PEAR1-EC Ab-induced platelet aggregation. Washed platelet aggregation by PEAR1-EC Ab (A) at 0.25 to 2 μg/mL, (B) at 0.5 μg/mL, in the presence of eptifibatide (10 μg/mL) or apyrase (0.4 U/mL) and aspirin (550μM). Aggregations with PEAR1-EC Ab in the presence of IV.3 (10 μg/mL), traces representative of at least 5 independent experiments. (C) Flow cytometric measurement as MFI of activated αIIbβ3 (PAC1) and P-selectin (CD62-P) on activation of washed platelets with PEAR1-EC Ab (5-10 μg/mL). Activated integrin and CD62-P exposure are expressed as percentage of positive platelets in the fluorescence histogram for one experiment, representative of 3 independent analyses. (D) Inhibition of aggregation by PP1 (10μM) versus DMSO, as indicated. (E) Phosphorylation of total tyrosine (P-Tyr, 1 and 5 minutes) and PEAR1 (PEAR1-P, 5 minutes). Phosphorylation was analyzed in platelets incubated with PP1 (10μM) or DMSO for 10 minutes before activation with PEAR1-EC Ab (7 μg/mL) in unstirred conditions, in the presence of aspirin (550μM), apyrase (0.4 U/mL) and eptifibatide (10 μg/mL). Total PEAR1 was determined after stripping and reprobing. Data are representative of 3 experiments.