Elevation of cGMP inhibits activation ofαIIbβ3 by VWF. (Ai) Human PRP (3 × 108 platelets/mL) or (ii) human washed platelets (3 × 108 platelets/mL) were stimulated with ristocetin alone (Rist; 1.5 mg/mL), ristocetin in the presence of VWF (VWF and rist at 10 μg/mL and 1 mg/mL, respectively), the NO donors glyco–SNAP-1 (100 μM) and sodium nitroprusside (SNP; 100 μM), or the PDE 5 inhibitor sildenafil (30 μM) for 5 minutes. The cGMP content was analyzed using a cGMP direct Biotrak kit (Amersham Biosciences). Results are presented as cGMP concentration/108 platelets. Error bars represent SEM. (B) Human PRP (3 × 108 platelets/mL) was stimulated with ristocetin (Rist; 1.5 mg/mL) simultaneously to the addition of the NO donor (i) glyco–SNAP-1 and (ii) the PDE 5 inhibitor sildenafil at the indicated concentrations or (iii) glyco–SNAP-1 (1 μM) and sildenafil (1 μM) alone or in combination. Experiments were conducted in aggregometer cuvettes with stirring at 37°C. Arrows indicate addition of agonist. (C) Human PRP (3 × 108 platelets/mL) was stimulated with threshold concentrations of ristocetin (Rist; 1 mg/mL) simultaneously with the NO donor glyco–SNAP-1 or the PDE 5 inhibitor sildenafil at the indicated concentration. Experiments were conducted in aggregometer cuvettes with stirring at 37°C. Arrows indicate addition of agonist. Results are representative of 5 experiments.
