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Annexin A5 (AnxA5) is a potent anticoagulant protein that crystallizes over phospholipid bilayers (PLBs), blocking their availability for coagulation reactions. Antiphospholipid antibodies disrupt AnxA5 binding, thereby accelerating coagulation reactions. This disruption may contribute to thrombosis and miscarriages in the antiphospholipid syndrome (APS). We investigated whether the antimalarial drug, hydroxychloroquine (HCQ), might affect this prothrombotic mechanism. Binding of AnxA5 to PLBs was measured with labeled AnxA5 and also imaged with atomic force microscopy. Immunoglobulin G levels, AnxA5, and plasma coagulation times were measured on cultured human umbilical vein endothelial cells and a syncytialized trophoblast cell line. AnxA5 anticoagulant activities of APS patient plasmas were also determined. HCQ reversed the effect of antiphospholipid antibodies on AnxA5 and restored AnxA5 binding to PLBs, an effect corroborated by atomic force microscopy. Similar reversals of antiphospholipid-induced abnormalities were measured on the surfaces of human umbilical vein endothelial cells and syncytialized trophoblast cell lines, wherein HCQ reduced the binding of antiphospholipid antibodies, increased cell-surface AnxA5 concentrations, and prolonged plasma coagulation to control levels. In addition, HCQ increased the AnxA5 anticoagulant activities of APS patient plasmas. In conclusion, HCQ reversed antiphospholipid-mediated disruptions of AnxA5 on PLBs and cultured cells, and in APS patient plasmas. These results support the concept of novel therapeutic approaches that address specific APS disease mechanisms.

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 (Rand et al, BLOOD, 112(5):1687–1695, 2008). We previously provided a prothrombotic mechanism for the aPL syndrome and showed that aPL IgG-β2GPI 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-β2GPI 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, 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.

Three members of a family with normal parents had a common constellation of findings that included absent corpus callosum and recurrent bacterial infections. The older male and female siblings both died from infection at an early age. The patient’s CBC was significant for elevated numbers of monocytes that were large and vacuolated. Her T- and B-cell function was normal. These preliminary findings suggested a defect in innate immunity. Evaluation of the patient’s peripheral blood monocytes by flow cytometry showed normal size and maturity. Phagocytosis and activation of peripheral blood derived macrophages by cytokines were also similar to controls. In contrast, the patient’s cultured macrophages were significantly more spread than those from normal individuals and contained a disordered actin cytoskeleton when cultured on fibronectin. The ability of the macrophage to transmigrate across an endothelial cell barrier was impaired. However examination by time-lapse videomicroscopy showed that the mutant macrophages had increased protrusional activity and movement. This is the first report describing a genetic macrophage motility disorder that results in an increased susceptibility to infection.