Dengue virus nonstructural protein NS1 binds to prothrombin/thrombin and inhibits prothrombin activation.

OBJECTIVES Dengue virus (DENV) infection may result in severe dengue hemorrhage fever (DHF). However the mechanisms to cause hemorrhage during DENV infection are not fully understood. The sera level of secreted DENV nonstructural protein 1 (NS1) is correlated with the development of DHF. However, whether secreted NS1 can interfere with coagulation and contribute to the hemorrhage in DHF is unknown. Since thrombin plays a very important role in the activation of coagulation, we investigated whether NS1 can bind to thrombin and affect its formation or activity. METHODS AND RESULTS We first demonstrated that NS1 could bind to thrombin and formed NS1/thrombin complex in dengue patients' sera by enzyme-linked immunosorbent assay (ELISA). The ability of NS1 binding to prothrombin or thrombin was further confirmed using recombinant NS1 (rNS1) by ELISA, co-immunoprecipitation, and rNS1-affinity column purification. Even though the binding of rNS1 to thrombin showed no effect on thrombin activity, rNS1 could inhibit prothrombin activation and prolong activated partial thromboplastin time (APTT) of human platelet poor plasma. CONCLUSION These results suggest secreted DENV NS1 may bind to prothrombin and inhibit it activation, which in turn, may contribute to the APTT prolongation and hemorrhage in DHF patients.

[1]  C. Gelfand,et al.  Thrombin induces broad spectrum proteolysis in human serum samples , 2009, Clinical chemistry and laboratory medicine.

[2]  Yee‐Shin Lin,et al.  Activation of coagulation and fibrinolysis during dengue virus infection , 2001, Journal of Medical Virology.

[3]  M. Diamond,et al.  Secreted NS1 of Dengue Virus Attaches to the Surface of Cells via Interactions with Heparan Sulfate and Chondroitin Sulfate E , 2007, PLoS pathogens.

[4]  M. Diamond,et al.  Antagonism of the complement component C4 by flavivirus nonstructural protein NS1 , 2010, The Journal of experimental medicine.

[5]  Duane J. Gubler,et al.  Dengue and Dengue Hemorrhagic Fever , 1998, Clinical Microbiology Reviews.

[6]  Pojen P. Chen,et al.  Antibodies to Serine Proteases in the Antiphospholipid Syndrome , 2010, Current rheumatology reports.

[7]  Yi-Ling Lin,et al.  High‐level expression of recombinant dengue viral NS‐1 protein and its potential use as a diagnostic antigen , 2001, Journal of medical virology.

[8]  Jacqueline L Deen,et al.  The WHO dengue classification and case definitions: time for a reassessment , 2006, The Lancet.

[9]  Y. Nemerson,et al.  Zymogens and cofactors of blood coagulation. , 1980, CRC critical reviews in biochemistry.

[10]  Eva Harris,et al.  Global spread and persistence of dengue. , 2008, Annual review of microbiology.

[11]  H. ten Cate,et al.  Is clinical outcome of dengue-virus infections influenced by coagulation and fibrinolysis? A critical review of the evidence. , 2003, The Lancet. Infectious diseases.

[12]  Yee‐Shin Lin,et al.  Molecular mimicry between virus and host and its implications for dengue disease pathogenesis , 2011, Experimental biology and medicine.

[13]  A. Nisalak,et al.  High circulating levels of the dengue virus nonstructural protein NS1 early in dengue illness correlate with the development of dengue hemorrhagic fever. , 2002, The Journal of infectious diseases.

[14]  P. Young,et al.  An Antigen Capture Enzyme-Linked Immunosorbent Assay Reveals High Levels of the Dengue Virus Protein NS1 in the Sera of Infected Patients , 2000, Journal of Clinical Microbiology.

[15]  Yee‐Shin Lin,et al.  Endothelial Cell Apoptosis Induced by Antibodies Against Dengue Virus Nonstructural Protein 1 Via Production of Nitric Oxide1 , 2002, The Journal of Immunology.

[16]  J. d'Alayer,et al.  Secreted dengue virus nonstructural protein NS1 is an atypical barrel-shaped high-density lipoprotein , 2011, Proceedings of the National Academy of Sciences.

[17]  Yee‐Shin Lin,et al.  Anti-dengue virus nonstructural protein 1 antibodies recognize protein disulfide isomerase on platelets and inhibit platelet aggregation. , 2009, Molecular immunology.

[18]  Yee‐Shin Lin,et al.  Deletion of the C-Terminal Region of Dengue Virus Nonstructural Protein 1 (NS1) Abolishes Anti-NS1-Mediated Platelet Dysfunction and Bleeding Tendency1 , 2009, The Journal of Immunology.

[19]  K Fujikawa,et al.  The coagulation cascade: initiation, maintenance, and regulation. , 1991, Biochemistry.

[20]  A. Basu,et al.  Vascular endothelium: the battlefield of dengue viruses , 2008, FEMS immunology and medical microbiology.

[21]  V. Deubel,et al.  Enzyme-Linked Immunosorbent Assay Specific to Dengue Virus Type 1 Nonstructural Protein NS1 Reveals Circulation of the Antigen in the Blood during the Acute Phase of Disease in Patients Experiencing Primary or Secondary Infections , 2002, Journal of Clinical Microbiology.

[22]  M. Diamond,et al.  Binding of Flavivirus Nonstructural Protein NS1 to C4b Binding Protein Modulates Complement Activation , 2011, The Journal of Immunology.

[23]  Trai-Ming Yeh,et al.  Correlation of serum levels of macrophage migration inhibitory factor with disease severity and clinical outcome in dengue patients. , 2006, The American journal of tropical medicine and hygiene.

[24]  S. Halstead,et al.  Association between sex, nutritional status, severity of dengue hemorrhagic fever, and immune status in infants with dengue hemorrhagic fever. , 2005, The American journal of tropical medicine and hygiene.

[25]  J. Lepault,et al.  Dengue Virus Type 1 Nonstructural Glycoprotein NS1 Is Secreted from Mammalian Cells as a Soluble Hexamer in a Glycosylation-Dependent Fashion , 1999, Journal of Virology.

[26]  J. Farrar,et al.  Hemostatic changes in Vietnamese children with mild dengue correlate with the severity of vascular leakage rather than bleeding. , 2009, The American journal of tropical medicine and hygiene.

[27]  Philippe Dussart,et al.  Evaluation of Two New Commercial Tests for the Diagnosis of Acute Dengue Virus Infection Using NS1 Antigen Detection in Human Serum , 2008, PLoS neglected tropical diseases.

[28]  M. Teixeira,et al.  Contribution of macrophage migration inhibitory factor to the pathogenesis of dengue virus infection , 2010, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.