Potent and rapid inhibition of thrombin-induced platelet activation by both cyclic nucleotide-based cGK activators and inhibitors. (A) cGK activators and inhibitors. Washed platelets (3 × 10⁸/mL) were preincubated with buffer, 5 μM SNP for 1 minute, or cGMP analogs (200 μM) as indicated for 10 minutes followed by a 1-minute incubation with thrombin (0.5 U/mL). Platelet activation was monitored by the analysis of P-selectin expression (FACS) and ERK phosphorylation (Western blots), cGK activation was monitored by VASP phosphorylation (P-VASP Western blots). Note that both cGK activators (SNP, 8-Br-PET-cGMP, 8-pCPT-cGMP) as well as cGK inhibitors (the stereoisomers Rp-8-Br-PET-cGMPS, Rp-8-pCPT-cGMPS) inhibit thrombin-induced platelet activation (P-selectin expression, ERK phosphorylation). The data shown are representative of 4 different experiments. (B) Concentration-dependent effects of Rp-8-Br-PET-cGMPS. Washed human platelets (3 × 10⁸/mL) were preincubated for 1 minute with 2 μM SNP or with Rp-8-Br-PET-cGMPS (concentration as indicated) followed by another 1-minute incubation with thrombin (0.5 U/mL). After stopping, the platelets were analyzed by FACS for P-selectin expression and by Western blot for VASP and ERK phosphorylation (P-VASP and P-ERK). Rp-8-Br-PET-cGMPS, starting from 1 μM concentration, significantly (28 ± 6%, P < .05, n = 4) inhibited P-selectin expression and ERK phosphorylation. Shown are representatives of 4 different experiments as means ± SEM.