Antiphospholipid syndrome (APS) is an autoimmune disease characterized by the persistent presence of antiphospholipid antibodies (aPLs) and recurrent thrombosis or fetal loss. The thrombophilic state has been partially related to the induction of a proinflammatory and procoagulant endothelial cell (EC) phenotype induced by anti–β₂-glycoprotein I (β₂-GPI) antibodies that bind β₂-GPI expressed on the EC surface. Anti–β₂-GPI antibody binding has been shown to induce nuclear factor-κB (NF-κB) translocation leading to a proinflammatory EC phenotype similar to that elicited by interaction with microbial products (lipopolysaccharide [LPS]) and proinflammatory cytokines (interleukin 1β [IL-1β], tumor necrosis factor α [TNF-α]). However, the upstream signaling events are not characterized yet. To investigate the endothelial signaling cascade activated by anti–β₂-GPI antibodies, we transiently cotransfected immortalized human microvascular endothelial cells (HMEC-1) with dominant-negative constructs of different components of the pathway (ΔTRAF2, ΔTRAF6, ΔMyD88) together with reporter genes (NF-κB luciferase and pCMV-β-galactosidase). Results showed that both human anti–β₂-GPI IgM monoclonal antibodies as well as polyclonal affinity-purified anti–β₂-GPI IgG display a signaling cascade comparable to that activated by LPS or IL-1. ΔTRAF6 and ΔMyD88 significantly abrogate antibody-induced as well as IL-1– or LPS-induced NF-κB activation, whereas ΔTRAF2 (involved in NF-κB activation by TNF) does not affect it. Moreover, anti– β₂-GPI antibodies and LPS followed the same time kinetic of IL-1 receptor–activated kinase (IRAK) phosphorylation, suggesting an involvement of the toll-like receptor (TLR) family. Our findings demonstrate that anti–β₂-GPI antibodies react with their antigen likely associated to a member of the TLR/IL-1 receptor family on the EC surface and directly induce activation.

Objective To investigate the in vitro ability of antiphospholipid antibodies (aPL) to bind human trophoblast cells and to affect gonadotropin secretion and invasiveness. Methods Antiphospholipid antibody IgG from women with recurrent miscarriages, β₂‐glycoprotein I (β₂GPI)–independent IgG aPL human monoclonal antibody (mAb)(519), and IgM anti‐β₂GPI human mAb (TM1G2) were investigated for their binding to trophoblasts cultured for various amounts of time, their ability to affect invasiveness of Matrigel‐coated filters, and their release of human chorionic gonadotropin (hCG). Results Polyclonal IgG aPL, as well as mAb 519 and TM1G2, bound to trophoblasts, the highest binding being found when cells displayed the greatest amount of syncytium formation. TM1G2 binding was found to be β₂GPI dependent. Both polyclonal and monoclonal aPL, but not the controls, significantly reduced hCG release and Matrigel invasiveness. Conclusion These findings suggest that aPL recognition of both anionic PL and adhered β₂GPI on trophoblast cell structures might represent a potential pathogenetic mechanism for defective placentation in women with the antiphospholipid syndrome.

