Established cGK activators and other cGMP analogs alone do not stimulate P-selectin expression and/or ERK phosphorylation in washed human platelets. (A) Regular conditions. Washed human platelets (3 × 10⁸/mL) were incubated with thrombin (0.5 U/mL) for 1 minute, and SNP (5 μM) or cGMP analogs (200 μM) for 1 or 10 minutes as indicated. Samples were then analyzed for P-selectin expression (FACS) and VASP/ERK phosphorylation (Western blots P-VASP and P-ERK). Thrombin-induced ERK-phosphorylation and P-selectin expression (mean fluorescence from 40 ± 12 in control to 582 ± 24, P < .01, n = 5, taken as 1) were used as positive controls for platelet activation. With respect to the effect of cGMP analogs on P-selectin expression, ERK phosphorylation, and VASP phosphorylation, corresponding pairs of cGK activators/cGK “inhibitors” (8-pCPT-cGMP/Rp-8-pCPT-cGMPS; 8-Br-PET-cGMP/Rp-8-Br-PET-cGMPS) or cyclic nucleotides/noncyclic nucleotides (8-Br-cGMP/8-Br-GMP; cGMP/GMP) were compared. Shown here are representatives of 5 different experiments as means ± SEM. (B) Stirring conditions. Washed human platelets (3 × 10⁸/mL) were incubated together with the substances indicated (U46619 [2 μM], cyclic nucleotides [200 μM], VWF + ristocetin [7.5μg/mL/0.5 mg/mL]) in a turbidometric platelet aggregometer without (0 rpm), or with (1000 rpm) stirring. After 1 minute of incubation time, SDS-stop solution was directly added to the cuvette, and platelet lysates were then analyzed for p38MAPK, P-VASP, and ERK phosphorylation by Western blots. Note that stirring of platelets alone increased p38, VASP, and ERK phosphorylation, but that only U46619 (used here as an established platelet activator) further enhanced ERK phosphorylation. Results are representative of 3 different experiments. Risto indicates ristocetin.