Effect of protein kinase G inhibitors on phosphorylation and aggregation. (A) Human washed platelets (5 × 10⁸/mL), preincubated for 5 minutes with vehicle (DMSO 0.1%), the PKC inhibitor Ro 31-8220 (10 μM), or the PKG inhibitors Rp-8-pCPT-cGMPS (0.5 mM), Rp-8-Br-PET-cGMPS (0.5 mM), and KT5823 (10 μM), were stimulated by VWF and ristocetin (10 μg/mL and 1 mg/mL, respectively), the phorbol ester PMA (300 nM), or the cGMP raising agents glyco–SNAP-1 (10 μM) and sildenafil (30 μM). Reactions were stopped by addition of equal volumes of sample buffer and proteins separated by SDS-PAGE and Western blotted for (i) Ser²³⁹-phosphorylated VASP and (ii-iii) phosphorylated p42/44 MAP kinase. Blots were subsequently stripped and reprobed with a pan p42/44 MAP kinase Ab to ensure equal amounts of protein had been loaded. Blots are representative of 4 experiments. (B) Human PRP (3 × 10⁸ platelets/mL) was stimulated with ristocetin (Rist; 1.5 mg/mL) simultaneously with the NO donor glyco–SNAP-1 (1 μM) and the PKG inhibitors Rp-8-pCPT-cGMPS (0.5 mM) and Rp-8-Br-PET-cGMPS (0.5 mM) either on their own or in combination. Experiments were conducted in aggregometer cuvettes with stirring at 37°C. Arrows indicate addition of agonist. Results are representative of 5 experiments. (C) Washed platelets were pooled from (i) 16 wild-type and (ii) 16 PKG null mice and suspended at a concentration of 2 × 10⁸ platelets/mL. Platelets were preincubated for 5 minutes with vehicle (DMSO 0.1%), the PKG inhibitors Rp-8-pCPT-cGMPS (0.5 mM) or Rp-8-Br-PET-cGMPS (0.5 mM), and stimulated by a threshold concentration of thrombin (0.1 IU/mL). Experiments were conducted in aggregometer cuvettes with stirring at 37°C. Arrows indicate addition of agonist.