Role of MRP4 in platelet activity in vitro. Washed-platelet aggregation was monitored by light transmission through a platelet suspension at a concentration of 3.5 × 10⁸ platelets per milliliter. (A) PAR4-ap (50 and 100 μM) induced platelet aggregation; results are expressed as the percentage of maximal aggregation (n ≥ 9) (**P < .01). (B) αIIbβ3 activation was evaluated in WT or MRP4^−/− washed platelets activated for 10 minutes with increasing concentrations of PAR4-ap in the presence of phycoerythrin-labeled JonA antibody. The experiment was performed without stirring to prevent platelet aggregation. The level of activated integrin is indicated by the percentage of JonA-positive platelets measured by flow cytometry (n ≥ 4) (*P < .05; **P < .01). (C) α-Granule secretion was measured by P-selectin (CD62P) plasma membrane expression in response to PAR4-ap (100 μM) and was analyzed using flow cytometry after platelet incubation with fluorescein isothiocyanate–labeled rat anti-mouse CD62P. P-selectin expression is expressed as the mean fluorescence intensity (MFI) (n ≥ 5) (*P < .05; **P < .01). (D) Dense granule secretion was measured by serotonin release in platelet supernatants after PAR4-ap (60 μM) induced activation (n ≥ 6) (*P < .05). (E-G) Agonist-induced platelet aggregation: platelets were incubated at 37°C under stirring and then activated with ADP in the presence of fibrinogen (5 μg/mL) (n = 13) (*P < .05) (E); with U46619 (n = 6) (**P < .01) (F); and with 50 and 100 ng/mL convulxin (n ≥ 6) (G). Each box represents the interquartile range with median maximal aggregation (horizontal line in the box); the whiskers represent the fifth to 95th percentiles. NS, not significant.