Reconstitution of the Platelet Glycoprotein Ib-IX Complex in Phospholipid Bilayer Nanodiscs
Reconstitution of the Platelet Glycoprotein Ib-IX Complex in Phospholipid Bilayer Nanodiscs
复制标题
磷脂双层纳米圆盘中血小板糖蛋白 Ib-IX 复合物的重建
DOI：
10.1021/bi201351d
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发表时间：
2011-12-13
期刊：
影响因子：
2.9
通讯作者：
Li, Renhao
中科院分区：
文献类型：
--
作者：
Yan, Rong;Mo, Xi;Li, Renhao
血小板质膜上表达的糖蛋白Ib-IX （GPIb-IX）复合体通过血小板与暴露在损伤血管壁上的血管性血友病因子（VWF）开始粘附参与血栓形成和止血。虽然大多数关于GPIb-IX复合物的知识是从血小板和表达GPIb-IX复合物的转染哺乳动物细胞的研究中获得的，但目前还没有一种体外膜系统可以对这种受体进行系统分析。磷脂双分子层纳米盘由膜支架蛋白包裹的磷脂块组成，是膜蛋白研究的重要工具。我们在这里展示了从人血小板中纯化的GPIb-IX复合体已经被重组到纳米盘中。纳米盘重组的GPIb-IX复合物能够结合多种构象敏感的单克隆抗体。在保乳素存在的情况下与VWF结合，表观K-d为0.73 +/- 0.07 nM。抗原表位与VWF结合位点重叠的抗gpib α抗体可抑制其与VWF的结合，而抗gpib β单克隆抗体ram 1则不能抑制其与VWF的结合。最后，纳米盘重构的GPIb-IX配合物显示出配体。结合活性类似于分离的GPIb α的胞外结构域。综上所述，GPIb-IX复合物具有纳米盘的天然构象，并且具有结合其天然配体的能力，因此纳米盘是进一步研究这种止血作用重要受体复合物的合适体外平台。
The glycoprotein Ib-IX (GPIb-IX) complex expressed on platelet plasma membrane is involved in thrombosis and hemostasis via the initiation of adhesion of platelets to von Willebrand factor (VWF) exposed at the injured vessel wall. While most of the knowledge of the GPIb-IX complex was obtained from studies on platelets and transfected mammalian cells expressing the GPIb-IX complex, there is not an in vitro membrane system that allows systematic analysis of this receptor. The phospholipid bilayer Nanodisc composed of a patch of phospholipid surrounded by membrane scaffold protein is an attractive tool for membrane protein study. We show here that the GPIb-IX complex purified from human platelets has been reconstituted into the Nanodisc. The Nanodisc-reconstituted GPIb-IX complex was able to bind various conformation-sensitive monoclonal antibodies. Furthermore, it bound to VWF in the presence of botrocetin with an apparent K-d of 0.73 +/- 0.07 nM. The binding to VWF was inhibited by anti-GPIb alpha antibodies with epitopes overlapping with the VWF-binding site, but not by anti-GPIb beta monoclonal antibody RAM.1. Finally, the Nanodisc-reconstituted GPIb-IX complex exhibited ligand. binding activity similar to that of the isolated extracellular domain of GPIb alpha. In conclusion, the GPIb-IX complex in Nanodiscs native-like Conformation and possesses the ability to bind its natural ligands, thus making a Nanodisc a suitable in vitro platform for further investigation of this hemostatically important receptor complex.