Role of cell surface protein disulphide isomerase in the platelet-selective action of S-nitrosoglutathione

Fang Xiao, Michael P. Gordge. University of Westminster, School of Life Sciences, 115 New Cavendish Street, W1W 6UW, London, United Kingdom.

Administration of S-nitrosoglutathione (GSNO) to patients is reported to inhibit platelets at doses that produce no vasodilation, suggesting a platelet-selective effect that might be useful in antithrombotic therapy (1), however the mechanism involved remains unknown. We previously showed that cell surface protein disulphide isomerase (csPDI) was required for rapid NO delivery into human platelets from NO donors (2). We have now explored expression and activity of csPDI on platelets and cells of the vascular wall, and their dependence on PDI for NO delivery from GSNO, to further define the platelet-selective properties of GSNO.

Washed human platelets were obtained by centrifugation and separation on Sepharose 2B. Human coronary artery endothelial (HCAEC) and smooth muscle cells (HCASMC) were purchased from PromoCell and cultured up to 3–5 passages. Expression of csPDI was analyzed using flow cytometry. csPDI reductase activity was monitored using synthetic pseudosubstrate dieosin glutathione disulfide (Di-E-GSSG) in a microplate fluorescence assay. To determine the effect of cell activation, cells were incubated with phorbol myristate acetate (PMA) or ionomycin (1-100 μM), prior to measurement of csPDI activity.

Intracellular delivery of NO from GSNO (0.1 - 100 μM) was measured fluorometrically using DAF-FM. In further experiments, csPDI on platelets, HCAEC and HCASMC was inhibited with 100 μM of phenylarsine oxide (PAO), prior to addition of 1 mM GSNO.

Flow cytometry revealed csPDI on all three cell types, but percentage positivity was higher on platelets (25.0% +/- 2.0) than on HCAEC (7.5% +/- 0.8) or HCASMC (11.9% +/- 0.9) (p < 0.01). csPDI reductase activity (measured as percentage of maximum fluorescence obtained using 10mM DTT) was also higher on platelets than on either vascular wall cell type (platelets 50.8% +/- 1.8, HCAEC 10.2% +/- 0.8, HCASMC 3.4% +/- 1.0, p < 0.01). Cellular activation with either PMA or ionomycin increased csPDI activity on both platelets (maximum rise 32% +/- 3.5 and 19% +/- 2.9 respectively) and smooth muscle cells (maximum rise 37% +/- 1.2 and 19% +/- 2.4, respectively) (p < 0.01 compared with untreated controls), but not on endothelium. Intracellular NO delivery from GSNO was greater in platelets, compared with HCAEC and HCASMC (p < 0.05 by 2 way ANOVA of dose response curves; EC50 values for platelets, HCAEC and HCASMC 10, 176 and 300 μM, respectively), and PAO caused a greater degree of inhibition of NO delivery in platelets than in vascular cells (Platelets 23% +/- 4.1, HCAEC 7% +/- 2.3, HCASMC 13% +/- 2.6, P < 0.05).

Platelets show higher activity of csPDI than endothelial or vascular smooth muscle cells, and this coincides with increased NO delivery from GSNO. Increased csPDI on platelets, compared with cells of vascular wall, may explain the platelet-selective actions of GSNO.

De Belder AJ, MacAllister R, Radomski MW, Moncada S, Vallance PJ (1994). Effects of S-nitroso-glutathione in the human forearm circulation: evidence for selective inhibition of platelet activation. Cardiovasc Res 28: 691-694.

Bell SE, Shah CM Gordge MP (2007) Protein disulphide isomerase mediates delivery of nitric oxide redox derivatives into platelets. Biochem J 403: 283-288

This work was supported by British Heart Foundation