An A1-A1 mutant with improved binding and inhibition of β2GPI/antibody complexes in antiphospholipid syndrome

β2 glycoprotein I (β2GPI) is the most common antigen for autoimmune antibodies in antiphospholipid syndrome (APS). Thrombosis is a clinical feature of APS. We created a molecule (A1-A1) that consists of two identical β2GPI-binding modules from ApoE receptor 2 (ApoER2). A1-A1 binds to β2GPI/antibody complexes, preventing their association with ApoER2 and anionic phospholipids, and reducing thrombus size in the mouse model of APS. Here, we describe a mutant of A1-A1 (mA1-A1ND) with improved affinity for β2GPI. mA1-A1ND inhibits the binding of β2GPI to cardiolipin in the presence of anti-β2GPI antibodies, and inhibits the binding to phospholipids in plasma samples of APS patients, affecting the clotting time. Reduction of the clotting time demonstrates the presence of soluble β2GPI/antibody complexes in patients' plasma. These complexes either already exist in patients' plasma or form rapidly in the proximity to phospholipids. All members of the low-density lipoprotein receptor family bind β2GPI. Modeling studies of A1 in a complex with domain V of β2GPI (β2GPI-DV) revealed two possible modes of interaction of a ligand-binding module from lipoprotein receptors with β2GPI-DV. In both orientations, the ligand-binding module interferes with binding of β2GPI to anionic phospholipids; however, it interacts with two different but overlapping sets of lysine residues in β2GPI-DV, depending on the orientation.