Role of secondary mediators in rhodocytin induced aggregation and protein phosphorylation. (Ai,iii) Platelets pretreated with or without inhibitors were stimulated with 30nM rhodocytin. Addition of agonist is indicated by an arrowhead. (ii) Dose responses to rhodocytin. Platelets pretreated with vehicle or 2 U/mL apyrase/10μM indomethacin are shown as mean and standard error of 3 independent experiments. Protein phosphorylation measurements (B-E) were performed in the presence of eptifibatide to prevent platelet aggregation. (B) Whole-cell lysates were prepared from control and rhodocytin-stimulated platelets with or without secondary mediators and indicated inhibitors. (C) CLEC-2 was immunoprecipitated from basal-, U46619/ADP-, and rhodocytin-stimulated platelet lysates, and tyrosine phosphorylation was assessed. (D) CLEC-2 was immunoprecipitated from rhodocytin-stimulated platelets pretreated with inhibitors, and tyrosine phosphorylation was assessed. (E) CLEC-2 was immunoprecipitated from rhodocytin-stimulated platelets pretreated with the indicated inhibitors with or without the addition of ADP/U46619, and tyrosine phosphorylation was assessed. Data shown are representative of 3 independent experiments.