Lutters B C, Meijers J C, Derksen R H, Arnout J, de Groot P G
Department of Haematology, University Medical Center, 3508 GA Utrecht, Utrecht University, 3508 TB Utrecht, The Netherlands.
J Biol Chem. 2001 Feb 2;276(5):3060-7. doi: 10.1074/jbc.M008224200. Epub 2000 Oct 25.
Anti-beta(2)-glycoprotein I antibodies are thought to cause lupus anticoagulant activity by forming bivalent complexes with beta(2)-glycoprotein I (beta(2)GPI). To test this hypothesis, chimeric fusion proteins were constructed of the dimerization domain (apple 4) of factor XI and beta(2)GPI. Both a covalent (apple 4-beta(2)GPI) and a noncovalent (apple 4-C321S-beta(2)GPI) chimer were constructed. As controls, apple 2-beta(2)GPI and apple 4-C321S-beta(2)GPI-W316S, in which beta(2)GPI-W316S is not able to bind to phospholipids, were made. In a phospholipid binding assay, apple 4-beta(2)GPI and apple 4-C321S-beta(2)GPI were able to bind to phospholipids with an affinity 35 times higher than that of plasma-derived beta(2)GPI and apple 2-beta(2)GPI. Apple 4-C321S-beta(2)GPI-W316S did not bind at all. Only apple 4-beta(2)GPI and apple 4-C321S-beta(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 microg/ml apple 4-beta(2)GPI or apple 4-C321S-beta(2)GPI was added. Addition of 200 microg/ml plasma-derived beta(2)GPI, apple 2-beta(2)GPI, or apple 4-C321S-beta(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-beta(2)GPI or apple 4-C321S-beta(2)GPI was achieved by the addition of monoclonal antibodies against beta(2)GPI. In conclusion, dimerization of beta(2)GPI explains the in vitro observed effects of beta(2)GPI-anti-beta(2)GPI antibody complexes.
抗β2糖蛋白I抗体被认为通过与β2糖蛋白I（β2GPI）形成二价复合物而导致狼疮抗凝活性。为了验证这一假设，构建了因子XI的二聚化结构域（苹果4）与β2GPI的嵌合融合蛋白。构建了共价（苹果4-β2GPI）和非共价（苹果4-C321S-β2GPI）嵌合体。作为对照，制备了苹果2-β2GPI和苹果4-C321S-β2GPI-W316S，其中β2GPI-W316S不能与磷脂结合。在磷脂结合试验中，苹果4-β2GPI和苹果4-C321S-β2GPI与磷脂结合的亲和力比血浆来源的β2GPI和苹果2-β2GPI高35倍。苹果4-C321S-β2GPI-W316S根本不结合。免疫荧光显示，只有苹果4-β2GPI和苹果4-C321S-β2GPI能够结合黏附的血小板。使用最敏感的凝血试验即凝血酶原时间，当加入200μg/ml苹果4-β2GPI或苹果4-C321S-β2GPI时，凝血时间大约加倍。加入200μg/ml血浆来源的β2GPI、苹果2-β2GPI或苹果4-C321S-β2GPI-W316S不影响凝血时间。加入额外的磷脂可纠正凝血时间，这表明存在狼疮抗凝活性。通过加入抗β2GPI单克隆抗体，苹果4-β2GPI或苹果4-C321S-β2GPI的凝血时间进一步延长。总之，β2GPI的二聚化解释了体外观察到的β2GPI-抗β2GPI抗体复合物的作用。
