Arthritis & Rheumatism, Volume 63,
November 2011 Abstract Supplement
Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Chicago, Illinois November 4-9, 2011.
Role of Apolipoprotein B100 and Oxidized Low-Density Lipoprotein in Anti-beta2 Glycoprotein I Induced Tissue Factor Expression on Monocytes.
Otomo1, Kotaro, Atsumi1, Tatsuya, Fujieda1, Yuichiro, Nakagawa1, Hisako, Kato1, Masaru, Amengual1, Olga, Horita1, Tetsuya
Department of Medicine II, Hokkaido University Graduate School of Medicine, Sapporo, Japan
Division of Proteomics, Kyusyu University Medical Institute of Bioregulation, Fukuoka, Japan
Department of Biochemistry, Hokkaido University Graduate School of Medicine, Sapporo, Japan
To explore plasma molecule involvement in the tissue factor expression induced by beta2 glycoprotein I dependent anticardiolipin antibody (aCL/b2GPI) on monocytes.
Unknown beta2-glycoprotein I (b2GPI) binding molecules in plasma were screened by a proteomics technique using immunoaffinity chromatography and mass spectrometric analysis. To identify b2GPI binding proteins, FLAG-tagged human b2GPI was constructed. The expression vector encoding FLAG-b2GPI was transfected into HEK293T cells, and the expression of full length FLAG-b2GPI in the culture supernatant was confirmed by immunoblotting. Human serum sample with FLAG-b2GPI was incubated and applied for affinity chromatography with anti -FLAG antibody-conjugated Sepharose beads. The purified fraction was subjected to SDS-PAGE, followed by a silver staining. Immunopurified proteins were analyzed by an online-nanoLC-MS/MS. Obtained MS/MS data were searched against nrNCBI database MASCOT algorithm.
Among many proteins detected in the spectrometry, ApoB100 was the only identified molecule as a candidate plasma protein. Since there was no significant binding between b2GPI and ApoB100 in ELISA, oxidized LDL, containing ApoB100 as well as ox-Lig1 (a known ligand of b2GPI) in its molecule, was considered as a b2GPI-binding molecule in plasma.
The involvement of oxidized LDL was further investigated in aCL/b2GPI induced tissue factor expression on monocytes. RAW264.7, a monocyte cell line, was incubated with a monoclonal aCL/b2GPI, WBCAL-1, in the presence/absence of oxidized LDL. Cells were lysed and TF mRNA was quantitated using Real Time PCR system. The presence of oxidized LDL (100mg/ml) and b2GPI markedly increased TF mRNA expression on RAW264.7 cells induced by WBCAL-1 (Fig). Oxidized LDL upregulated TF mRNA induction as well by purified IgG from APS patients with high titre of aCL/b2GPI.
Oxidized LDL was detected as a major b2GPI binding plasma molecule by proteomics analysis. The presence of oxidized LDL upregulated aCL/b2GPI induced TF expression on monocytes, suggesting the involvement of oxidized LDL in the pathophysiology of thrombosis in patients with APS.
Figure 1. RAW264.7 cells were incubated with monoclonal aCL/b2GPI (WBCAL-1) in the presence of oxidized LDL. TF mRNA was quantitated by Real Time PCR.
To cite this abstract, please use the following information:
Otomo, Kotaro, Atsumi, Tatsuya, Fujieda, Yuichiro, Nakagawa, Hisako, Kato, Masaru, Amengual, Olga, et al; Role of Apolipoprotein B100 and Oxidized Low-Density Lipoprotein in Anti-beta2 Glycoprotein I Induced Tissue Factor Expression on Monocytes. [abstract]. Arthritis Rheum 2011;63 Suppl 10 :15