Platelet nitric oxide synthase is activated by tyrosine dephosphorylation: possible role for SHP‐1 phosphatase

Abstract

Summary. Background: Endothelial nitric oxide synthase (eNOS) activity in endothelial cells is regulated by posttranslational phosphorylation of critical serine, threonine and tyrosine residues in response to a variety of stimuli. However, the post-translational regulation of eNOS in platelets is poorly defined. Objectives: We investigated the role of tyrosine phosphorylation in the regulation of platelet eNOS activity. Methods: Tyrosine phosphorylation of eNOS and interaction with the tyrosine phosphatase SHP-1 were investigated by coimmunoprecipitation and immunoblotting. An in vitro immunoassay was used to determine eNOS activity together with the contribution of protein tyrosine phosphorylation. Results: We found platelet eNOS was tyrosine phosphorylated under basal conditions. Thrombin induced a dose-and timedependent increase in eNOS activity without altering overall level of tyrosine phosphorylation, although we did observe evidence of minor tyrosine dephosphorylation. In vitro tyrosine dephosphorylation of platelet eNOS using a recombinant protein tyrosine phosphatase enhanced thrombin-induced activity compared to thrombin alone, but had no effect on endothelial eNOS activity either at basal or after stimulation with bradykinin. Having shown that dephosphorylation could modulate platelet eNOS activity we examined the role of potential protein phosphatases important for platelet eNOS activity. We found SHP-1 protein tyrosine phosphatase, coassociated with platelet eNOS in resting platelets, but does not associate with eNOS in endothelial cells. Stimulation of platelets with thrombin increased SHP-1 association with eNOS, while inhibition of SHP-1 abolished the ability of thrombin to induce elevated eNOS activity. Conclusions: Our data suggest a novel role for tyrosine dephosphorylation in platelet eNOS activation, which may be mediated by SHP-1.

The Endothelial Tyrosine Phosphatase SHP-1 Plays an Important Role for Vascular Haemostasis in TNFα-Induced InflammationIn Vivo

“…This is of special interest especially in the light of a previous study where platelets isolated from “motheaten viable mice”, which have a defect in the SHP-1 gene, were hyporesponsive to GPVI stimulation suggesting a physiological role for SHP-1 in lowering the threshold for activation by GPVI [27]. In addition, a role for SHP-1 in the regulation of platelet eNOS has been reported [37]. To elucidate whether the observed prothrombotic effects in vivo could be due to direct effects of SHP-1 inhibition by sodium stibogluconate in platelets we performed in vitro (human PRP) and ex vivo (mouse blood) aggregation studies.…”

Unresolved roles of platelet nitric oxide synthase

“…Treatment of platelets with sodium orthovanadate, a tyrosine phosphatase inhibitor, increased tyrosine phosphorylation of NOS, resulting in a concomitant decrease in basal enzyme activity and blunting of thrombin‐induced cGMP formation [61]. Consistent with this, dephosphorylation of NOS from thrombin‐stimulated platelets increased enzyme activity, suggesting that cellular phosphatases play a role in NOS activation [62]. Furthermore, we found that platelet eNOS formed a complex with SHP‐1 phosphatase, and that SHP‐1 regulated NO synthesis in response to thrombin [62].…”

“…Initial studies suggested that phosphorylation of unknown serine/threonine and tyrosine residues controls the activity of NOS in collagen‐stimulated platelets [67]. Using phosphospecific NOS antibodies, only phosphorylation at ser 1177 has been demonstrated in platelet NOS, but only in response to insulin [48] and adenosine [50] and not platelet agonists [61,62]. Insulin induces phosphorylation of platelet NOS on ser 1177 through activation of PI3kinase and AMP activated protein kinase (AMPK), and this was associated with increased cGMP levels [48].…”

Reactive Nitrogen Species and Hydrogen Sulfide as Regulators of Protein Tyrosine Phosphatase Activity

“…Platelet stimulation by thrombin further increases SHP-1 association with eNOS. Association of SHP-1 with eNOS was confirmed in both resting and thrombin-activated platelets, but it was not 2204 HENEBERG detected in the endothelial cells (68). Platelet treatment with 0.1-1.0 mM sodium orthovanadate was shown to increase tyrosine phosphorylation of eNOS, leading to the decrease of basal eNOS activity and the blunting of thrombin-induced guanosine 3¢,5¢-monophosphate (cGMP) formation; the vanadate-induced changes were abolished when the platelets were preincubated with the Src kinase inhibitor PP2, but not by the PKC inhibitor Ro-31-8220 or PI3K inhibitor wortmannin (61).…”