Abstract
Factor (F) Xa reactive IgG isolated from patients with antiphospholipid syndrome (APS) display higher avidity binding to FXa with greater coagulant effects compared to systemic lupus erythematosus (SLE) non APS IgG. FXa signalling via activation of protease-activated receptors (PAR) leads to increased intracellular calcium (Ca2+). Therefore, we measured alterations in Ca2+ levels in human umbilical vein endothelial cells (HUVEC) following FXa-mediated PAR activation and investigated whether FXa reactive IgG from patients with APS or SLE/APS- alter these responses. We observed concentration-dependent induction of Ca2+ release by FXa that was potentiated by APS-IgG and SLE/APS- IgG compared to healthy control subjects’ IgG, and FXa alone. APS-IgG and SLE/APS- IgG increased FXa mediated NFκB signalling and this effect was fully-retained in the affinity purified anti-FXa IgG sub-fraction. Antagonism of PAR-1 and PAR-2 reduced FXa-induced Ca2+ release. Treatment with a specific FXa inhibitor, hydroxychloroquine or fluvastatin significantly reduced FXa-induced and IgG-potentiated Ca2+ release. In conclusion, PAR-1 and PAR-2 are involved in FXa-mediated intracellular Ca2+ release in HUVEC and FXa reactive IgG from patients with APS and/or SLE potentiate this effect. Further work is required to explore the potential use of IgG FXa reactivity as a novel biomarker to stratify treatment with FXa inhibitors in these patients.
Highlights
Pathogenic antiphospholipid antibodies interact with various cells including monocytes, endothelial cells (EC) and trophoblast cells leading to the recruitment of cell surface receptors and activation of intracellular signalling pathways[1]
We have shown that FXa stimulation of human umbilical vein endothelial cells (HUVEC) is mediated via protease-activated receptors (PAR)-1 and PAR-2 dependent signalling and that this response is enhanced by IgG from FXa reactive antibody positive patients with antiphospholipid syndrome (APS) as well as systemic lupus erythematosus (SLE)/APS- and can be blocked by a specific FXa proteolytic inhibitor, antistasin, HCQ and fluvastatin
We developed a method to purify the specific aFXa sub-fraction of these antibodies, demonstrated that they fully-retain binding to FXa and have FXa dependent functional effects upon FXa-PAR mediated signalling in EC
Summary
Pathogenic antiphospholipid antibodies (aPL) interact with various cells including monocytes, endothelial cells (EC) and trophoblast cells leading to the recruitment of cell surface receptors and activation of intracellular signalling pathways[1] These interactions give rise to the major clinical manifestations of vascular thrombosis and/or pregnancy morbidity that define the antiphospholipid syndrome (APS). In those experiments, we did not study the effects of IgG on the actions exerted by FXa on cells via PARs
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