Dimers of β2-Glycoprotein I Mimic the in Vitro Effects of β2-Glycoprotein I-Anti-β2-glycoprotein I Antibody Complexes

Abstract

Anti-β2-glycoprotein I antibodies are thought to cause lupus anticoagulant activity by forming bivalent complexes with β 2-glycoprotein I (β2GPI). To test this hypothesis, chimeric fusion proteins were constructed of the dimerization domain (apple 4) of factor XI and β2GPI. Both a covalent (apple 4-β 2GPI) and a noncovalent (apple 4-C321S-β2GPI) chimer were constructed. As controls, apple 2-β2GPI and apple 4-C321S-β2GPI-W316S, in which β2GPI-W316S is not able to bind to phospholipids, were made. In a phospholipid binding assay, apple 4-β2GPI and apple 4-C321S-β2GPI were able to bind to phospholipids with an affinity 35 times higher than that of plasma-derived β2GPI and apple 2-β2GPI. Apple 4-C321S-β2GPI-W316S did not bind at all. Only apple 4-β2GPI and apple 4-C321S-β2GPI were able to bind to adhered platelets as shown by immunofluorescence. Using the prothrombin time, which was the most responsive coagulation assay, the clotting time was approximately doubled when 200 μg/ml apple 4-β2GPI or apple 4-C321S-β2GPI was added. Addition of 200 μg/ml plasma-derived β2GPI, apple 2-β2GPI, or apple 4-C321S-β2GPI-W316S did not affect clotting time. Clotting time could be corrected with the addition of extra phospholipids, which is indicative for lupus anticoagulant activity. An additional increase in clotting times for apple 4-β2GPI or apple 4-C321S-β2GPI was achieved by the addition of monoclonal antibodies against β 2GPI. In conclusion, dimerization of β2GPI explains the in vitro observed effects of β2GPI-anti-β 2GPI antibody complexes.