Inhibition of thrombin-stimulated calcium transients and U46619-induced platelet activation in human platelets by both cGK activators and inhibitors. (A) Calcium transients. Fura-2–loaded washed human platelets were resuspended in HEPES buffer and preincubated (1 minute) either with vehicle (Control), 5 μM SNP (i), 100 μM 8-Br-PET-cGMP or 100 μM Rp-8-Br-PET-cGMPS (ii), or 100 μM 8-pCPT-cGMP or 100 μM Rp-8-pCPT-cGMPS (iii). Then, platelets were stimulated with thrombin (0.2 U/mL) in the presence of 1 mM CaCl₂ and 10 mM MgCl₂, and the time-dependent increase of fluorescence was monitored. Abscissa values are arbitrary units of Fura-2 fluorescence intensity. The data are representative of 3 different experiments. (B) U46619-induced platelet stimulation. Washed platelets (3 × 10⁸/mL) were preincubated with buffer, SNP (5 μM) or forskolin (5 μM) for 1 minute, or cGMP analogs (200 μM) for 10 minutes followed by 1-minute incubation with U46619 (2 μM). Platelet activation was monitored by the analysis of P-selectin expression (FACS) and ERK phosphorylation. Activation of cGMP- and cAMP-dependent protein kinases by SNP and forskolin, respectively, was monitored by VASP phosphorylation. Note the inhibition of P-selectin expression and ERK phosphorylation by both cGK activators and inhibitors. Shown are representatives of 3 different experiments as means ± SEM.