The EMI domain of PEAR1 in platelet aggregation and signaling. (A) PEAR1 phosphorylation during platelet activation by thrombin (0.1 U/mL, 30s) in the absence or presence of eptifibatide (20 μg/mL), after immunoprecipitation of PEAR1-P with the 4G10 platinum antiphosphotyrosine antibody and Western blotting for PEAR1. (B) Washed platelet aggregation by collagen and TRAP-6 in the presence of GST or GST-EMI (6 μg/mL). Aggregation traces are representative of at least 3 independent experiments. (C) PEAR1 phosphorylation in platelets aggregated with collagen after 1 minute, in the presence of GST or GST-EMI. (D) Tyr-phosphorylation of washed human platelets, activated with collagen in the presence of GST (−) or GST-EMI (+), 5 minutes after start of the aggregation. (E) Flow cytometric measurement as MFI of activated αIIbβ3 (PAC-1) on activation of washed unstirred platelets with thrombin and convulxin (CVX) in the presence of GST or GST-EMI. (F) Rap1-GTP after TRAP-6 stimulation of platelets (10 or 60 seconds), with or without stirring, as indicated, in the presence of GST (−) or GST-EMI (+).