Downregulation of platelet signaling induced by PAR4-AP. (A) Aggregation of washed platelets for P1 and P2 was initiated by PAR4-AP (50 μM) for 3 minutes. Aggregation was expressed as the percentage change in light transmission, with the value of the blank (buffer without platelets) set at 100%. Tracings are representative of at least 2 experiments per patient. (B) Quantification of activated αIIbβ3 at the surface of washed platelets was assessed by flow cytometry by binding of the specific mAb PAC1 to control, P1, and P2 platelets upon activation by PAR4-AP (100 to 200 μM). (C) Quantification of P-selectin expression at the surface of washed platelets was assessed by flow cytometry by binding of the specific anti-P-selectin antibody to control, P1, and P2 platelets upon activation by PAR4-AP (100 to 200 μM). These results are representative of 2 experiments for each patient. (D) Ca²⁺ mobilization was assessed in unstirred platelets preincubated with the cytosolic Ca²⁺ fluorescent probe Oregon green BAPTA-AM after stimulation by PAR4-AP (100 and 200 μM) by flow cytometry in conditions of no external Ca²⁺ (1mM EGTA). (E-F) Phosphorylation of PKC substrates and Src-Y418 of control and P1 platelets activated by PAR4-AP (0 to 300 µM) was assessed by immunoblotting using an anti-PKC substrates (p-S) and anti-Src-p-Y418, respectively. Results are representative of 2 experiments. Quantification of Src phosphorylation (Src-p-Y418) was the average of the 2 experiments.