Arnout J, Wittevrongel C, Vanrusselt M, Hoylaerts M, Vermylen J
Center for Molecular and Vascular Biology, University of Leuven, Belgium.
Thromb Haemost. 1998 Jan;79(1):79-86.
The precise mechanism by which Beta-2-glycoprotein I (beta2-GPI-) dependent lupus anticoagulants lengthen phospholipid-dependent clotting reactions is still poorly understood. In order to study this, murine monoclonal antibodies (moabs) against human beta2GPI were raised. Eight of the 21 anti-beta2GPI moabs, obtained from 2 fusions, fulfilled the criteria for lupus anticoagulant (LA) activity as tested with a variety of sensitive screening assays and confirmatory tests. Seven moabs did not influence any clotting test. The LA positive moabs were found to compete for similar or closely spaced epitopes on immobilized beta2GPI. Two moabs with potent LA activity (moabs 22 F 6 and 22 B 3) and 1 moab without LA activity (moab 16 B 3) were selected to study the interaction between antibody, beta2GPI and phospholipid. Interactions were investigated by real-time biospecific interaction analysis (BIA) based on plasmon surface resonance technology on a BIA-core instrument using a sensor chip coated with phospholipid. When 22 F 6, the moab with the most pronounced LA activity, was allowed to interact with the phospholipid surface at concentrations between 0 and 400 nmol/l, no appreciable binding could be detected. Likewise, no binding could be measured when beta2GPI at concentrations between 0 and 400 nmol/l was passed over the phospholipid coated sensor chip. Combinations of beta2GPI and 22 F 6 resulted in significant binding. Similar results were obtained with 22 B 3, another moab with LA activity. A LA negative Moab, 16 B 3, did not cause binding of antibody-beta2GPI complexes. Fab' fragments, derived from moab 22 F 6, inhibited the binding of beta2GPI-22 F 6 and beta2GPI-22 B 3 in a concentration dependent way, indicating that only bivalent beta2GPI-antibody complexes bind with high affinity to phospholipids. Fab' fragments, derived from moab 22 F 6, also inhibited the LA effect of moabs 22 F 6 and 22 B 3 in diluted plasma. In summary, these experiments indicate that the beta2GPI-dependent LA effect depends on the formation of bivalent beta2GPI-antibody complexes on phospholipid surfaces.
β2糖蛋白I（β2-GPI）依赖性狼疮抗凝物延长磷脂依赖性凝血反应的确切机制仍未完全明了。为研究此机制，制备了针对人β2GPI的鼠单克隆抗体（mAb）。从2次融合获得的21种抗β2GPI mAb中，有8种通过多种敏感筛选试验和确证试验检测，符合狼疮抗凝物（LA）活性标准。7种mAb不影响任何凝血试验。发现LA阳性mAb竞争固定化β2GPI上相似或紧密相邻的表位。选择2种具有强LA活性的mAb（mAb 22 F 6和22 B 3）和1种无LA活性的mAb（mAb 16 B 3）研究抗体、β2GPI与磷脂之间的相互作用。基于表面等离子体共振技术，在BIA-core仪器上，使用包被磷脂的传感器芯片，通过实时生物特异性相互作用分析（BIA）研究相互作用。当具有最显著LA活性的mAb 22 F 6在0至400 nmol/L浓度下与磷脂表面相互作用时，未检测到明显结合。同样，当0至400 nmol/L浓度的β2GPI通过包被磷脂的传感器芯片时，也未检测到结合。β2GPI与22 F 6的组合导致显著结合。另一种具有LA活性的mAb 22 B 3也得到类似结果。LA阴性mAb 16 B 3未引起抗体-β2GPI复合物的结合。源自mAb 22 F 6的Fab′片段以浓度依赖性方式抑制β2GPI-22 F 6和β2GPI-22 B 3的结合，表明只有二价β2GPI-抗体复合物以高亲和力与磷脂结合。源自mAb 22 F 6的Fab′片段也抑制mAb 22 F 6和22 B 3在稀释血浆中的LA效应。总之，这些实验表明β2GPI依赖性LA效应取决于磷脂表面二价β2GPI-抗体复合物的形成。
