Kinetics of tyrosine phosphorylation of α-actinin during platelet activation and inhibition of SHP-1 by PTPI-1. Washed human platelets were stimulated with PAR1-AP (25μM) or PAR4-AP (150μM) for the time indicated. (A) Tyrosine-phosphorylated proteins were immunoprecipitated then immunoblotted with anti–α-actinin antibody. Immunoblots were analyzed by scanning densitometry and were quantified with ImageJ. (B) Washed human platelets were incubated at room temperature for 2 minutes in the presence of PTPI-1 (50μM). The platelets were then stimulated with PAR1-AP (25μM) for the time indicated. αIIbβ3 or tyrosine-phosphorylated proteins were immunoprecipitated then immunoblotted with anti–α-actinin antibody. (C) FITC–PAC-1 was added to the activated platelets after stimulation and incubated for 30 seconds to obtain the PAC-1 binding velocity at the time indicated. Error bars represent SEMs of 3 experiments.