Kolyada Alexey, Barrios David A, Beglova Natalia
Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, 02215, USA.
Antibodies (Basel). 2017 Jun;6(2). doi: 10.3390/antib6020008. Epub 2017 Jun 2.
Upregulation of the procoagulant activity of monocytes by antibodies to beta2- glycoprotein I (β2GPI) is one of the mechanisms contributing to thrombosis in antiphospholipid syndrome. Current knowledge about receptors responsible for the upregulation of procoagulant activity by β2GPI/anti-β2GPI complexes and their binding sites on β2GPI is far from complete. We quantified the procoagulant activity expressed by phorbol 12-myristate 13-acetate (PMA)- differentiated U937 cells by measuring clotting kinetics in human plasma exposed to stimulated cells. Cells stimulated with anti-β2GPI were compared to cells treated with dimerized domain V of β2GPI (β2GPI-DV) or point mutants of β2GPI-DV. We demonstrated that dimerized β2GPI-DV is sufficient to induce procoagulant activity in monocytes. Using site-directed mutagenesis, we determined that the phospholipid-binding interface on β2GPI is larger than previously thought and includes Lys308 in β2GPI-DV. Intact residues in two phospholipid-binding loops of β2GPI-DV were important for the potentiation of procoagulant activity. We did not detect a correlation between the ability of β2GPI-DV variants to bind ApoER2 and potentiation of the procoagulant activity of cells. The region on β2GPI inducing procoagulant activity in monocytes can now be narrowed down to β2GPI-DV. The ability of β2GPI-DV dimers to come close to cell membrane and attach to it is important for the stimulation of procoagulant activity.
抗β2糖蛋白I（β2GPI）抗体上调单核细胞的促凝活性是抗磷脂综合征中导致血栓形成的机制之一。目前关于负责β2GPI /抗β2GPI复合物上调促凝活性的受体及其在β2GPI上的结合位点的知识还远远不够完整。我们通过测量暴露于刺激细胞的人血浆中的凝血动力学，对佛波酯12-肉豆蔻酸酯13-乙酸酯（PMA）分化的U937细胞表达的促凝活性进行了定量。将用抗β2GPI刺激的细胞与用β2GPI的二聚化结构域V（β2GPI-DV）或β2GPI-DV的点突变体处理的细胞进行比较。我们证明二聚化的β2GPI-DV足以诱导单核细胞中的促凝活性。使用定点诱变，我们确定β2GPI上的磷脂结合界面比以前认为的更大，并且包括β2GPI-DV中的Lys308。β2GPI-DV的两个磷脂结合环中的完整残基对于促凝活性的增强很重要。我们没有检测到β2GPI-DV变体结合载脂蛋白E受体2（ApoER2）的能力与细胞促凝活性增强之间的相关性。现在可以将β2GPI上诱导单核细胞促凝活性的区域缩小到β2GPI-DV。β2GPI-DV二聚体接近细胞膜并附着于其上的能力对于促凝活性的刺激很重要。
