title: Reconstitution of the platelet glycoprotein Ib-IX complex in phospholipid bilayer Nanodiscs.
abstract: 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-GPIbα antibodies with epitopes overlapping with the VWF-binding site, but not by anti-GPIbβ monoclonal antibody RAM.1. Finally, the Nanodisc-reconstituted GPIb-IX complex exhibited ligand binding activity similar to that of the isolated extracellular domain of GPIbα. In conclusion, the GPIb-IX complex in Nanodiscs adopts a 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.
authors.affiliation: Ministry of Health Key Laboratory of Thrombosis and Haemostasis, Jiangsu Institute of Hematology, First Affiliated Hospital of Soochow University, Suzhou, China.
authors.forename: Rong
authors.initials: R
authors.lastname: Yan
authors.forename: Xi
authors.initials: X
authors.lastname: Mo
authors.forename: Angel M
authors.initials: AM
authors.lastname: Paredes
authors.forename: Kesheng
authors.initials: K
authors.lastname: Dai
authors.forename: Francois
authors.initials: F
authors.lastname: Lanza
authors.forename: Miguel A
authors.initials: MA
authors.lastname: Cruz
authors.forename: Renhao
authors.initials: R
authors.lastname: Li
mesh_terms: D000911:Antibodies, Monoclonal / Q000378:metabolism; D001792:Blood Platelets / Q000737:chemistry; D003435:Crotalid Venoms / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry*; D036103:Nanotechnology; D010743:Phospholipids / Q000737:chemistry; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D014841:von Willebrand Factor / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D003435:Crotalid Venoms; D008051:Lipid Bilayers; D010743:Phospholipids; D019038:Platelet Glycoprotein GPIb-IX Complex; C023592:glycocalicin; D014841:von Willebrand Factor; C030961:botrocetin
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