Effects of PKG and PKA inhibitors on 8-pCPT-cGMP–induced phosphorylation of VASP in human platelets. Washed platelets were preincubated with (A) PKG inhibitor Rp-pCPT-cGMPS (0.2 and 0.5 mM) or PKA inhibitor Rp-Br-cAMPS (0.2 and 0.5 mM); and (B) PKA inhibitor, myristoylated PKI peptide (14-22) (5 μM) for 10 minutes. Platelets were also preincubated with buffer or DMSO as controls. Platelets were further treated with 8-pCPT-cGMP (20 μM) at 37°C in the platelet aggregometer for 10 minutes and solubilized in SDS-PAGE sample buffer. Phosphorylation of VASP was then detected by immunoblotting as described in Figure 1. Pictures shown in the figure are representative of 3 independent experiments. The bar graph depicts the quantitative results from 3 experiments obtained by scanning the 16C2 reactive bands and quantifying using NIH Image software. (C) Washed platelets were preincubated with Rp-pCPT-cGMPS (0.2 mM) or Rp-Br-cAMPS (0.2 mM) for 10 minutes. Platelets were then treated with 100 μM 8-pCPT-cGMP at 37°C for 10 minutes. Phosphorylation of VASP at Ser²³⁹ was detected by immunoblotting with the monoclonal antibody 16C2. Phosphorylation of VASP at Ser¹⁵⁷ was detected by immunoblotting with the monoclonal antibody 5C6.