Effect of PAR1 and PAR4 activation on tyrosine phosphorylation of PKCδ. Washed and aspirin-treated platelets were stimulated with thrombin (1.0 U/mL), SFLLRN 50 μM, AYPGKF 500 μM, and convulxin 100 ng/mL (A), with either increasing concentrations (B-C) or varying times (D-E) of agonists as indicated at 37°C. The stimulation times for thrombin, SFLLRN, AYPGKF, and convulxin were 60, 20, 60, and 60 seconds, respectively. PKCδ was immunoprecipitated as described, and the samples were analyzed for tyrosine phosphorylation by Western blotting using the monoclonal phosphotyrosine (4G10) antibody. Equal lane loading was assured by probing the samples with PKCδ antibody. The Western blot shown is representative of experiments done using platelets from 3 different donors.