Effects of simvastatin on (A) cyclic nucleotides (B) nitrate formations, (C) Ser157-vasodilator-stimulated phosphoprotein (VASP) and (D) phospholipase Cγ2 phosphorylation as well as (E and F) platelet aggregation in the presence of inhibitors of cyclic nucleotides in washed platelets. Platelets were incubated with prostaglandin E1 (PGE1, 10 μM), nitroglycerin (NTG, 10 μM), simvastatin (30 and 50 μM), or 0.5% DMSO. Cells were collected, and subcellular extracts were analyzed for (A) cyclic nucleotides, (B) nitrate formations (C) Ser157-VASP, and (D) phospholipase Cγ2 phosphorylations as described in "Methods". For platelet aggregation study, washed platelets were preincubated with simvastatin (50 μM) in the absence or presence of (E) ODQ (20 μM) or (F) SQ22536 (100 μM), followed by the addition of collagen (1 μg/ml). Data are presented as the means ± S.E.M. (n = 3-4); *P < 0.05, **P < 0.01, and ***P < 0.001, compared to the control group; #P < 0.05, compared to the without SQ22536 groups. ≠P < 0.05, compared to the collagen plus simvastatin group. The profiles (E and F) are representative examples of four similar experiments.