Role of Divalency in the High-Affinity Binding of Anticardiolipin Antibody−β2-Glycoprotein I Complexes to Lipid Membranes

Abstract

β₂-Glycoprotein I (β₂GPI) is an essential cofactor for the binding to lipids of anticardiolipin antibodies (ACA), isolated from patients with anti-phospholipid syndrome. We used ellipsometry to study the binding of β₂GPI and the β₂GPI-mediated binding of ACA to planar membranes composed of phosphatidylcholine (PC) and 5−20 mol % phosphatidylserine (PS). No binding of β₂GPI was observed to neutral (PC) membranes. Maximal binding of β₂GPI was 3.2−3.6 pmol·cm^-2. Affinity decreased strongly with decreasing PS content; increasing the NaCl and CaCl₂ concentrations also led to a decrease in affinity. At physiologic conditions (10 mol % PS, 120 mM NaCl, and 3 mM CaCl₂), a K_d of 14 μM was observed. Binding constants were insensitive to the chemical composition of the negatively charged phospholipid headgroup. ACA (1.25−10 μg·mL^-1) caused a 30−40-fold enhancement of β₂GPI binding to PS/PC membranes (20 mol % PS), resulting in the binding of about 2 pmol·cm^-2 divalent ACA−(β₂GPI)₂ complexes at 100 nM β₂GPI. In the absence of β₂GPI, binding of ACA was negligible. Ad- and desorption kinetics of ACA−β₂GPI complexes indicate that the initial monovalent association of ACA to membrane-bound β₂GPI is rapidly followed by formation of divalent ACA−(β₂GPI)₂ complexes. Experiments with monovalent Fab¹ fragments of ACA showed no appreciable effect on the β₂GPI binding to lipid, substantiating the notion that divalent interactions are essential for the high-affinity binding of ACA−β₂GPI. The anticoagulant effect of ACA is rationalized by the observation that binding of ACA−β₂GPI complexes to the PSPC membrane severely restricts the adsorption of blood coagulation factor Xa.