The role of superoxide anion in the inhibitory effect of SIN-1 in thrombin-activated human platelet adhesion.
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 x 10(8)platelets/ml) were added to adhere. Exposure of non-activated and thrombin-activated platelets to SIN-1 (0.001-100 microM) 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 alpha-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 alpha-actinin nitration.
Cardoso
Marcia H M
MH
Department of Pharmacology, Faculty of Medical Sciences, State University of Campinas, P.O. Box 6111, 13084-971 Campinas, Brazil.
Morganti
Rafael P
RP
Lilla
Sergio
S
Murad
Ferid
F
De Nucci
Gilberto
G
Antunes
Edson
E
Marcondes
Sisi
S
D001792:Blood Platelets / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D001798:Blood Proteins / Q000378:metabolism; D002448:Cell Adhesion / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D005340:Fibrinogen / Q000378:metabolism; D016166:Free Radical Scavengers / Q000494:pharmacology; D006162:Guanylate Cyclase / Q000037:antagonists & inhibitors; D006801:Humans; D008981:Molsidomine / Q000031:analogs & derivatives* / Q000494:pharmacology; D009566:Nitrates / Q000378:metabolism; D030421:Peroxynitrous Acid / Q000494:pharmacology; D012995:Solubility; D013481:Superoxides / Q000494:pharmacology*; D013917:Thrombin / Q000494:pharmacology*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
D001798:Blood Proteins; D004791:Enzyme Inhibitors; D016166:Free Radical Scavengers; D009566:Nitrates; D013481:Superoxides; D030421:Peroxynitrous Acid; C002744:3-nitrotyrosine; D014443:Tyrosine; C002385:linsidomine; D005340:Fibrinogen; D008981:Molsidomine; D013917:Thrombin; D006162:Guanylate Cyclase