The role of superoxide anion in the inhibitory effect of SIN-1 in thrombin-activated human platelet adhesion

• Published in: European journal of pharmacology Vol. 627; no. 1; pp. 229 - 234
• Main Authors: Cardoso, Marcia H.M., Morganti, Rafael P., Lilla, Sergio, Murad, Ferid, De Nucci, Gilberto, Antunes, Edson, Marcondes, Sisi
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 10-02-2010; Elsevier
• Subjects: 3-Morpholinosydnonimine (SIN-1); Biological and medical sciences; Blood Platelets - cytology; Blood Platelets - drug effects; Blood Platelets - metabolism; Blood Proteins - metabolism; Cell Adhesion - drug effects; Enzyme Inhibitors - chemistry; Enzyme Inhibitors - pharmacology; Fibrinogen - metabolism; Free Radical Scavengers - pharmacology; Guanylate Cyclase - antagonists & inhibitors; Humans; Medical sciences; Molsidomine - analogs & derivatives; Molsidomine - pharmacology; Nitrates - metabolism; Nitration; Peroxynitrite; Peroxynitrous Acid - pharmacology; Pharmacology. Drug treatments; Platelets; Solubility; Superoxide anion; Superoxides - pharmacology; Thrombin - pharmacology; Tyrosine - analogs & derivatives; Tyrosine - metabolism; Nitration; Superoxide anion; Peroxynitrite; Platelets; 3-Morpholinosydnonimine (SIN-1); Human; Serine endopeptidases; Enzyme; Thrombin; Adhesion; Peptidases; Anions; Hyperoxides; Platelet; Hydrolases
• ISSN: 0014-2999; 1879-0712
• DOI: 10.1016/j.ejphar.2009.10.060

Reactive oxygen species have an important role in the control of platelet activity. Superoxide anion (O
2
−) is a free radical that can be converted into other reactive oxygen species such as peroxynitrite (ONOO
−) that is formed from the reaction between O
2
− and nitric oxide (NO). There are conflicting data on ONOO
− effects in platelets because it presents pro- or anti-aggregatory actions. 3-morpholinosydnonimine (SIN-1) co-generates NO and O
2
−, yielding ONOO
−. Therefore, the present study aimed to investigate the mechanisms involved in the inhibition of human platelet adhesion by SIN-1. Microtiter plates were coated with human fibrinogen, after which washed platelets (6
×
10
8
platelets/ml) were added to adhere. Exposure of non-activated and thrombin-activated platelets to SIN-1 (0.001–100
μM) concentration-dependently inhibited adhesion, which was accompanied by marked increases in the cyclic GMP levels. In non-activated platelets, the soluble guanylate cyclase inhibitor ODQ prevented the SIN-1-induced cGMP elevations and adhesion inhibition. In thrombin-activated platelets, ODQ fully prevented the SIN-1-induced cGMP elevations, but only partly prevented the adhesion inhibition. The O
2
− and ONOO
− scavengers superoxide dismutase (SOD) and -(−)epigallocatechin gallate, respectively, had minimal effects in non-activated platelets. The inhibition of activated platelets by SIN-1 was reversed by SOD and partly reduced by ECG. Western blot analysis of SIN-1-treated platelets showed a single 105
kDa-nitrated band. Nanospray LC-MS-MS identified the protein containing 3-nitrotyrosine residues as human α-actinin-1-cytoskeletal isoform. Our data show that platelet adhesion inhibition by SIN-1 in activated platelets involves cGMP-independent mechanism through O
2
− generation. Superoxide anion signaling pathway includes ONOO
− formation and α-actinin nitration.