ACC DKI platelets display increased dense granule secretion and thromboxane generation on collagen stimulation. (A) Washed platelets were stimulated with thrombin or collagen at the indicated concentrations, and light transmission was measured (Chrono-Log). Aggregation is expressed as the maximal percentage of light transmitted. The dashed line represents separate analyses. The results are expressed as means ± SEM (at least 4 experiments for each condition). (B) αIIbβ3 activation (binding of JON/A) was analyzed by flow cytometry in washed ACC WT and DKI platelets stimulated with thrombin for 8 minutes or collagen for 30 minutes at the indicated concentrations. The dashed line represents separate analyses. The results are expressed as mean fluorescence intensity (MFI) ± SEM (at least 4 experiments for each condition). (C-D) Washed ACC WT and DKI platelets were stimulated with thrombin or collagen at the indicated concentrations in the presence of Luciferase-Luciferin reagent, and ATP release was measured in a Lumi-aggregometer. (C) The dashed line represents separate analyses. The results are expressed as mean amount of ATP released (nmoles) ± SEM (at least 4 experiments for each condition). #P ≤ .05 between ACC WT and DKI platelets. The data underwent 2-way ANOVA. (D) Representative traces of ATP secretion after 0.5 µg/mL, 1.5 µg/mL, and 2.5 µg/mL collagen stimulation. (E) Washed platelets (30 × 10³/µL) were stimulated with 30 mU/mL thrombin or 5 µg/mL collagen for 5 minutes, and serotonin (5-HT) was measured in the supernatant by ELISA kit. The dashed line represents separate analyses. The results are normalized to ACC WT-stimulated platelets and are expressed as means ± SEM (at least 3 experiments for each condition). #P ≤ .05 between ACC WT and DKI platelets. The data underwent 2-way ANOVA. (F) Washed platelets (7.5 × 10³/µL) were centrifuged, the pellet was lysed, and serotonin (5-HT) was assayed in the lysate. The results are expressed as means ± SEM (n = 3). (G) Washed platelets were centrifuged, the pellet was lysed, and ADP and ATP content assayed in the lysate by reverse-phase high-performance liquid chromatography. Results are expressed as means ± SEM (n = 3). (H) Washed platelets were stimulated with 100 mU/mL thrombin alone or preincubated or not for 45 minutes with 1 mM aspirin (ASA), and stimulated with 5 µg/mL collagen. TXA₂ was measured in the supernatant by ELISA. The dashed line represents separate analyses. The results are expressed as means ± SEM (at least 3 experiments for each condition). #P ≤ .05 between ACC WT and DKI platelets. The data underwent 2-way ANOVA. (I) Washed platelets were preincubated or not for 45 minutes with 1 mM ASA and stimulated with 5 µg/mL collagen. Serotonin (5-HT) was measured in the supernatant by ELISA. The results are expressed as means ± SEM (at least 3 experiments for each condition). #P ≤ .05 between ACC WT and DKI platelets. The data were assessed by 2-way ANOVA. (J) Washed platelets were preincubated with 30 µM ticagrelor or the corresponding vehicle (dimethyl sulfoxide) for 30 minutes and stimulated with 5 µg/mL collagen for 5 minutes. TXA₂ was measured in the supernatant by ELISA. The results are expressed as means ± SEM (n = 4). *P ≤ .05 relative to respective untreated platelets, #P ≤ .05 between ACC WT and ACC DKI platelets. The data underwent 2-way ANOVA. See also supplemental Figures 1-5.