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-b 2 glycoprotein I activity by an affinity column. The two IgG-APS induced significantly smaller thrombi and fewer WBC adhering to endothelial cells in LPS2/2 mice than in LPS+/+ mice. IgG-APS induced higher TF activity in carotid artery homogenates of LPS+/+ mice than in LPS2/2 mice. The prevalence of Asp299Gly and Thr399Ile tlr4 polymorphisms was significantly lower than in controls. Conclusions: These findings in LPS2/2 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 (aPL)/anti- 2 glycoprotein I (anti- 2 GPI) antibodies stimulates tissue factor (TF) expression within vasculature and in blood cells, thereby leading to increased thrombosis. Several cellular receptors have been proposed to mediate these effects, but no convincing evidence for the involvement of a specific one has been provided. We investigated the role of Apolipoprotein E receptor 2 (ApoER2) on the pathogenic effects of a patient-derived polyclonal aPL IgG preparation (IgG-APS), a murine anti- 2 GPI monoclonal antibody (E7) and of a constructed dimeric  2 GPI I (dimer), which in vitro mimics  2 GPI-antibody immune complexes, using an animal model of thrombosis, and ApoER2-deficient (؊/؊) mice. In wild type mice, IgG-APS, E7 and the dimer increased thrombus formation, carotid artery TF activity as well as peritoneal macrophage TF activity/expression. Those pathogenic effects were significantly reduced in ApoER2 (؊/؊) mice. In addition, those effects induced by the IgG-APS, by E7 and by the dimer were inhibited by treatment of wild-type mice with soluble binding domain 1 of ApoER2 (sBD1). Altogether these data show that ApoER2 is involved in pathogenesis of antiphospholipids antibodies. (Blood. 2011;117(4):1408-1414) IntroductionThe association between persistently present antiphospholipid (aPL) antibodies and the clinical manifestations of thrombosis or pregnancy morbidity is known as the antiphospholipid syndrome (APS). 1 aPL antibodies are heterogeneous and recognize a wide variety of plasma proteins with phospholipid-binding properties, such as prothrombin 2 and  2 glycoprotein I ( 2 GPI). 3,4 aPL antibodies directed against  2 GPI, a plasma protein without known physiologic function, are considered the most pathologically relevant antibodies.There is strong experimental evidence that anti- 2 GPI antibodies have thrombogenic properties. In studies on endothelial cell activation, 5-8 authors have shown the induction of a prothrombotic and proinflammatory phenotype upon exposure to anti- 2 GPI antibodies, indicated by expression of tissue factor (TF) and increased surface expression of adhesion molecules, such as intercellular adhesion molecule-1, vascular-cell adhesion molecule-1, and E-selectin. Activation of monocytes by anti- 2 GPI antibodies leads to TF expression as well. 9 Furthermore, anti- 2 GPI antibodies, or recombinant dimers of  2 GPI that mimic  2 GPI-antibody immune complexes, increase platelet deposition to extracellular matrix components in in vitro flow models. 10 Injection of anti- 2 GPI antibodies in murine 11 or hamster 12 thrombosis models leads to increased thrombus formation.Several receptors were postulated to mediate the prothrombotic cellular effects of anti- 2 GPI antibodies. The interaction between annexin A2 and  2 GPI-antibody immune complexes has been reported to lead to endothelial cell activation. 13 It seems unlikely, however, that annexin A2 is able to convey activation signals across the cell membrane because this phospholipid-binding protei...
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.
Purpose To examine the prevalence of isolated IgA anti-β2Glycoprotein I (anti-β2GPI) positivity and the association of these antibodies, and a subgroup that bind specifically to domain IV/V of β2GPI, with clinical manifestations of the Antiphospholipid Syndrome (APS) in three patients groups. The pathogenicity of IgA anti-β2GPI was also evaluated in a mouse model of thrombosis. Methods Patients with systemic lupus erythematosus (SLE) from a multiethnic, multicenter cohort (LUpus in MInorities, NAture versus nurture [LUMINA]) (n=558), patients with SLE from the Hopkins Lupus Cohort (n=215), and serum samples referred to the Antiphospholipid Standardization Laboratory (APLS) (n=5,098) were evaluated. IgA anti-β2GPI titers and binding to domain IV/V of β2GPI were examined by enzyme-linked immunosorbent assay (ELISA). CD1 mice were inoculated with purified IgA anti- β2GPI antibodies, and surgical procedures and ELISAs were performed to evaluate thrombus development and tissue factor (TF) activity. Results A total of 198 patients were found to be positive for IgA anti-β2GPI isotype, and 57 patients were positive exclusively for IgA anti-β2GPI antibodies. Of these, 13 of 23 patients (56.5%) in the LUMINA cohort, 17 of 17 patients (100%) in the Hopkins cohort, and 10 of 17 patients (58.9%) referred to APLS had at least one APS-related clinical manifestation. Fifty-four percent of all the IgA anti-β2GPI positive serum samples reacted with domain IV/V of anti-β2GPI, and 77% of those had clinical features of APS. Isolated IgA anti-β2GPI positivity was associated with an increased risk for arterial thrombosis (p<0.001), venous thrombosis (p=0.015) and all thrombosis (p<0.001). The association between isolated IgA anti-β2GPI and arterial thrombosis (p=0.0003) and all thrombosis (p=0.0003) remained significant after adjusting for other risk factors for thrombosis. In vivo mouse studies demonstrated that IgA anti-β2GPI antibodies induced significantly larger thrombi and higher TF levels compared to controls. Conclusion Isolated IgA anti-β2GPI positive titers may identify additional patients with clinical features of APS. Testing for these antibodies when other antiphospholipid (aPL) tests are negative and APS is suspected is recommended. IgA anti-β2GPI antibodies directed to domain IV/V of β2GPI represent an important subgroup of clinically relevant antiphospholipids.
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