Elsevier

Molecular Immunology

Volume 47, Issues 2–3, December 2009, Pages 398-406
Molecular Immunology

Anti-dengue virus nonstructural protein 1 antibodies recognize protein disulfide isomerase on platelets and inhibit platelet aggregation

https://doi.org/10.1016/j.molimm.2009.08.033Get rights and content

Abstract

Hemorrhagic syndrome is a hallmark of severe dengue diseases. We previously suggested a mechanism of molecular mimicry in which antibodies against dengue virus (DV) nonstructural protein 1 (NS1) cross-react with platelets. In the present study, we demonstrate that protein disulfide isomerase (PDI) on the platelet surface is recognized by anti-DV NS1 antibodies. Anti-DV NS1 obtained from hyperimmunized mouse sera inhibited PDI activity and platelet aggregation, and both inhibitory effects were prevented when anti-DV NS1 antibodies were preabsorbed with PDI. Anti-PDI antibodies bound to a peptide consisting of amino acid residues 311–330 (P311–330) of NS1. This peptide was a predicted epitope analyzed by homologous sequence alignments between DV NS1 and PDI. The platelet binding activities of anti-PDI and anti-DV NS1 antibodies were both reduced by P311–330 preabsorption. Similar to the findings using anti-DV NS1, antibodies against P311–330 bound to PDI and platelets, followed by inhibition of PDI activity and platelet aggregation. Furthermore, the cross-reactivity of dengue hemorrhagic fever patient sera with platelets was reduced when patient sera were preabsorbed with PDI or P311–330. Dengue hemorrhagic fever patient sera also inhibited platelet aggregation, while PDI or P311–330 reduced this inhibitory effect. In conclusion, anti-DV NS1 antibodies cross-react with PDI on platelet surface causing inhibition of platelet aggregation, which may provide implications in dengue disease pathogenesis.

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.

Section snippets

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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