title: Phenotype changes resulting in high-affinity binding of von Willebrand factor to recombinant glycoprotein Ib-IX: analysis of the platelet-type von Willebrand disease mutations.
abstract: To maintain hemostasis under shear conditions, there must be an interaction between the platelet glycoprotein (GP) Ib-IX receptor and the plasma ligand von Willebrand factor (vWf). In platelet-type von Willebrand disease (Pt-vWD), hemostasis is compromised. Two mutations in the GPIbalpha polypeptide chain have been identified in these patients-a glycine-233 to valine change and a methionine-239 to valine change. For this investigation, these mutant proteins have been expressed in a Chinese hamster ovary cell model system. Ligand-binding studies were performed at various concentrations of ristocetin, and adhesion assays were performed under flow conditions. The Pt-vWD mutations resulted in a gain-of-function receptor. vWf binding was increased at all concentrations of ristocetin examined, and adhesion on a vWf matrix was enhanced in terms of cell tethering, slower rolling velocity, and decreased detachment with increasing shear rate. Two other mutations were also introduced into the GPIbalpha chain. One mutation, encompassing both the Pt-vWD mutations, created an increase in the hydrophobicity of this region. The second mutation, involving a valine-234 to glycine change, decreased the hydrophobicity of this region. Both mutations also resulted in a gain-of-function receptor, with the double mutation producing a hyperreactive receptor for vWf. These data further support the hypothesis that ligand binding is regulated by conformational changes in the amino-terminal region of GPIbalpha, thereby influencing the stability of the GPIbalpha-vWf interaction.
authors.affiliation: Department of Haematology, Prince of Wales Hospital, Sydney, Australia. s.tait@unsw.edu.au
authors.forename: A S
authors.initials: AS
authors.lastname: Tait
authors.forename: S L
authors.initials: SL
authors.lastname: Cranmer
authors.forename: S P
authors.initials: SP
authors.lastname: Jackson
authors.forename: I W
authors.initials: IW
authors.lastname: Dawes
authors.forename: B H
authors.initials: BH
authors.lastname: Chong
mesh_terms: D000818:Animals; D016466:CHO Cells; D002448:Cell Adhesion; D002449:Cell Aggregation; D006224:Cricetinae; D006487:Hemostasis; D009154:Mutation; D010641:Phenotype; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000235:genetics* / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D012310:Ristocetin / Q000494:pharmacology; D014842:von Willebrand Diseases / Q000235:genetics* / Q000378:metabolism; D014841:von Willebrand Factor / Q000378:metabolism*
chemical_list: D019038:Platelet Glycoprotein GPIb-IX Complex; D011994:Recombinant Proteins; D014841:von Willebrand Factor; D012310:Ristocetin