 μM). Results were expressed as the amount of ATP release by platelets from at least 3 independent experiments, and statistical significance was determined by unpaired Student t test (***P < .001). (H) Aggregation of washed human platelets induced by thrombin (100 mU/mL) with or without incubation with apyrase (5 U/mL) and apelin-13 (10 μM). The relative percentage ± SEM of 3 independent experiments is expressed, and statistical significance was determined by 1-way ANOVA followed by Tukey test (**P < .01).