title: Essential role of the p38 mitogen-activated protein kinase pathway in tissue factor gene expression mediated by the phosphatidylserine-dependent antiprothrombin antibody.
abstract: OBJECTIVE
  The aim of this study was to investigate the effects of phosphatidylserine-dependent antiprothrombin antibody (aPS/PT) on the expression of tissue factor (TF) and the signal transduction pathway in procoagulant cells.
  
  
  METHODS
  Peripheral blood mononuclear cells (PBMCs) from a healthy donor, murine monocyte RAW264.7 cells and human umbilical vein endothelial cells (HUVECs) were treated with either IgG fractions obtained from APS patients who were positive for aPS/PT or a murine monoclonal aPS/PT antibody, 231D, in the presence of prothrombin. The levels of TF mRNA were measured using real-time PCR. TF function, as measured by procoagulant activity, was determined with a clotting assay. 231D binding on the surface of treated cells was determined by flow cytometric analysis. Screening for phosphorylation of intracellular signalling proteins was conducted using an array assay. Phosphorylation of p38 MAPK was quantitatively analysed with ELISA, and SB203580 was used as a specific inhibitor of p38 MAPK. Specific siRNA for p38 MAPK was used for the knockdown assay.
  
  
  RESULTS
  The IgG fractions from APS patients and 231D induced TF mRNA overexpression and shortening of coagulation time in cells in the presence of prothrombin. The 231D moiety induced phosphorylation of p38 MAPK after binding to the cell surface of RAW264.7 cells. SB203580 or p38 siRNA significantly hampered TF overexpression.
  
  
  CONCLUSIONS
  Expression of TF in procoagulant cells was induced by aPS/PT via p38MAPK phosphorylation. This phenomenon may be correlated with the thrombogenicity of APS.
authors.affiliation: Department of Medicine II, Hokkaido University Graduate School of Medicine, N15 W7, Kita-ku, Sapporo 060-8638, Japan. kenoku@med.hokudai.ac.jp.
authors.forename: Kenji
authors.initials: K
authors.lastname: Oku
authors.forename: Olga
authors.initials: O
authors.lastname: Amengual
authors.forename: Polona
authors.initials: P
authors.lastname: Zigon
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Horita
authors.forename: Shinsuke
authors.initials: S
authors.lastname: Yasuda
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Atsumi
mesh_terms: D000328:Adult; D000368:Aged; D000818:Animals; D016736:Antiphospholipid Syndrome / Q000097:blood / Q000276:immunology*; D001777:Blood Coagulation / Q000276:immunology; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000276:immunology; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D007963:Leukocytes, Mononuclear / Q000276:immunology; D020935:MAP Kinase Signaling System / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008875:Middle Aged; D009000:Monocytes / Q000276:immunology; D010718:Phosphatidylserines / Q000276:immunology*; D010766:Phosphorylation / Q000276:immunology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011516:Prothrombin / Q000276:immunology*; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D013925:Thromboplastin / Q000096:biosynthesis* / Q000235:genetics; D014471:Umbilical Veins / Q000378:metabolism; D015854:Up-Regulation / Q000276:immunology; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D007074:Immunoglobulin G; D010718:Phosphatidylserines; D047428:Protein Kinase Inhibitors; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011516:Prothrombin; D013925:Thromboplastin; D048051:p38 Mitogen-Activated Protein Kinases
keywords: antiphospholipid syndrome; antiprothrombin antibody; p38 MAPK; procoagulant cell activation; tissue factor