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Ribophorin II is involved in the tissue factor expression mediated by phosphatidylserine-dependent antiprothrombin antibody on monocytes.
- Source :
- Rheumatology; Jun2016, Vol. 55 Issue 6, p1117-1126, 10p, 1 Diagram, 1 Chart, 4 Graphs
- Publication Year :
- 2016
-
Abstract
- Objective. Phosphatidylserine-dependent, also called aPS-PT, recognizes the phosphati dylserine-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. Conclusion. 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. [ABSTRACT FROM AUTHOR]
- Subjects :
- ANTIPHOSPHOLIPID syndrome
CELL culture
CHI-squared test
ENZYME-linked immunosorbent assay
FISHER exact test
FLOW cytometry
GENE expression
IMMUNOGLOBULINS
LIQUID chromatography
MASS spectrometry
MEMBRANE proteins
MONOCLONAL antibodies
POLYMERASE chain reaction
RESEARCH funding
RNA
THROMBOSIS
PROTEOMICS
DATA analysis software
DESCRIPTIVE statistics
PROTHROMBIN time
IN vitro studies
DISEASE complications
Subjects
Details
- Language :
- English
- ISSN :
- 14620324
- Volume :
- 55
- Issue :
- 6
- Database :
- Complementary Index
- Journal :
- Rheumatology
- Publication Type :
- Academic Journal
- Accession number :
- 115473289
- Full Text :
- https://doi.org/10.1093/rheumatology/kew005