Molecular Immunology 47 (2009) 398–406 Contents lists available at ScienceDirect Molecular Immunology journal homepage: www.elsevier.com/locate/molimm Anti-dengue virus nonstructural protein 1 antibodies recognize protein disulfide isomerase on platelets and inhibit platelet aggregation Hsien-Jen Cheng a , Huan-Yao Lei b , Chiou-Feng Lin c , Yueh-Hsia Luo a , Shu-Wen Wan a , Hsiao-Sheng Liu b , Trai-Ming Yeh d , Yee-Shin Lin b,e,∗ a Institute of Basic Medical Sciences, National Cheng Kung University Medical College, Tainan, Taiwan b Department of Microbiology and Immunology, National Cheng Kung University Medical College, Tainan, Taiwan c Institute of Clinical Medicine, National Cheng Kung University Medical College, Tainan, Taiwan d Department of Medical Laboratory Science and Biotechnology, National Cheng Kung University Medical College, Tainan, Taiwan e Center for Gene Regulation and Signal Transduction Research, National Cheng Kung University, Tainan, Taiwan article info Article history: Received 28 June 2009 Received in revised form 13 August 2009 Accepted 28 August 2009 Available online 12 October 2009 Keywords: Dengue virus Nonstructural protein 1 Protein disulfide isomerase Antibodies Platelet aggregation Cross-reactive epitope 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 con- sisting 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. © 2009 Elsevier Ltd. All rights reserved. 1. 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, includ- ing 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). ∗ Corresponding author at: Department of Microbiology and Immunology, National Cheng Kung University Medical College, 1 University Road, Tainan 701, Taiwan. Fax: +886 6 2082705. E-mail address: yslin1@mail.ncku.edu.tw (Y.-S. Lin). Hemorrhagic syndrome, a feature of DHF/DSS, is a hematologic abnormality resulting from multiple factors, including thrombocy- topenia, 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, 2007; Lin et al., 2001, 2002, 2003, 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 sur- face (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 0161-5890/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.molimm.2009.08.033