Enhanced nitric oxide and cyclic GMP formation plays a role in the anti-platelet activity of simvastatin

Abstract

Background and purpose: It has been found that 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) exert various vascular protective effects, beyond their cholesterol-lowering property, including inhibition of platelet-dependent thrombus formation. The objective of the present study was to determine whether the nitric oxide (NO)/cyclic GMP-mediated processes in platelets contribute to the anti-aggregatory activity of simvastatin.

Experimental approach: After rabbit platelets were incubated with simvastatin for 5 min, aggregation was induced and the platelet aggregation, nitric oxide synthase activity, guanylyl cyclase activity, NO and cyclic GMP formation were measured appropriately.

Key results: Treatment with simvastatin concentration-dependently inhibited platelet aggregation induced by collagen or arachidonic acid with an IC(50) range of 52-158 microM. We also demonstrated that simvastatin (20-80 microM) concentration-dependently further enhanced collagen-induced NO and cyclic GMP formation through increasing NOS activity (from 2.64+/-0.12 to 3.52+/-0.21-5.10+/-0.14 micromol min(-1) mg protein(-1)) and guanylyl cyclase activity (from 142.9+/-7.2 to 163.5+/-17.5-283.8+/-19.5 pmol min(-1) mg protein(-1)) in the platelets. On the contrary, inhibition of platelet aggregation by simvastatin was markedly attenuated (by about 50%) by addition of a nitric oxide synthase inhibitor, a NO scavenger or a NO-sensitive guanylyl cyclase inhibitor. The anti-aggregatory effects of simvastatin were significantly increased by addition of a selective inhibitor of cyclic GMP phosphodiesterase.

Conclusions and implications: Our findings indicate that enhancement of a NO/cyclic GMP-mediated process plays an important role in the anti-aggregatory activity of simvastatin.

Figure 1

Effects of simvastatin and combination with L-NAME or PTIO on platelet aggregation. (a) In the presence of L-arginine (100 μM), washed platelets were preincubated with DMSO or simvastatin (20–200 μM) for 5 min. Then, collagen (10 μg ml⁻¹) or AA (100 μM) was added to trigger platelet aggregation. (b) In the presence of L-arginine, washed platelets were preincubated with simvastatin (20–80 μM) or L-NAME (100 μM) and simvastatin (20–80 μM) for 5 min. Then, collagen was added to trigger platelet aggregation. (c) In the presence of L-arginine, washed platelets were preincubated with simvastatin (60 or 80 μM) or simvastatin (60 or 80 μM)+PTIO (50 μM) for 5 min, followed by addition of collagen. ^*P<0.05, ^**P<0.01 and ^***P<0.001, as compared with the respective simvastatin or collagen-treated platelets. Percent inhibition of platelet aggregation is presented as means±s.e.mean (n=6). AA, arachidonic acid; DMSO, dimethyl sulphoxide; L-NAME, N^G-nitro-L-arginine methyl ester; PTIO, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide.

Figure 2

Effects of simvastatin on NO release and NOS activity in platelets. (a) In the presence of L-arginine (100 μM), typical traces of NO release from platelets shown in the presence of DMSO alone (a) or in the presence of collagen (10 μg ml⁻¹) (b) or in the presence of simvastatin (60 μM) and collagen (10 μg ml⁻¹) (c) or in the presence of simvastatin (80 μM) and collagen (10 μg ml⁻¹) (d). These profiles are representative of five similar experiments. (b) In the presence of L-arginine, washed platelets were preincubated with simvastatin (20–80 μM) or DMSO for 5 min, followed by addition of collagen for 6 min. The homogenized precipitated platelets were used for NOS activity assay by measuring the NOx formation after a 15-min incubation at 37 °C with lysed platelets in reaction buffer. Data from washed platelets only suspended in Tyrode's solution without addition of collagen and other drugs are shown as the resting group. ^**P<0.01 and ^***P<0.001, as compared with the resting group; ^#P<0.001 compared with the collagen-stimulated platelets. All data are presented as means±s.e.mean (n=5). DMSO, dimethyl sulphoxide; NO, nitric oxide; NOS, nitric oxide synthase.

Figure 3

Effects of simvastatin on cyclic GMP formation and guanylyl cyclase activity in collagen-stimulated platelets. (a) In the presence of L-arginine (100 μM), washed platelets were preincubated with simvastatin (20–80 μM) or DMSO for 5 min, followed by addition of collagen (10 μg ml⁻¹) for 6 min. Then, the supernatant and precipitated platelets were collected for cyclic GMP and guanylyl cyclase activity assay, respectively. The incubation was stopped by immediately boiling for 3 min and the concentrations of cyclic GMP in supernatant were determined. (b) For guanylyl cyclase activity assay, the levels of cyclic GMP were measured following a 20-min incubation at 37 °C with platelet cytosol (1 μg) in reaction buffer. Data from washed platelets only suspended in Tyrode's solution without addition of collagen and other drugs are shown as the resting group. ^*P<0.05, ^**P<0.01 and ^***P<0.001, as compared with the resting group. All data were presented as means±s.e.mean (n=5). DMSO, dimethyl sulphoxide; cyclic GMP, 3′,5′-cyclic guanosine monophosphate.

Figure 4

Effects of ODQ and zaprinast on the inhibitory effect of simvastatin on platelet aggregation. In the presence of L-arginine (100 μM), washed platelets were preincubated with simvastatin (60 μM) alone or in the presence of ODQ (1 μM) or zaprinast (5 μM) for 5 min, and then collagen (10 μg ml⁻¹) was added to trigger platelet aggregation. ^*P<0.05 and ^**P<0.01, as compared with the simvastatin and collagen-treated platelets. Percent inhibition of platelet aggregation is presented as the means±s.e.mean (n=5). ODQ, 1H-[1,2,4] oxadiazolo [4,3-a]quinoxalin-1-one.