Gain-of-function GPIb ELISA assay for VWF activity in the Zimmerman Program for the Molecular and Clinical Biology of VWD

Blood. 2011 Feb 10;117(6):e67-74. doi: 10.1182/blood-2010-08-299016. Epub 2010 Dec 10.

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.

Publication types

  • Evaluation Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • Blood Chemical Analysis / methods
  • Case-Control Studies
  • Enzyme-Linked Immunosorbent Assay / methods*
  • Humans
  • In Vitro Techniques
  • Membrane Glycoproteins / genetics
  • Membrane Glycoproteins / metabolism*
  • Mutant Proteins / chemistry
  • Mutant Proteins / genetics
  • Mutant Proteins / metabolism
  • Platelet Glycoprotein GPIb-IX Complex
  • Platelet Membrane Glycoproteins / genetics
  • Platelet Membrane Glycoproteins / metabolism
  • Polymorphism, Genetic
  • Protein Binding
  • Protein Multimerization
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Ristocetin / metabolism
  • von Willebrand Disease, Type 1 / blood
  • von Willebrand Disease, Type 1 / diagnosis
  • von Willebrand Disease, Type 1 / genetics
  • von Willebrand Disease, Type 2 / blood
  • von Willebrand Disease, Type 2 / diagnosis
  • von Willebrand Disease, Type 2 / genetics
  • von Willebrand Disease, Type 3 / blood
  • von Willebrand Disease, Type 3 / diagnosis
  • von Willebrand Disease, Type 3 / genetics
  • von Willebrand Diseases / blood*
  • von Willebrand Diseases / diagnosis*
  • von Willebrand Diseases / genetics
  • von Willebrand Factor / analysis*
  • von Willebrand Factor / chemistry
  • von Willebrand Factor / metabolism

Substances

  • Membrane Glycoproteins
  • Mutant Proteins
  • Platelet Glycoprotein GPIb-IX Complex
  • Platelet Membrane Glycoproteins
  • Recombinant Proteins
  • adhesion receptor
  • von Willebrand Factor
  • von Willebrand factor receptor
  • Ristocetin