The role of Munc13-4 and ADP/P2Y₁₂ signaling in platelet α-granule secretion. Washed platelets were prepared from WT and Unc13d^Jinx mice. Indomethacin (10 µM) was added to remove the effect of P2Y₁₂-dependent regulation of thromboxane A₂ synthesis. Platelets were stimulated with PAR4-AP (AYPGKF-NH₂ / PAR4-AP; 300 µM) for 10 minutes in the presence of fluorescein isothiocyanate-labeled anti-CDP62P antibodies. CD62P surface expression was determined by flow cytometry. (A) Shows representative histograms of platelets stimulated with PAR4-AP in the absence or presence of exogenous ADP (10 µM). (B) Shows mean data (±S.E.). In some experiments, platelets were pretreated with the P2Y₁ antagonist, MRS2279 (10 µM), or the P2Y₁₂ antagonist, AR-C69913MX (1 µM), prior to stimulation. n = 5-14; **P < .01; ***P < .001 (2-way analysis of variance, Bonferroni posttest). In (C), platelets were stimulated with PAR4-AP and/or ADP. Platelets were pelleted by centrifugation and released PF4 in the supernatant was quantified by enzyme-linked immunosorbent assay. Released PF4 is expressed as the percentage of total PF4 in unstimulated platelets. n = 4; **P < .01; ***P < .001 (2-way analysis of variance, Bonferroni posttest).