title: Ribophorin II is involved in the tissue factor expression mediated by phosphatidylserine-dependent antiprothrombin antibody on monocytes.
abstract: OBJECTIVE
  Phosphatidylserine-dependent, also called aPS-PT, recognizes the phosphatidylserine-prothrombin complex, which is associated with APS. We have previously reported that aPS-PT induces tissue factor (TF) expression on monocytes through the p38 mitogen-activated protein kinase pathway. However, the cell surface interaction between prothrombin and aPS-PT, which is involved in the activation of cell-signalling pathways, has remained unknown. The objective of this study was to identify membrane proteins involved in the binding of prothrombin and aPS-PT to monocyte surfaces as well as the induction of TF expression.
  
  
  METHODS
  RAW264.7 cells with FLAG-tagged prothrombin were incubated and separated using affinity chromatography with anti-FLAG antibody-conjugated Sepharose beads. Immunopurified proteins were then analysed by an online nano-liquid chromatography-tandem mass spectrometry. The binding between prothrombin and the identified protein, ribophorin II (RPN2), was analysed by ELISA and surface plasmon resonance. To elucidate the role of RPN2 in TF expression, the TF mRNA level in RAW264.7 cells treated with RPN2 small interfering RNA was determined by quantitative real-time PCR (qPCR).
  
  
  RESULTS
  RPN2 was identified as a candidate molecule involved in the binding of prothrombin to the cell surface. The binding between prothrombin and RPN2 was confirmed by ELISA and surface plasmon resonance. RAW264.7 cells treated with RPN2 small interfering RNA showed significant reduction of the TF expression mediated by prothrombin and a mouse monoclonal aPS-PT.
  
  
  CONCLUSIONS
  We identified that RPN2 is one of the prothrombin-binding proteins on monocyte surfaces, suggesting that RPN2 is involved in the pathophysiology of thrombosis in patients with APS.
authors.affiliation: Division of Rheumatology, Endocrinology and Nephrology, Hokkaido University Graduate School of Medicine, Sapporo, at3tat@med.hokudai.ac.jp.
authors.forename: Yuichiro
authors.initials: Y
authors.lastname: Fujieda
authors.affiliation: Division of Rheumatology, Endocrinology and Nephrology, Hokkaido University Graduate School of Medicine, Sapporo.
authors.forename: Olga
authors.initials: O
authors.lastname: Amengual
authors.affiliation: Division of Proteomics, Multi-scale Research Center for Prevention of Medical Science, Medical Institute of Bioregulation, Kyushu University, Fukuoka.
authors.forename: Masaki
authors.initials: M
authors.lastname: Matsumoto
authors.affiliation: Laboratory of Biomolecular Science, Faculty of Pharmaceutical Sciences, Hokkaido University and.
authors.forename: Kimiko
authors.initials: K
authors.lastname: Kuroki
authors.affiliation: Department of Biochemistry, Hokkaido University Graduate School of Medicine, Sapporo, Japan.
authors.forename: Hidehisa
authors.initials: H
authors.lastname: Takahashi
authors.affiliation: Division of Rheumatology, Endocrinology and Nephrology, Hokkaido University Graduate School of Medicine, Sapporo.
authors.forename: Michihito
authors.initials: M
authors.lastname: Kono
authors.affiliation: Division of Rheumatology, Endocrinology and Nephrology, Hokkaido University Graduate School of Medicine, Sapporo.
authors.forename: Takashi
authors.initials: T
authors.lastname: Kurita
authors.affiliation: Division of Rheumatology, Endocrinology and Nephrology, Hokkaido University Graduate School of Medicine, Sapporo.
authors.forename: Kotaro
authors.initials: K
authors.lastname: Otomo
authors.affiliation: Division of Rheumatology, Endocrinology and Nephrology, Hokkaido University Graduate School of Medicine, Sapporo.
authors.forename: Masaru
authors.initials: M
authors.lastname: Kato
authors.affiliation: Division of Rheumatology, Endocrinology and Nephrology, Hokkaido University Graduate School of Medicine, Sapporo.
authors.forename: Kenji
authors.initials: K
authors.lastname: Oku
authors.affiliation: Division of Rheumatology, Endocrinology and Nephrology, Hokkaido University Graduate School of Medicine, Sapporo.
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Bohgaki
authors.affiliation: Division of Rheumatology, Endocrinology and Nephrology, Hokkaido University Graduate School of Medicine, Sapporo.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Horita
authors.affiliation: Division of Rheumatology, Endocrinology and Nephrology, Hokkaido University Graduate School of Medicine, Sapporo.
authors.forename: Shinsuke
authors.initials: S
authors.lastname: Yasuda
authors.affiliation: Laboratory of Biomolecular Science, Faculty of Pharmaceutical Sciences, Hokkaido University and.
authors.forename: Katsumi
authors.initials: K
authors.lastname: Maenaka
authors.affiliation: Department of Biochemistry, Hokkaido University Graduate School of Medicine, Sapporo, Japan.
authors.forename: Shigetsugu
authors.initials: S
authors.lastname: Hatakeyama
authors.affiliation: Division of Proteomics, Multi-scale Research Center for Prevention of Medical Science, Medical Institute of Bioregulation, Kyushu University, Fukuoka.
authors.forename: Keiichi I
authors.initials: KI
authors.lastname: Nakayama
authors.affiliation: Division of Rheumatology, Endocrinology and Nephrology, Hokkaido University Graduate School of Medicine, Sapporo.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Atsumi
mesh_terms: D000818:Animals; D017152:Antibodies, Antiphospholipid / Q000276:immunology / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D009000:Monocytes / Q000276:immunology / Q000378:metabolism*; D010718:Phosphatidylserines / Q000378:metabolism; D011516:Prothrombin / Q000276:immunology; D000067996:RAW 264.7 Cells; D012333:RNA, Messenger / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D013925:Thromboplastin / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases
chemical_list: D017152:Antibodies, Antiphospholipid; D008565:Membrane Proteins; D010718:Phosphatidylserines; D012333:RNA, Messenger; C034241:ribophorin; D011516:Prothrombin; D013925:Thromboplastin; D048051:p38 Mitogen-Activated Protein Kinases
keywords: anti-phospholipid antibodies; anti-phospholipid syndrome; lupus anticoagulant; prothrombin