Problem-Women with antiphospholipid antibodies (aPL) are at risk for recurrent miscarriage, preeclampsia and preterm labor. aPL target the placenta directly by binding to Beta 2 -Glycoprotein I (β 2 GPI) expressed on the surface of trophoblast cells. The objective of this study was to determine the effects of aPL on trophoblast function and the mechanisms involved.Method of study-First trimester trophoblast were treated with anti-β 2 GPI monoclonal antibodies and patient-derived aPL, after which cell survival and function was evaluated.Results-We report that anti-β 2 GPI antibodies trigger an inflammatory response in trophoblast, characterized by increased secretion of IL-8, MCP-1, GRO-α and IL-1β, and that this occurs in a TLR-4/MyD88-dependent manner. At high concentrations, these antibodies also induce caspasemediated cell death. This was attenuated upon disabling of the MyD88 pathway, suggesting that anti-β 2 GPI-induced inflammatory mediators compromise trophoblast survival by acting in an autocrine/ paracrine manner. Enhanced IL-8, GRO-α and IL-1β secretion also occured when trophoblast were incubated with antibodies from patients with antiphospholipid syndrome. Heparin, which acts as a pro-survival factor in human trophoblast, attenuated the anti-β 2 GPI antibody-mediated cell death, and also the pro-inflammatory response, but only at high concentrations.Conclusions-These findings demonstrate that aPL triggers a placental inflammatory response via the TLR-4/MyD88 pathway, which in turn compromises trophoblast survival. Thus, the TLR-4/ MyD88 pathway may provide a new therapeutic target to improve pregnancy outcome in antiphospholipid syndrome patients.

Objective. Pathogenic antiphospholipid antibodies (aPL) bind the self antigen N-terminal domain (domain I) of ␤ 2 -glycoprotein I (␤ 2 GPI), with residues G40-R43 being important. However, peptides homologous to other regions of domain I have also been shown to bind aPL. Furthermore, there are no published reports of the effects of altering R39, which has greater surface exposure than the G40-R43 residues.Methods. We used a novel, efficient method of production and purification of human domain I by Escherichia coli to create multiple mutants of domain I. These domain I mutants were then screened for binding to a range of polyclonal IgG purified from patients with antiphospholipid syndrome, using both solid-phase and fluid-phase assays.Results. E coli-expressed purified domain I selectively bound IgG derived from patients with antiphospholipid syndrome. In region R39-R43, the R39S mutation had the greatest effect in terms of reducing binding to a panel of aPL in the fluid phase (mean ؎ SD inhibition 14 ؎ 18.5% versus 44.1 ؎ 31.7% for G40E and 62.9 ؎ 25.7% for wild-type domain I). Conversely, altering both D8 and D9 to S8 and G9, respectively, had the effect of enhancing binding to aPL in the fluid phase. Adding the remainder of the domain I-II interlinker resulted in enhanced binding over wild-type in the solid phase but not the fluid phase.Conclusion. The binding of aPL to ␤ 2 GPI domain I is complex and likely to involve discontinuous epitopes that include R39 in addition to G40-R43, the domain I-II interlinker, and possibly D8 and D9. Domain I variants with enhanced binding to aPL compared with wild-type domain I may aid in the development of novel therapies.

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Summary. Objectives: In the antiphospholipid syndrome (APS), the immunodominant epitope for the majority of circulating pathogenic antiphospholipid antibodies (aPLs) is the N-terminal domain I (DI) of b 2 -glycoprotein I. We have previously shown that recombinant DI inhibits the binding of aPLs in fluid phase to immobilized native antigen, and that this inhibition is greater with the DI(D8S/D9G) mutant and absent with the DI(R39S) mutant. Hence, we hypothesized that DI and DI(D8S/D9G) would inhibit aPL-induced pathogenicity in vivo. Methods: C57BL/6 mice (n = 5, each group) were injected with purified IgG derived from APS patients (IgG-APS, 500 lg) or IgG from normal healthy serum (IgG-NHS) and either recombinant DI, DI(R39S), DI(D8S/D9G), or an irrelevant control peptide (at 10-40 lg). Outcome variables measured were femoral vein thrombus dynamics in treated and control groups following standardized vessel injury, expression of vascular cell adhesion molecule-1 (VCAM-1) on the aortic endothelial surface, and tissue factor (TF) activity in murine macrophages. Results: IgG-APS significantly increased thrombus size as compared with IgG-NHS. The IgG-APS thrombus enhancement effect was abolished in mice pretreated with recombinant DI (P £ 0.0001) and DI(D8S/D9G) (P £ 0.0001), but not in those treated with DI(R39S) or control peptide. This inhibitory effect by DI was dose-dependent, and at lower doses DI(D8S/D9G) was a more potent inhibitor of thrombosis than wild-type DI (P £ 0.01). DI also inhibited IgG-APS induction of VCAM-1 on the aortic endothelial surface and TF production by murine macrophages. Conclusion: Our findings in this proof-of-concept study support the development of recombinant DI or the novel variant DI(D8S/D9G) as a potential future therapeutic agent for APS.

PROBLEM  Women with antiphospholipid antibodies (aPL) are at risk of miscarriage and pre-eclampsia, obstetrical disorders associated with reduced trophoblast invasion and spiral artery transformation. aPL target the placenta by binding beta(2) -glycoprotein I (β(2) GPI) on the trophoblast. In this study, we determined whether aPL alter the trophoblast secretion of angiogenic factors and evaluated the effect of low molecular weight heparin (LMWH) on this response. METHOD OF STUDY  First-trimester trophoblast was treated with anti-β(2) GPI antibodies with or without LMWH. Angiogenic factor secretion was measured by enzyme-linked immunosorbent assay. RESULTS  Trophoblast cells produced more vascular endothelial growth factor (VEGF), placenta growth factor (PlGF), and soluble endoglin following exposure to anti-β(2) GPI Abs, and this occurred in both a MyD88-dependent and MyD88-independent manner. LMWH was unable to reverse the effects of the anti-β(2) GPI Abs on trophoblast VEGF secretion, but enhanced PlGF. Strikingly, LMWH upregulated soluble fms-like tyrosine kinase receptor-1 (sFlt-1) secretion independently of aPL. CONCLUSION  This study demonstrates that aPL perturb the secretion of trophoblast angiogenic factors. LMWH does not reverse this effect but exacerbates sFlt-1 secretion, a potent anti-angiogenic factor. These findings may help to explain why women with antiphospholipid syndrome, who are treated with heparin to prevent early pregnancy loss, remain at increased risk of developing late obstetrical complications, such as pre-eclampsia.