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 (O2−) is a free radical that can be converted into other reactive oxygen species such as peroxynitrite (ONOO−) that is formed from the reaction between O2− 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 O2−, 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 × 108 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 O2− 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 O2− generation. Superoxide anion signaling pathway includes ONOO− formation and α-actinin nitration.