Apelin-13 inhibits platelet aggregation that expresses apelin and APJ receptor. (A) Quantity of apelin determined by enzyme immunoassay in cell lysates and media of resting “none-stimulated” (NS) human (left) and mouse (right) platelets and after activation by thrombin (1 U/mL). The quantity of apelin was expressed in picograms per 109 platelets ± standard deviation of at least 3 independent platelet samples. ***P < .001, Student t test. (B) Representative immunoblot of APJ in human (left) and mouse (right) cells. The molecular weight and the APJ-expressing human U87 and murine CT-26 cells (positive controls) are indicated. (C) Immunofluorescence analysis of APJ and apelin expression in platelets. Controls were stained using specific APJ or apelin antibody with the secondary antibodies only and were negative. Original magnification ×100. (D-E) Aggregation of washed human (D) and mouse (E) platelets induced by indicated concentration of collagen, thrombin, ADP plus fibrinogen (100 μg/mL), or U-46619, with or without incubation with apelin-13 (10 μM; Apl). 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; Ctl), set at 100%. (F) Thrombin-induced aggregation of human platelets preincubated with PBS; apelin-13 (10 μM), F13A (100 μM), F13A (100 μM) plus apelin-13 (10 μM), MM54 (100 μM), or MM54 (100 μM) plus 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 < .05; **P < .01; ***P < .001). KD, KDa; KO, knockout; PBS, phosphate-buffered saline; SEM, standard error of the mean.
