The antiphospholipid (aPL) syndrome is an autoimmune disorder marked by vascular thrombosis and/or recurrent pregnancy losses that is treated with anticoagulant medications. The binding of aPL IgG-β2-glycoprotein I (β2GPI) complexes to phospholipid membranes has been postulated to play a central role in the aPL disease process. Recently, we reported that the antimalarial drug, hydroxychloroquine (HCQ), interferes with the formation of aPL IgG-β2GPI complexes on phospholipid bilayers (
Methods: IgG fractions were purified from plasmas of 3 aPL patients and 3 normal disease-free controls and used at the concentration of 0.5 mg/ml. HCQ concentrations of 1 μg/mL and 1 mg/mL - the lowest and highest concentrations used in our previous studies - were utilized. The binding of AnxA5 to 70% phosphatidylcholine/30% phosphatidylserine (PC/PS) bilayers, cultured BeWo trophoblasts, and cultured human umbilical-vein endothelial cells (HUVECs) was measured with fluorescence-tagged AnxA5 and with immunoassays. For experiments with the PS/PC, β2GPI was used at the concentration of 5 μg/ml; for experiments with cultured cells the β2GPI was supplied by bovine serum in the culture media. We also measured the coagulation times of plasmas that were overlaid on the cultured cells.
Results: aPL IgGs reduced the binding of AnxA5 to phospholipid bilayers (0.10±0.03 μg/cm2 for aPL IgGs versus 0.25±0.01 μg/cm2 for control IgGs, n=3 pairs of different IgGs, p=0.0001). Addition of HCQ (1 mg/mL) restored AnxA5 binding (0.23±0.03 μg/cm2 for HCQ-treated aPL IgGs versus 0.24±0.03 μg/cm2 for HCQ-treated control IgGs, p=0.71). aPL IgGs reduced the levels of AnxA5 on BeWo trophoblasts (2.5±0.4 ng/well versus 4.1±0.7 ng/well for control IgGs, n=3, p=0.02). Addition of HCQ restored the levels of AnxA5 on the aPL IgG-treated trophoblasts (4.6±1.1 ng/well for 1 μg/mL HCQ and 5.7±0.8 ng/well for 1 mg/mL HCQ, versus 4.6±1.3 ng/well and 4.7±0.6 ng/well for the control IgG-treated trophoblasts, p=0.97 and 0.16 respectively).
aPL IgGs reduced the levels of AnxA5 on HUVECs (1.2±0.1 ng/well versus 1.9±0.2 ng/well for control IgGs, n=3, p=0.003). Addition of HCQ also restored the levels of AnxA5 on the aPL IgG-treated HUVECs (1.8±0.4 ng/well for 1 μg/mL HCQ and 1.9±0.1 ng/well for 1 mg/mL HCQ, versus 2.1±0.4 ng/well and 2.2±0.3 ng/well for the control IgG-treated cells, p=0.50 and 0.12 respectively).
Addition of aPL IgGs accelerated the coagulation time of plasma overlaid on BeWo trophoblasts (172±4 sec versus 231±7 sec for control IgGs, n=3, p=0.001). Addition of HCQ prolonged plasma coagulation time on the aPL IgG-treated trophoblasts (256±17 sec for 1 μg/mL HCQ and 242±3 sec for 1 mg/mL HCQ, versus 233±5 sec and 235±27 sec for control IgG-treated trophoblasts, p=0.10 and 0.68 respectively).
aPL IgGs accelerated coagulation time of plasma overlaid on HUVECs (101±11 sec versus 135±4 sec for control IgGs, n=3, p=0.02). Addition of HCQ prolonged plasma coagulation time on the aPL IgG-treated HUVECs (130±3 sec for 1 μg/mL HCQ and 134±13 sec for 1 mg/mL HCQ, versus 133±17 sec and 138±14 sec for control IgG-treated trophoblasts, p=0.78 and 0.74 respectively).
Conclusions: HCQ protects the binding of AnxA5 to reconstituted phospholipid bilayers, BeWo trophoblasts and HUVECS against reduction by aPL IgG-β2GPI complexes. HCQ also reverses the acceleration of plasma coagulation that is induced by aPL IgGβ2GPI complexes. These results provide a novel mechanism to explain the reduction of thrombosis by HCQ that has been reported in experimental animal models of aPL syndrome and support the possibility of innovative non-anticoagulant approaches to treating the aPL disease process in humans.
Disclosures: No relevant conflicts of interest to declare.