TOLL-LIKE RECEPTOR 4 AS A CELL MEMBRANE RECEPTOR FOR BETA2 GLYCOPROTEIN I

Background: Antiphospholipid Syndrome (APS) is characterized by recurrent fetal losses and arterial/venous thrombosis in the presence of aPL1. aPL are directed against anionic phospholipid binding proteins: mainly prothrombin and beta2 glycoprotein I (β2GPI). The complex formation between circulating aPL and β2GPI expressedon the cell membranes induces signalling and triggers cell activation2,3. The exact nature of the EC receptors for β2GPI is still matter of research: heparan sulphate, Annexin A2 and apoER2 have been suggested to be candidate molecules4. Furthermore there is also evidence that aPL trigger TLR-4 dependent cell signalling. Objectives: To address the role of TLR4 as cell membrane receptor for β2GPI and its involvement in anti-β2GPI Abs-mediated EC activation pathway. Methods: We performed: i) in vitro experiments with or without blocking antibodies directed to Annexin A2 and TLR-4; ii) small interfering RNA experiments for silencing TLR-4 and Annexin A2 expression in suitably transfected HUVEC; iii) confocal microscopy studies to visualize in real time the binding and internalization of β2GPI and aPL in living MOCK and TLR-4-transfected (TLR-4+) CHO cells. Results: TLR-4 silencing by siRNA significantly inhibited both binding and activation of aPL-exposed HUVEC, while Annexin A2 silencing only affected binding. Fluoresceinated β2GPI bound to TLR-4+ CHO more strongly and persistently as compared to MOCK CHO. Membrane binding was followed within minutes by internalization in granular structures in TLR-4+ cells only. Experiments are being set up to evaluate the effects of anti-β2GPI Abson this event. Conclusions: Our results suggest that more than one receptor may be involved in aPL-mediated EC activation. However, TLR-4 appears particularly relevant as it seems necessary for β2GPI internalization in cells and Annexin A2, lacking a cytoplasmic tail, is unable to transduce signals. Understanding in detail the mechanisms of β2GPI and β2GPI-aPL complex interaction with plasma membrane will lead to identify new strategies for treatment and prevention of pro-thrombotic and pro-inflammatory state in APS. References: Miyakis, S., et al. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS). J. Thromb. Haemost. 2006: 4, 295-306. Giannakopoulos, B., et al. Current concepts on the pathogenesis of the antiphospholipid syndrome. Blood 2007: 109, 422-430. de Laat, B., et al. Mechanisms of disease: antiphospholipid antibodies-from clinical association to pathologic mechanism. Nat. Clin. Pract. Rheumatol. 2008: 4, 192-199. Pierangeli SS, et al. Antiphospholipid antibodies and the antiphospholipid syndrome: pathogenic mechanisms. Semin Thromb Hemost. 2008: 34, 236-50.