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

Abstract

Beta2-glycoprotein I (β2GPI) is the most common antigen for autoimmune antibodies in antiphospholipid syndrome (APS). Thrombosis is a clinical feature of APS. We made a molecule (A1-A1) that consists of two identical β2GPI-binding modules from ApoE receptor 2. A1-A1 binds to β2GPI/anti-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 β2GPI/antibody complexes in plasma samples of APS patients affecting the clotting time. Inhibition of the clotting time demonstrates the presence of soluble β2GPI/anti-β2GPI antibody complexes in patients’ plasma. These complexes either already exist in patients’ plasma or form rapidly in the vicinity of phospholipids. All members of the LDL receptor family can bind β2GPI. Modelling studies of A1 in a complex with domain V of β2GPI (β2GPI-DV) revealed two possible orientations of a ligand-binding module from lipoprotein receptors on β2GPI-DV. In both orientations, the ligand-binding module interferes with the binding of β2GPI to anionic phospholipids, however it interacts with two different though overlapping sets of lysine residues in β2GPI-DV, depending on the orientation.

Dimerized Domain V of Beta2-Glycoprotein I Is Sufficient to Upregulate Procoagulant Activity in PMA-Treated U937 Monocytes and Require Intact Residues in Two Phospholipid-Binding Loops

“…Information, detailed at the amino acid resolution, on how β2GPI interacts with cells is limited to ApoER2 and anionic phospholipids [ 29 , 30 , 32 , 34 , 53 ]. Domain V of β2GPI contains residues critical for the binding to ApoER2 and anionic phospholipids ( Figure 4 ).…”

“…We made point mutants of the HA-tagged domain V of β2GPI (HA-DV) with the goal of dissecting the contribution of ApoER2 and anionic phospholipids to potentiation of procoagulant activity in monocytes treated with dimerized HA-DV. The selected residues were Lys308 and Lys282, which are involved in the binding of β2GPI domain V to ApoER2 [ 34 , 53 ] and the residues in two phospholipid-binding loops ( Figure 4 ). One of the phospholipid-binding loops contains basic residues Lys284, Lys286 and Lys287, and the other loop is composed of a hydrophobic sequence between the residues Leu313 and Trp316 [ 29 , 30 , 32 ].…”

Entangling COVID-19 associated thrombosis into a secondary antiphospholipid antibody syndrome: Diagnostic and therapeutic perspectives (Review)

“…Because β2-GP1 is present in the blood at high concentration, 4 µ M ( 66 ), it is important that a potential drug binds predominantly to pathological β2-GP1/antibody complexes. The A1-A1 molecule has been shown to be effective in several rodent models of APS ( 64 , 67 , 68 ).…”

Soluble analog of ApoER2 targeting beta2‐glycoprotein I in immune complexes counteracts hypertension in lupus‐prone mice with spontaneous antiphospholipid syndrome

“…For the treatment, A1‐A1, an Asn36/Asp mutant of A1‐A1 and LA6 in 25 m m Hepes, 150 m m NaCl, 0.7 m m CaCl2, pH 7.4, were delivered via osmotic minipumps (models 2002 and 2004, Alzet, Cupertino, CA, USA) implanted subcutaneously. A1‐A1 and LA6 were used for a 2‐week treatment and the Asn36/Asp mutant of A1‐A1 for a 4‐week treatment.…”