Phosphorylation of VASP induced by forskolin and effects of cGMP on platelet cAMP levels. (A) Washed platelets were stimulated for increasing lengths of time with 1 μM of forskolin. (B) Platelets were preincubated with Rp-pCPT-cGMPS (0.2 and 0.5 mM) or Rp-Br-cAMPS (0.2 and 0.5 mM) for 10 minutes prior to stimulation with forskolin (1 μM) for 5 minutes. Phosphorylation of VASP was then detected by immunoblotting as described in Figure 1. Data shown are representative of at least 3 independent experiments. (C) Washed platelets (3 × 10⁸/mL) in Tyrode solution were incubated with 8-pCPT-cGMP (0.1 mM) or 8-bromo-cGMP (0.1 mM) for 5 minutes at 37°C. The reaction was stopped by addition of equal volumes of ice-cold 12% (wt/vol) trichloroacetic acid. Platelet cAMP levels then were determined using a cAMP enzyme immunoassay kit from Amersham-Pharmacia Biotech. To exclude and correct possible influence of cGMP analogs on cAMP enzyme immunoassay analysis, cAMP standards also were analyzed in the presence of identical concentrations of the cGMP analogs. Shown in the figure are data (mean ± SD) from 3 samples. Please note that cAMP levels in 8-bromo-cGMP– or 8-pCPT-cGMP–treated platelets were significantly higher than control platelets (P < .001) as determined by Students t test.