The Roles Of ANXA2 On APS IgG/β₂GPI-Induced Tissue Factor Expression In THP-1 Cells

Objective:To explore the functions of annexin A2(ANXA2) in antiphospholipid syndrome patients' IgG(APS IgG)/β₂-glycoproteinâ… (β₂GPI)-induced tissue factor(TF) expression on monocyte drived THP-1 cells.Methods:â‘ The pIRES₂-eGFP-ANXA2 eukaryotic expression vector carrying human ANXA2 cDNA was constructed and analyzed by restriction and sequencing.The recombinant pIRES₂-eGFP-ANXA2 plasmid was transfected into 293T cells in the mediation of liposome to get the high-ANXA2-expression cells.â‘¡Four different short hairpin RNAs(shRNA) targeting ANXA2 gene were cloned into pGCSIL-GFP vector and identificated with PCR and sequencing.The effective interference sequence was selected by Western blotting.The recombinant vector was co-transfected into 293T cells to get RNAi lentivirus LV-RNAi-ANXA2.Then the lentivirus harvested from 293T cells were transferred into THP-1 cells to block ANXA2 expression on cells.â‘¢The purifiedβ₂GPI and IgG from antiphospholipid syndrome(APS) patients were incubated in media with THP-1 cells,LV-RNAi-ANXA2-transferred THP-lcells,and 293T cells transfected with pIRES₂-eGFP-ANXA2 for a certain time.TF mRNA and TF activity on these cells were investigated.â‘£THP-1 lysates was incubated withβ₂GPI and immunoprecipitated using anti-β₂GPI antibody,and the coprecipitates was identified by Western blotting with anti-ANXA2 antibody,in order to analyze the interaction ofβ₂GPI with ANXA2.Results:â‘ Eukaryotic expression vector pIRES₂-eGFP-ANXA2 was correctly constructed and analyzed by restriction and sequencing. ANXA2 expression was highly increased both at mRNA and protein levels on 293T cells after transfection.â‘¡The RNA interference(RNAi) sequences targeting human ANXA2 were successfully inserted into the lentiviral vector and the high-performance RNAi sequence was sieved out. The recombinant lentivirus was harvested from 293T cells with a viral titer of 3×10⁹ TU/ml.THP-1 cells transfected with LV-RNAi-ANXA2 showed almost lockout of ANXA2 both at mRNA and protein levels.The best MOI for THP-1 cells was 100.â‘¢APS IgG/β₂GPI compound significantly increased TF mRNA expression and TF activity on THP-1 cell.While down-regulation of ANXA2 in THP-1 cells decreased TF mRNA and activity levels with the similar stimulation.293T cells transfected with pIRES₂-eGFP-ANXA2 and stimulated byβ₂GPI/anti-β₂GPI compound,could express high level of TF mRNA.â‘£ANXA2 was detected in the immune complexes of THP-1 cells lysates by co-immunoprecipitation with anti-β₂GPI andβ₂GPI,indicating thatβ₂GPI can interact directly with ANXA2. Conclusions:ANXA2 acts as the receptor of APS IgGβ₂GPI compound,medicates TF expression on THP-1 cells induced by this compound,which is contributed to the thrombotic diathesis in APS.