Assessment of α_IIb β₃ activation and engagement in washed platelets from control or SERCA3^−/− mice and aggregation to ADP. (A) Quantitation of activated α_IIbβ₃ integrin at the surface of washed platelets was assessed by flow cytometry by binding of the specific mAb JON/A to control (black bars) or SERCA3^−/− (gray bars) platelets upon activation with thrombin at 40 or 100 mU/mL. The same experiments were conducted in the presence of 5 U/mL apyrase (noted “+ Apy”) or after addition of 10 µM ADP (“+ ADP”). Statistical significance was established with 1-way ANOVA followed by Tukey’s multiple comparison test; ns, not significant; **P < .01. (B) Assessment of the role of α_IIbβ₃ engagement in dense granule secretion in SERCA3^−/− platelets. Platelets stimulated with either 40 or 100 mU/mL of thrombin were subjected to aggregation, in the absence (-) or the presence (+) of the blocking mAb Leo.H4 (20 µg/mL) specific for mouse α_IIbβ₃, as well as in the absence (-) or the presence (+) of 10 µM ADP. Secretion was assessed by ATP measurement in the supernatant. Using WT as control, statistical significance was established with 1-way ANOVA followed by Tukey’s multiple comparison test; ns, not significant; **P < .01; ***P < .001. (C) Aggregation to ADP was assessed at 0.25, 0.5, 1, 2.5, 5, and 10 µM in control (WT) or SERCA3^−/− platelet-rich plasma.