Role of divalency in the high-affinity binding of anticardiolipin antibody-beta 2-glycoprotein I complexes to lipid membranes.
beta 2-Glycoprotein I (beta 2GPI) 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 beta 2GPI and the beta 2GPI-mediated binding of ACA to planar membranes composed of phosphatidylcholine (PC) and 5-20 mol % phosphatidylserine (PS). No binding of beta 2GPI was observed to neutral (PC) membranes. Maximal binding of beta 2GPI 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 Kd of 14 microM was observed. Binding constants were insensitive to the chemical composition of the negatively charged phospholipid headgroup. ACA (1.25-10 micrograms.mL-1) caused a 30-40-fold enhancement of beta 2GPI binding to PS/PC membranes (20 mol % PS), resulting in the binding of about 2 pmol.cm-2 divalent ACA-(beta 2GPI)2 complexes at 100 nM beta 2GPI. In the absence of beta 2GPI, binding of ACA was negligible. Ad- and desorption kinetics of ACA-beta 2GPI complexes indicate that the initial monovalent association of ACA to membrane-bound beta 2GPI is rapidly followed by formation of divalent ACA-(beta 2GPI)2 complexes. Experiments with monovalent Fab1 fragments of ACA showed no appreciable effect on the beta 2GPI binding to lipid, substantiating the notion that divalent interactions are essential for the high-affinity binding of ACA-beta 2GPI. The anticoagulant effect of ACA is rationalized by the observation that binding of ACA-beta 2GPI complexes to the PSPC membrane severely restricts the adsorption of blood coagulation factor Xa.
Willems
G M
GM
Cardiovascular Research Institute Maastricht, University of Limburg, The Netherlands.
Janssen
M P
MP
Pelsers
M M
MM
Comfurius
P
P
Galli
M
M
Zwaal
R F
RF
Bevers
E M
EM
D000818:Animals; D017153:Antibodies, Anticardiolipin / Q000378:metabolism* / Q000494:pharmacology; D001667:Binding, Competitive; D001777:Blood Coagulation / Q000502:physiology; D002122:Calcium Chloride / Q000494:pharmacology; D002417:Cattle; D065427:Factor Xa Inhibitors; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000494:pharmacology; D007700:Kinetics; D008051:Lipid Bilayers / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding; D012965:Sodium Chloride / Q000494:pharmacology; D053482:beta 2-Glycoprotein I
D017153:Antibodies, Anticardiolipin; D065427:Factor Xa Inhibitors; D006023:Glycoproteins; D007140:Immunoglobulin Fab Fragments; D008051:Lipid Bilayers; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D010743:Phospholipids; D053482:beta 2-Glycoprotein I; D012965:Sodium Chloride; D002122:Calcium Chloride