Role of PKC-θ in α_IIbβ₃ activation and α-granule secretion downstream of GPVI and PARs. (A) Isolated platelets from PKC-θ^−/− mice (■) and WT littermates (□) were stimulated with GPVI agonist CRP (10 and 20 μg/mL) and PAR4 agonsit AYPGKF (100 and 200 μM) to test the JON/A binding to activated α_IIbβ₃ receptors. (B) WT and PKC-θ^−/− murine platelets and (C) washed human platelets with or without PKC-θ RACK peptide were stimulated with CRP (10 and 20 μg/mL) as well as AYPGKF (100 and 200 μM) and PAR1 agonist SFLLRN (5 μM; only in human platelets) for 15 minutes at 37°C in nonstirring condition in the presence of FITC-labeled anti–P-selectin (CD62)-antibody. Reactions were terminated by fixing the platelets with PFA and then analyzed by flow cytometry. Each bar is the average of 3 experiments plus or minus SD from 3 different donors. (D) Dot plots of dual-color labeling for JON/A binding and CD62 expression on murine platelets are representative data of panels A and B.