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Protein disulfide-isomerase mediates delivery of nitric oxide redox derivatives into platelets.

ABSTRACT: S-nitrosothiol compounds are important mediators of NO signalling and can give rise to various redox derivatives of NO: nitrosonium cation (NO+), nitroxyl anion (NO-) and NO* radical. Several enzymes and transporters have been implicated in the intracellular delivery of NO from S-nitrosothiols. In the present study we have investigated the role of GPx (glutathione peroxidase), the L-AT (L-amino acid transporter) system and PDI (protein disulfide-isomerase) in the delivery of NO redox derivatives into human platelets. Washed human platelets were treated with inhibitors of GPx, L-AT and PDI prior to exposure to donors of NO redox derivatives (S-nitrosoglutathione, Angeli's salt and diethylamine NONOate). Rapid delivery of NO-related signalling into platelets was monitored by cGMP accumulation and DAF-FM (4-amino-5-methylamino-2'7'-difluorofluorescein) fluorescence. All NO redox donors produced both a cGMP response and DAF-FM fluorescence in target platelets. NO delivery was blocked by inhibition of PDI in a dose-dependent manner. In contrast, inhibition of GPx and L-AT had only a minimal effect on NO-related signalling.PDI activity is therefore required for the rapid delivery into platelets of NO-related signals from donors of all NO redox derivatives. GPx and the L-AT system appeared to be unimportant in rapid NO signalling by the compounds used in the present study. This does not, however, exclude a possible role during exposure of cells to other S-nitrosothiol compounds, such as S-nitrosocysteine. These results further highlight the importance of PDI in mediating the action of a wide range of NO-related signals.

SUBMITTER: Bell SE

PROVIDER: S-EPMC1874238 | biostudies-other | 2007 Apr

REPOSITORIES: biostudies-other

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Project description:Essentials Nitric oxide synthesis controls protein disulfide isomerase (PDI) function. Nitric oxide (NO) modulation of PDI controls endothelial thrombogenicity. S-nitrosylated PDI inhibits platelet function and thrombosis. Nitric oxide maintains vascular quiescence in part through inhibition of PDI. SUMMARY: Background Protein disulfide isomerase (PDI) plays an essential role in thrombus formation, and PDI inhibition is being evaluated clinically as a novel anticoagulant strategy. However, little is known about the regulation of PDI in the vasculature. Thiols within the catalytic motif of PDI are essential for its role in thrombosis. These same thiols bind nitric oxide (NO), which is a potent regulator of vessel function. To determine whether regulation of PDI represents a mechanism by which NO controls vascular quiescence, we evaluated the effect of NO on PDI function in endothelial cells and platelets, and thrombus formation in vivo. Aim To assess the effect of S-nitrosylation on the regulation of PDI and other thiol isomerases in the vasculature. Methods and results The role of endogenous NO in PDI activity was evaluated by incubating endothelium with an NO scavenger, which resulted in exposure of free thiols, increased thiol isomerase activity, and enhanced thrombin generation on the cell membrane. Conversely, exposure of endothelium to NO+ carriers or elevation of endogenous NO levels by induction of NO synthesis resulted in S-nitrosylation of PDI and decreased surface thiol reductase activity. S-nitrosylation of platelet PDI inhibited its reductase activity, and S-nitrosylated PDI interfered with platelet aggregation, ?-granule release, and thrombin generation on platelets. S-nitrosylated PDI also blocked laser-induced thrombus formation when infused into mice. S-nitrosylated ERp5 and ERp57 were found to have similar inhibitory activity. Conclusions These studies identify NO as a critical regulator of vascular PDI, and show that regulation of PDI function is an important mechanism by which NO maintains vascular quiescence.

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Protein disulfide-isomerase mediates delivery of nitric oxide redox derivatives into platelets.

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