Anti-dengue virus nonstructural protein 1 antibodies recognize protein disulfide isomerase on platelets and inhibit platelet aggregation
Introduction
Dengue virus (DV) infection causes diseases ranging from mild dengue fever to severe dengue hemorrhagic fever or dengue shock syndrome (DHF/DSS) (Henchal and Putnak, 1990, Gubler, 1998, Clyde et al., 2006, Halstead, 2007). A number of mechanisms are involved in the pathogenesis of DHF/DSS progression, including antibody-dependent enhancement of infection (Halstead et al., 1984, Littaua et al., 1990, Mady et al., 1991, Morens, 1994, Anderson et al., 1997, Huang et al., 2006) and host abnormal immune responses to viral infection (Avirutnan et al., 1998, Green et al., 1999, Mathew et al., 1999, King et al., 2000, Lei et al., 2001, Ubol et al., 2008).
Hemorrhagic syndrome, a feature of DHF/DSS, is a hematologic abnormality resulting from multiple factors, including thrombocytopenia, coagulopathy, and vasculopathy related with dysfunction of platelets and endothelial cells (Rothman and Ennis, 1999, Green and Rothman, 2006). We and others have suggested a mechanism of molecular mimicry, in which dengue patient sera or antibodies (Abs) directed against DV nonstructural protein 1 (NS1) can cross-react with platelets and endothelial cells (Falconar, 1997, Falconar, 2007, Lin et al., 2001, Lin et al., 2002, Lin et al., 2003, Lin et al., 2008). Anti-platelet autoantibodies elicited by DV NS1 caused thrombocytopenia and mortality in mice (Sun et al., 2007). We have demonstrated that anti-DV NS1 Abs reduce platelet aggregation (Lin et al., 2008). However, the mechanism of anti-DV NS1-mediated inhibition of platelet aggregation remains unclear.
Protein disulfide isomerase (PDI), traditionally thought to be an endoplasmic reticulum protein, can also be expressed on the cell surface (Turano et al., 2002). PDI is localized to the platelet surface (Chen et al., 1995, Essex et al., 1995, Burgess et al., 2000) and is involved in regulation of integrin-mediated platelet aggregation (Essex and Li, 1999, Essex et al., 2001, Lahav et al., 2002, Manickam et al., 2008). Those studies also showed that anti-PDI Abs blocked platelet adhesion and aggregation. In the present study, we demonstrated that platelet membrane PDI can be recognized by anti-DV NS1 Abs. The PDI enzymatic activity and ADP-stimulated platelet aggregation were reduced by anti-DV NS1. The DV NS1 amino acid residues 311–330 (P311–330) represent a dominant epitope sharing sequence homology with the thioredoxin domain of PDI.
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Antibodies and reagents
PDI protein was obtained from TaKaRa (Japan) or ProSpec-Tany TechnoGene Ltd. (Israel). RNase A and cCMP were purchased from Sigma (St. Louis, MO, USA). Anti-PDI was obtained from Stressgen (Victoria, BC, Canada). Horseradish peroxidase (HRP)-conjugated goat anti-mouse, anti-rabbit and anti-human IgG, and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse and anti-human IgG were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). Synthetic peptides of NS1 amino
PDI in platelet membrane extract is recognized by anti-DV NS1 antibodies
Using Western blot analysis, we found that the proteins in platelet membrane extract recognized by anti-DV NS1 were predominantly 55–60 kDa proteins (Fig. 1A). PDI (58 kDa), which is present on the platelet surface and involved in platelet aggregation (Manickam et al., 2008), was examined for its possible role as an autoantigen recognized by anti-DV NS1 Abs. We showed that purified PDI protein can be directly recognized by anti-DV NS1 Abs, and, conversely, purified DV NS1 can be recognized by
Discussion
The hemorrhagic abnormalities of DHF/DSS include coagulopathy, vasculopathy, and thrombocytopenia. The presence of anti-platelet autoantibodies in dengue patient sera correlated with the severity of disease and thrombocytopenia (Oishi et al., 2003, Saito et al., 2004). The generation of anti-platelet Abs was also found in DV-infected mice (Huang et al., 2000). Anti-platelet autoantibodies were, at least in part, due to Abs against DV NS1 (Falconar, 1997, Sun et al., 2007, Lin et al., 2008). In
Conflict of interest
The authors have no financial conflict of interest.
Acknowledgements
We thank Dr. Robert Anderson for critical reading of this manuscript. This work was supported by Grants NSC97-3112-B006-004 from the National Research Program for Genomic Medicine, National Science Council, Taiwan, and NHRI-CN-CL96015 from the National Health Research Institutes, Taiwan.
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