Cyclic GMP-independent mechanisms contribute to the inhibition of platelet adhesion by nitric oxide donor: a role for alpha-actinin nitration

Abstract

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.

Fig. 1.

Inhibitory effects of SNP on human platelet adhesion to fibrinogen. Platelets (6 × 10⁶ platelets per well), in the absence or presence of thrombin (50 milliunits/ml), were incubated with SNP (0.001–1 mM) for 15 min (A) or 60 min (B) before platelet adhesion measurement. Results represent the mean ± SEM of three separate experiments, each performed in triplicate. ∗, P < 0.05 and ∗∗, P < 0.01, compared with the respective adhesion in the absence of thrombin. #, P < 0.05 and ##, P < 0.01, compared with the respective adhesion in 15 min.

Fig. 2.

Effect of the soluble guanylate cyclase inhibitor ODQ on cGMP levels (Upper) and inhibition of platelet adhesion (Lower) by SNP at 15 (A) and 60 (B) min. Nonactivated or thrombin (50 milliunits/ml)-activated platelets (1.2 × 10⁸ platelets/ml) were incubated with SNP (0.1 and 1 mM) for 15 or 60 min in the presence or absence of ODQ (10 μM). Dashed lines represent basal levels of cGMP. The results are shown as the means ± SEM (n = 3). ∗, P < 0.05, ∗∗, P < 0.01, and ∗∗∗, P < 0.001, compared with the experiments in the absence of ODQ.

Fig. 3.

Effect of SOD on inhibition of platelet adhesion by SNP at 15 (A) and 60 (B) min. Nonactivated or thrombin (50 milliunits/ml)-activated platelets (1.2 × 10⁸ platelets/ml) were incubated with SNP (0.1 and 1 mM) for either 15 or 60 min in the presence of SOD (100 units/ml). Results represent the means ± SEM (n = 3). ∗, P < 0.05 and ∗∗, P < 0.01, compared with the adhesion in absence of SOD.

Fig. 4.

Effect of ECG on inhibition of platelet adhesion by SNP at 15 (A) and 60 (B) min. Nonactivated or thrombin (50 milliunits/ml)-activated platelets (1.2 × 10⁸ platelets/ml) were incubated with SNP (0.1 and 1 mM) for either 15 min or 60 min in the presence of ECG (5 μM). Results represent the mean ± SEM (n = 3). ∗∗, P < 0.01 compared to the adhesion in absence of ECG.

Fig. 5.

Detection of nitrated protein in human platelets. Lines 1, 2, and 3 represent untreated platelets, and platelets incubated with SNP at 0.1 and 1 mM, respectively. Position of molecular marker (in kDa) is indicated on the left. (Lower) The graph shows densitometric analysis of nitrated protein bands in platelets from three different individuals. Dashed line represents background density. ∗, P < 0.05 and ∗∗, P < 0.01 compared with respective control. ##, P < 0.01 compared with nonactivated platelets.

Fig. 6.

Purification of nitrated protein in washed activated platelets. Coomassie-stained SDS-polyacrylamide gel of proteins contained in fractions from elution of Sephacryl S-200 HR column (A) and hydroxyapatite column (B). Western blotting analysis using monoclonal anti-nitrotyrosine antibody indicated the presence of a nitrated band in fractions 22 and 23, which were pooled and loaded onto a hydroxyapatite column. Numbers 1 and 2 represent the bound proteins eluted with 100 and 200 mM sodium phosphate. The arrowhead indicates the protein band excised and analyzed by using capillary liquid chromatography/electrospray ionization-MS/MS. Line H represents the proteins contained in the total material before chromatography (30 μg of protein per line). Position of the molecular marker (in kDa) is indicated on the left.