PEAR1 activation induces sustained PI3K signaling. (A) Aggregation of washed platelets by PEAR1-EC Ab (7 μg/mL), in the presence of apyrase (0.4 U/mL) and aspirin (550μM), with added DMSO or LY294002 (50μM). (B) Corresponding blots for Akt phosphorylation (Akt-P vs total Akt) for platelets activated with PEAR1-EC Ab in the absence or presence of LY294002. (C) Akt phosphorylation (Akt-P vs total Akt) was analyzed in platelets incubated with PP1 (10μM) or DMSO for 10 minutes before activation with PEAR1-EC Ab (2 μg/mL) in unstirred conditions, in the presence of aspirin (550μM), apyrase (0.4 U/mL), and eptifibatide (10 μg/mL) for 5 minutes. (D) Washed platelet aggregation with a “low dose” of PEAR1-EC Ab (1 μg/mL) with LY294002 (50μM), added during early aggregation, as indicated. The inset shows the corresponding Akt phosphorylation during the aggregation at corresponding time points. (E) Washed platelet aggregation by PEAR1-EC Ab (1 μg/mL) supplemented with aspirin (550μM) and apyrase (0.4 U/mL), for 12 minutes. The inset shows the corresponding Akt phosphorylation at the end of the aggregation (12 minutes). (F) Washed platelet aggregation with a high dose of PEAR1-EC Ab (7 μg/mL) with LY294002 (50μM), added during late aggregation, as indicated. The insert shows the corresponding Akt phosphorylation at the end of the aggregation (12 minutes). (G) Flow cytometric measurement as MFI of activated αIIbβ3 (PAC1) on activation of washed platelets with PEAR1-EC Ab (7 μg/mL), in the presence of apyrase (0.4 U/mL) and aspirin (550μM), with or without LY294002 (50μM). Vertical lines indicate a repositioned gel lane.