title: Functional expression of single chain glycoprotein Ib alpha on the surface of COS cells and BHK cells.
abstract: The GpIb-IX complex constitutes the major receptor for von Willebrand factor (vWF) on the surface of blood platelets. The vWF-binding site has been mapped to one of the three constituent chains, GpIb alpha. Surface expression of GpIb alpha depends on the correct intracellular assembly with the GpIb beta and GpIX chains. We have now grafted a portion of the extracellular domain of GpIb alpha containing the vWF binding site onto the transmembrane/intracellular domain of the single chain surface molecule ICAM-1. Transient transfection of this chimeric protein in COS cells resulted in surface expression as assessed by immunostaining of live cells. Similar results were obtained after stable transfection into BHK cells. Purified vWF bound to the surface of transfected cells in the presence of ristocetin and botrocetin with a Kd of 52 ng/ml, comparable to the Kd for fixed platelets (65.5 ng/ml). This study indicates that functional expression of the vWF-binding domain of GpIb alpha on the surface of mammalian cells can be obtained in the absence of GpIb beta and GpIX. Furthermore, this model system simplifies existing methods for the assessment of the functional consequences of mutations in GpIb alpha as found in pseudo-von Willebrand disease and Bernard-Soulier syndrome.
authors.affiliation: Department of Haematology, University Hospital Utrecht, The Netherlands. j.vd.velde@digd.azu.nl
authors.forename: E J
authors.initials: EJ
authors.lastname: Petersen
authors.forename: E
authors.initials: E
authors.lastname: Posthumus
authors.forename: J J
authors.initials: JJ
authors.lastname: Sixma
mesh_terms: D000818:Animals; D019556:COS Cells; D002460:Cell Line; D006224:Cricetinae; D003435:Crotalid Venoms / Q000494:pharmacology; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D007668:Kidney; D008647:Mesocricetus; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012310:Ristocetin / Q000494:pharmacology; D014162:Transfection; D014841:von Willebrand Factor / Q000378:metabolism*
chemical_list: D003435:Crotalid Venoms; D019038:Platelet Glycoprotein GPIb-IX Complex; D011993:Recombinant Fusion Proteins; D014841:von Willebrand Factor; D018799:Intercellular Adhesion Molecule-1; D012310:Ristocetin; C030961:botrocetin