Hydroxychloroquine Protects the Annexin A5 Anticoagulant Shield from Disruption by Antiphospholipid Antibodies

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-ß₂-glycoprotein I (ß2GPI) 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-ß2GPI complexes on phospholipid bilayers (Rand et al, BLOOD, 112(5):1687-1695, 2008).  We previously provided a prothrombotic mechanism for the aPL syndrome and  showed that  aPL IgG-ß2GPI complexes disrupt the binding of annexin A5 (AnxA5) to phospholipid bilayers and its assembly into an ordered 2-dimensional crystalline  anticoagulant shield. This creates defects in the integrity of the crystals that  expose phospholipids for assembly of coagulation enzyme-substrate complexes. We therefore investigated whether HCQ might protect AnxA5 and its anticoagulant effect from disruption by aPL-ß2GPI complexes.

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, ß2GPI was used at the concentration of 5 µg/ml; for experiments with cultured cells the ß2GPI 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-ß2GPI complexes. HCQ also reverses the acceleration of plasma coagulation that is induced by aPL IgG-ß2GPI 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.