Objective To investigate the ability of statins, the inhibitors of the hydroxymethylglutaryl–coenzyme A reductase enzyme, to affect endothelial cell activation induced by anti–β₂‐glycoprotein I (anti‐β₂GPI) antibodies in vitro. Methods Human umbilical vein endothelial cell (HUVEC) activation was evaluated as U937 monocyte adhesion, E‐selectin, and intercellular adhesion molecule 1 (ICAM‐1) expression by cell enzyme‐linked immunosorbent assay and as interleukin‐6 (IL‐6) messenger RNA (mRNA) expression by RNA protection assay. E‐selectin–specific nuclear factor κB (NF‐κB) DNA‐binding activity was evaluated by the gel‐shift assay. HUVECs were activated by polyclonal affinity‐purified IgG, human monoclonal IgM anti‐β₂GPI antibodies, human recombinant IL‐1β, tumor necrosis factor α, or lipopolysaccharide (LPS). Results Fluvastatin reduced, in a concentration‐dependent manner (1–10 μM), the adhesion of U937 to HUVECs and the expression of E‐selectin and ICAM‐1 induced by anti‐β₂GPI antibodies as well as by cytokines or LPS. Another lipophilic statin, simvastatin, display similar effects but to a lesser extent than fluvastatin. The inhibition of E‐selectin expression exerted by fluvastatin was related to the impairment of NF‐κB binding to DNA. Moreover, the drug attenuated the expression of IL‐6 mRNA in HUVEC exposed to anti‐β₂GPI antibodies or cytokines. Incubation of HUVECs with mevalonate (100 μM), concomitantly with fluvastatin, greatly prevented the inhibitory effect of statin. Conclusion Endothelial activation mediated by anti‐β₂GPI antibody can be inhibited by statins. Because of the suggested role of endothelial cell activation in the pathogenesis of antiphospholipid syndrome (APS), our data provide, for the first time, a rationale for using statins as an additional therapeutic tool in APS.

Objective: A study was undertaken to investigate the in vivo pathogenic role of Toll-like receptor 4 (TLR-4) in the antiphospholipid syndrome (APS) by studying the thrombogenic antiphospholipid (aPL) activity in lipopolysaccharide (LPS) non-responsive (LPS–/–) mice and the association between tlr4 gene polymorphisms and APS in patients. Methods: IgGs from two patients with APS, one with aPL negative systemic lupus erythematosus (SLE) and one with normal human serum (NHS), were evaluated for thrombosis, tissue factor (TF) activity and endothelial cell activation in LPS–/– mice displaying a tlr4 spontaneous mutation vs LPS responsive (LPS+/+) mice. Human tlr4 Asp299Gly and Thr399Ile polymorphisms were evaluated by allele-specific PCR in 110 patients with APS with arterial/venous thrombosis and in 220 controls of the same ethnic origin. Results: IgG-APS produced significantly larger thrombi and more leucocytes (WBC) adhering to endothelial cells in the cremaster muscle microcirculation of LPS+/+ mice than IgG-NHS or aPL negative SLE-IgG. These effects were abrogated after absorption of the anti-β₂glycoprotein I activity by an affinity column. The two IgG-APS induced significantly smaller thrombi and fewer WBC adhering to endothelial cells in LPS−/− mice than in LPS+/+ mice. IgG-APS induced higher TF activity in carotid artery homogenates of LPS+/+ mice than in LPS−/− mice. The prevalence of Asp299Gly and Thr399Ile tlr4 polymorphisms was significantly lower than in controls. Conclusions: These findings in LPS−/− mice and the reduction in the “protective” polymorphism in patients with APS with thrombosis suggest that TLR-4 is involved in the interaction of aPL with endothelial cells in vivo.

Antiphospholipid antibodies (Abs) are associated with thrombosis and are a risk factor for recurrent pregnancy loss and obstetric complications in patients with the antiphospholipid syndrome. It is generally accepted that the major autoantigen for aPL Abs is β₂ glycoprotein I, which mediates the binding of aPL Abs to target cells (i.e., endothelial cells, monocytes, platelets, trophoblasts, etc.) leading to thrombosis and fetal loss. This article addresses molecular events triggered by aPL Abs on endothelial cells, platelets, and monocytes and complement activation, as well as a review of the current knowledge with regard to the putative receptor(s) recognized by aPL Abs on target cells as well as novel mechanisms that involve fibrinolytic processes. A section is devoted to the description of thrombotic and inflammatory processes that lead to obstetric complications mediated by aPL Abs. Based on experimental evidence using in vitro and in vivo models, new targeted therapies for treatment and/or prevention of thrombosis and pregnancy loss in antiphospholipid syndrome are proposed.