title: Gain-of-function GPIb ELISA assay for VWF activity in the Zimmerman Program for the Molecular and Clinical Biology of VWD.
abstract: von Willebrand disease (VWD) is a common bleeding disorder, but diagnosis is sometimes challenging because of issues with the current von Willebrand factor (VWF) assays, VWF antigen (VWF:Ag) and VWF ristocetin cofactor activity (VWF:RCo), used for diagnosis. We evaluated 113 healthy controls and 164 VWD subjects enrolled in the T.S. Zimmerman Program for the Molecular and Clinical Biology of VWD for VWF:Ag, VWF:RCo, and a new enzyme-linked immunosorbent assay (ELISA)-based assay of VWF-glycoprotein Ib (GPIb) interactions using a gain-of-function GPIb construct (tGPIbα(235Y;239V)) as a receptor to bind its ligand VWF in an assay independent of ristocetin (VWF:IbCo ELISA). Healthy controls, type 1, 2A, 2M, and 2N subjects had VWF:RCo/VWF:Ag ratios similar to the ratio obtained with VWF:IbCo ELISA/VWF:Ag. Type 2B VWD subjects, however, had elevated VWF:IbCo ELISA/VWF:Ag ratios. Type 3 VWD subjects had undetectable (< 1.6 U/dL) VWF:IbCo ELISA values. As previously reported, VWF:RCo/VWF:Ag ratio was decreased with a common A1 domain polymorphism, D1472H, as was direct binding to ristocetin for a 1472H A1 loop construct. The VWF:IbCo ELISA, however, was not affected by D1472H. The VWF:IbCo ELISA may be useful in testing VWF binding to GPIb, discrimination of type 2 variants, and in the diagnosis of VWD as it avoids some of the pitfalls of VWF:RCo assays.
authors.affiliation: Department of Pediatrics, Division of Hematology/Oncology, Medical College of Wisconsin, Milwaukee, WI, USA. vflood@mcw.edu
authors.forename: Veronica H
authors.initials: VH
authors.lastname: Flood
authors.forename: Joan Cox
authors.initials: JC
authors.lastname: Gill
authors.forename: Patricia A
authors.initials: PA
authors.lastname: Morateck
authors.forename: Pamela A
authors.initials: PA
authors.lastname: Christopherson
authors.forename: Kenneth D
authors.initials: KD
authors.lastname: Friedman
authors.forename: Sandra L
authors.initials: SL
authors.lastname: Haberichter
authors.forename: Raymond G
authors.initials: RG
authors.lastname: Hoffmann
authors.forename: Robert R
authors.initials: RR
authors.lastname: Montgomery
mesh_terms: D019943:Amino Acid Substitution; D001774:Blood Chemical Analysis / Q000379:methods; D016022:Case-Control Studies; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D066298:In Vitro Techniques; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019038:Platelet Glycoprotein GPIb-IX Complex; D010980:Platelet Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D011110:Polymorphism, Genetic; D011485:Protein Binding; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012310:Ristocetin / Q000378:metabolism; D056725:von Willebrand Disease, Type 1 / Q000097:blood / Q000175:diagnosis / Q000235:genetics; D056728:von Willebrand Disease, Type 2 / Q000097:blood / Q000175:diagnosis / Q000235:genetics; D056729:von Willebrand Disease, Type 3 / Q000097:blood / Q000175:diagnosis / Q000235:genetics; D014842:von Willebrand Diseases / Q000097:blood* / Q000175:diagnosis* / Q000235:genetics; D014841:von Willebrand Factor / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism
chemical_list: D008562:Membrane Glycoproteins; D050505:Mutant Proteins; D019038:Platelet Glycoprotein GPIb-IX Complex; D010980:Platelet Membrane Glycoproteins; D011994:Recombinant Proteins; C094990:adhesion receptor; D014841:von Willebrand Factor; C028254:von Willebrand factor receptor; D012310:Ristocetin