Cyclic GMP-independent mechanisms contribute to the inhibition of platelet adhesion by nitric oxide donor: a role for alpha-actinin nitration.
The nitric oxide-mediated actions are mostly due to cyclic GMP (cGMP) formation, but cGMP-independent mechanisms, such as tyrosine nitration, have been suggested as potential signaling pathways modulating the NO-induced responses. However, the mechanisms that lead to tyrosine nitration in platelets are poorly studied, and the protein targets of nitration have not been identified in these cells. Therefore, we have used the model of platelet adhesion to fibrinogen-coated plates to investigate the cGMP-independent mechanisms of the NO-donor sodium nitroprusside (SNP) that leads to inhibition of platelet adhesion. SNP concentration-dependently inhibited platelet adhesion, as observed at 15-min and 60-min adhesion. Additionally, SNP markedly increased the cGMP levels, and the soluble guanylate inhibitor ODQ nearly abolished the SNP-mediated cGMP elevations in all experimental conditions used. Nevertheless, ODQ failed to affect the adhesion inhibition obtained with 1.0 mM SNP at 15 min. On the other hand, superoxide dismutase or peroxynitrite (ONOO(-)) scavenger epigallocatechin gallate significantly reversed the inhibition of platelet adhesion by SNP (1 mM, 15 min). Western blot analysis in SNP (1 mM, 15 min)-treated platelets showed a single tyrosine-nitrated protein with an apparent mass of approximately 105 kDa. Nanospray LC-MS/MS identified the human alpha-actinin 1 cytoskeletal isoform (P12814) as the protein contained in the nitrated SDS gel band. Thus, tyrosine nitration of alpha-actinin, through ONOO(-) formation, may be a key modulatory mechanism to control platelet adhesion.
Department of Pharmacology, Faculty of Medical Sciences, State University of Campinas, Brazil. s_marcondes@uol.com.br
Sisi
S
Marcondes
Marcia H M
MH
Cardoso
Rafael P
RP
Morganti
Sara M
SM
Thomazzi
Sergio
S
Lilla
Ferid
F
Murad
Gilberto
G
De Nucci
Edson
E
Antunes
D000185:Actinin / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D001792:Blood Platelets / Q000737:chemistry / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D002392:Catechin / Q000031:analogs & derivatives / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000378:metabolism*; D005340:Fibrinogen / Q000378:metabolism; D006162:Guanylate Cyclase / Q000037:antagonists & inhibitors / Q000378:metabolism; D006801:Humans; D009566:Nitrates / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000378:metabolism / Q000494:pharmacology*; D009599:Nitroprusside / Q000494:pharmacology; D010973:Platelet Adhesiveness / Q000187:drug effects*; D012995:Solubility; D013482:Superoxide Dismutase / Q000037:antagonists & inhibitors / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism
D009566:Nitrates; D020030:Nitric Oxide Donors; D000185:Actinin; D009599:Nitroprusside; D014443:Tyrosine; D002392:Catechin; D005340:Fibrinogen; D013482:Superoxide Dismutase; D013917:Thrombin; D006162:Guanylate Cyclase; D006152:Cyclic GMP; C057580:gallocatechol