Platelet adhesion to PEAR1 extracellular domain. (A,B,D) Washed human platelets were plated by incubation for the indicated times at 37°C in wells precoated with PEAR1 extracellular domain (50 μg/mL) or (E) fibrinogen (200 μg/mL). (A) Kinetics of the adhesion of platelets stimulated or not with TRAP-6 (50μM). B) Role of αIIbβ3 and amplification pathways (ADP/ATP, TXA2) during platelet adhesion to PEAR1 for platelets activated with TRAP-6 (50μM). Platelets were either treated with apyrase (0.4 U/mL) and aspirin (550μM) before TRAP-6 stimulation or with eptifibatide (10 μg/mL) after TRAP-6 stimulation. Platelet adhesion was measured at 60 minutes. (C) Schematic representation of the modular architecture of PEAR1. Human PEAR1 is composed of an extracellular domain (comprising 1 EMI domain and 15 EGF-like domains), a transmembrane domain (TM, gray box) and a cytoplasmic domain (comprising 5 proline-rich domains). The sequence of the EMI domain N22-P103 is fused to GST (GST-EMI). (D) Platelet adhesion at 60 minutes in the presence or the absence of either GST or GST-EMI (6 μg/mL), with or without platelet activation by TRAP-6 (50μM). (E) Effect of GST-EMI (6 μg/mL) on platelet adhesion to fibrinogen after TRAP-6 stimulation (t test: *P < .05; **P < .01; ***P < .001).