thromboplastin and benzyloxycarbonylleucyl-leucyl-leucine-aldehyde

Our previous study has shown that Toll-like receptor 4 (TLR4) and its signalling pathway contribute to anti-β₂-glycoprotein I/β₂-glycoprotein I (anti-β₂GPI/β₂GPI)-induced tissue factor (TF) expression in human acute monocytic leukaemia cell line THP-1 and annexin A2 (ANX2) is involved in this pathway. However, its downstream molecules have not been well explored. In this study, we have established that interleukin-1 receptor-associated kinases (IRAKs) and tumour necrosis factor receptor-associated factors (TRAFs) are crucial downstream molecules of anti-β₂GPI/β₂GPI-induced TLR4 signaling pathway in THP-1 cells and explored the potential mechanisms of their self-regulation. Treatment of THP-1 cells with anti-β₂GPI/β₂GPI complex induced IRAKs and TRAFs expression and activation. Anti-β₂GPI/β₂GPI complex firstly induced expression of IRAK4 and IRAK1, then IRAK1 phosphorylation and last IRAK3 upregulation. In addition, anti-β₂GPI/β₂GPI complex simultaneously and acutely enhanced mRNA levels of TRAF6, TRAF4 and zinc finger protein A20 (A20), while chronically increased A20 protein level. Moreover, anti-β₂GPI/β₂GPI complex-induced IRAKs and TRAFs expression and activation were attenuated by knockdown of ANX2 by infection with ANX2-specific RNA interference lentiviruses (LV-RNAi-ANX2) or by treatment with paclitaxel, which inhibits TLR4 as an antagonist of myeloid differentiation protein 2 (MD-2) ligand. Furthermore, both IRAK1/4 inhibitor and a specific proteasome inhibitor MG-132 could attenuate TRAFs expression as well as TF expression induced by anti-β₂GPI/β₂GPI complex. In conclusion, our results indicate that IRAKs and TRAFs play important roles in anti-β₂GPI/β₂GPI-stimulated TLR4/TF signaling pathway in THP-1 cells and contribute to the pathological processes of antiphospholipid syndrome (APS).

Topics: Annexin A2; Antibodies; Antigen-Antibody Complex; Antiphospholipid Syndrome; beta 2-Glycoprotein I; Cell Line, Tumor; Enzyme Inhibitors; Gene Expression Regulation; Humans; Interleukin-1 Receptor-Associated Kinases; Leupeptins; Monocytes; Paclitaxel; RNA, Small Interfering; Signal Transduction; Thromboplastin; TNF Receptor-Associated Factor 4; TNF Receptor-Associated Factor 6; Toll-Like Receptor 4; Tumor Necrosis Factor Receptor-Associated Peptides and Proteins

It has been shown that endothelial cell (EC) activation and tissue factor (TF) upregulation in EC and monocytes by antiphospholipid antibodies (aPL Abs) leads to a prothrombotic state and involves translocation of nuclear factor-kappa B (NF-kappaB). Here we examined the effects of an NF-kappaB inhibitor on aPL-induced thrombosis, TF activity, and EC in vivo. We treated CD1 mice with IgG from a patient with antiphospholipid syndrome (IgG-APS) or with control IgG (IgG-NHS). The adhesion of leukocytes (number of white blood cells) to EC in cremaster muscle (as an indication of EC activation) as well as the size of an induced thrombus in the femoral vein of the mice were examined. Some mice in each group were infused with 10 microM MG132 (an inhibitor of NF-kappaB). TF activity was determined using a chromogenic assay in homogenates of carotid arteries and in peritoneal cells of mice. In vivo, IgG-APS increased significantly the number of white blood cells adhering to ECs (4.7 +/- 2.2) when compared to control mice (1.5 +/- 0.8), and these effects were significantly reduced when mice were pretreated with MG132 (0.8 +/- 0.2). IgG-APS increased significantly the thrombus size and MG132 inhibited that effect (93%). Treatment of the mice with IgG-APS also induced significantly increased TF function in peritoneal cells and in homogenates of carotid arteries. Pretreatment of the mice with MG132 abrogated those effects significantly. Mice injected with IgG-APS or with IgM-APS with or without the inhibitor had medium-high titers of anticardiolipin antibodies in serum at the time of the surgical procedures. The data show that prothrombotic and proinflammatory properties of IgG-APS and IgM-APS are downregulated in vivo by an NF-kappaB inhibitor. These findings may be important in designing new modalities of targeted therapies to treat thrombosis in patients with APS.

Topics: Animals; Antibodies, Antiphospholipid; Antiphospholipid Syndrome; Cell Adhesion; Endothelial Cells; Humans; Leupeptins; Male; Mice; Middle Aged; NF-kappa B; Thromboplastin; Thrombosis