The inhibitory effects of apelin are dependent on apelin-13, but not on apelin-36. (A) Tail-bleeding time in WT mice injected with PBS (●), apelin-13 (50 nmol/kg; ○), apelin-36 (500 nmol/kg; △), apelin-DM (500 nmol/kg; ▪), apelin-36 plus apelin-13 (▲), or apelin-DM plus apelin-13 (□). Each symbol represents 1 individual, and statistical significance was determined by 2-tailed Mann-Whitney test (**P < .001). (B) Thrombin-induced aggregation of human platelets preincubated with PBS (black), apelin-13 (10 μM; white), apelin-36 (10 μM; dark gray), apelin-DM (100 μM; light gray), apelin-36 plus apelin-13 (angled lines), or apelin-DM plus apelin-13 (vertical lines). (C) [Ca²⁺] in human platelets preincubated with PBS (black), apelin-13 (10 μM; white), apelin-36 (100 μM; dark gray), apelin-DM (100 μM; light gray), apelin-36 plus apelin-13 (angled lines), or apelin-DM plus apelin-13 (vertical lines). In panels B and C, the relative percentage ± SEM of 3 independent experiments is expressed, and statistical significance was determined by 1-way ANOVA followed by Tukey test (*P < .05; **P < .001). (D) Schematic representation of the proposed mechanism regulating platelet activity by apelin in the presence of agonists. After interaction of apelin with its platelet APJ receptor, the latter mediates cGMP production through an NO-dependent mechanism that increased the level of cGMP produced in thrombin-activated platelets, which in turn inhibits platelet activation/aggregation. Apelin also inhibits Ca²⁺ mobilization induced by thrombin and collagen receptor activation. The inhibition of TXA₂ synthesis by apelin is also involved in the inhibition of platelet activity because U-46619, an analog of TXA₂, mediated platelet activity in the presence of apelin. ADP-induced platelet activation is not affected by apelin. GTP, guanosine triphosphate; PARs, protease-activated receptors; TPα, TXA₂ receptor α isoform; P2Y1, P2Y purinoceptor 1.