title: Cytoplasmic truncation of glycoprotein Ib alpha weakens its interaction with von Willebrand factor and impairs cell adhesion.
abstract: The interaction of the platelet glycoprotein (GP) Ib-IX-V complex with von Willebrand factor (VWF) is a critical step in the adhesion of platelets to the subendothelial matrix following endothelial cell damage, particularly under arterial flow conditions. In the human GP Ib-IX-V complex, the recognition of VWF appears to be mediated entirely by GP Ibalpha, the largest of four GP Ib-IX-V polypeptides. The goal of the present study was to investigate the involvement of the cytoplasmic domain of GP Ibalpha in the GP Ib-IX-VWF interaction under both static conditions and in the presence of high fluid shear stress. Using Chinese hamster ovary (CHO) cells that express GP Ibbeta, GP IX, and either wild-type GP Ibalpha or GP Ibalpha mutants missing various lengths of the cytoplasmic domain, we evaluated adhesion and flow-driven cell rolling on immobilized VWF in a parallel-plate flow chamber. Cells expressing GP Ibalpha polypeptides with truncations of 6-82 amino acids rolled faster than cells expressing wild-type GP Ibalpha. Cells that expressed polypeptides with intact actin-binding protein 280 binding sites (truncated to residue 582 of 610) rolled more slowly than those expressing GP Ibalpha with longer truncations. The rolling velocity of cells expressing truncated GP Ibalpha mutants increased with decreasing VWF coating density. In addition, a fraction of the truncated cells exhibited saltatory translocation at the lower VWF densities. Studies measuring the GP Ibalpha-VWF bond strength of three of the mutants using laser tweezers showed that progressive deletion of the cytoplasmic domain led to progressive weakening of the strength of individual GP Ibalpha-VWF bonds.
authors.affiliation: Cox Laboratory for Biomedical Engineering, Rice University, Houston, Texas 77030, USA.
authors.forename: Alicia J
authors.initials: AJ
authors.lastname: Schade
authors.forename: Maneesh
authors.initials: M
authors.lastname: Arya
authors.forename: Shan
authors.initials: S
authors.lastname: Gao
authors.forename: Reyhan
authors.initials: R
authors.lastname: Diz-Küçükkaya
authors.forename: Bahman
authors.initials: B
authors.lastname: Anvari
authors.forename: Larry V
authors.initials: LV
authors.lastname: McIntire
authors.forename: José A
authors.initials: JA
authors.lastname: López
authors.forename: Jing-fei
authors.initials: JF
authors.lastname: Dong
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001666:Binding Sites, Antibody / Q000235:genetics; D016466:CHO Cells; D002448:Cell Adhesion / Q000235:genetics; D002460:Cell Line; D002465:Cell Movement / Q000235:genetics; D006224:Cricetinae; D003593:Cytoplasm / Q000235:genetics* / Q000378:metabolism*; D015194:Diffusion Chambers, Culture; D006801:Humans; D007834:Lasers; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics / Q000276:immunology; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology / Q000378:metabolism; D017384:Sequence Deletion; D033081:Shear Strength; D014841:von Willebrand Factor / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D019038:Platelet Glycoprotein GPIb-IX Complex; D011994:Recombinant Proteins; D014841:von Willebrand Factor