Inhibition of cAK and cGK by cyclic nucleotide analogs. (A) Inhibition of VASP phosphorylation by inhibitors of cAK and cGK. Washed platelets (3 × 10⁸/mL) were preincubated with 200 μM and 500 μM Rp-8-Br-Pet-cGMPS (cGMPS) and Rp-8-Br-cAMPS (cAMPS), respectively, and 5 μM myristoylated PKI for 10 minutes. Then, cells were incubated with 2 μM forskolin (2 minutes), 1 μM SNAP (5 minutes), or 20 μM 8-Br-PET-cGMP (10 minutes). VASP phosphorylation was detected by immunoblotting. (B) Activation of MAP kinases by PKI. Washed platelets (3 × 10⁸/mL) were incubated with 5 μM or 50 μM myristoylated PKI for 12 minutes. Phosphorylation of ERK (P-ERK) and p38 (P-p38) was detected by immunoblotting. Data shown are representative of at least 3 independent experiments. (C) FACS analysis of PKI-stimulated platelets. Washed platelets (3 × 10⁸/mL) were incubated with myristoylated PKI for 12 minutes and then analyzed by flow cytometry. Forward scatter (FSC-H) and side scatter (SSC-H) distributions of resting platelets (left panel) and platelets preincubated with 50 μM PKI (right panel) are shown. Note the scatter change of PKI-incubated platelets indicating the formation of platelet aggregates (arrow). Data shown are representative of 3 independent experiments.