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

Anti-β 2 -glycoprotein I antibodies are thought to cause lupus anticoagulant activity by forming bivalent complexes with β 2 -glycoprotein I (β 2 GPI). To test this hypothesis, chimeric fusion proteins were constructed of the dimerization domain (apple 4) of factor XI and β 2 GPI. Both a covalent (apple 4-β 2 GPI) and a noncovalent (apple 4-C321S-β 2 GPI) chimer were constructed. As controls, apple 2-β 2 GPI and apple 4-C321S-β 2 GPI-W316S, in which β 2 GPI-W316S is not able to bind to phospholipids, were made. In a phospholipid binding assay, apple 4-β 2 GPI and apple 4-C321S-β 2 GPI were able to bind to phospholipids with an affinity 35 times higher than that of plasma-derived β 2 GPI and apple 2-β 2 GPI. Apple 4-C321S-β 2 GPI-W316S did not bind at all. Only apple 4-β 2 GPI and apple 4-C321S-β 2 GPI 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-β 2 GPI or apple 4-C321S-β 2 GPI was added. Addition of 200 μg/ml plasma-derived β 2 GPI, apple 2-β 2 GPI, or apple 4-C321S-β 2 GPI-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-β 2 GPI or apple 4-C321S-β 2 GPI was achieved by the addition of monoclonal antibodies against β 2 GPI. In conclusion, dimerization of β 2 GPI explains the in vitro observed effects of β 2 GPI-anti-β 2 GPI antibody complexes.