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

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

Veronica H Flood  1 , Joan Cox Gill, Patricia A Morateck, Pamela A Christopherson, Kenneth D Friedman, Sandra L Haberichter, Raymond G Hoffmann, Robert R Montgomery
Affiliations

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

Veronica H Flood et al. Blood. .

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.

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Figures

Figure 1
Figure 1
VWF assays in healthy control subjects. Results for the VWF:Ag, VWF:RCo, and VWF:IbCo ELISA for 113 healthy control subjects enrolled in the ZPMCB-VWD. The line indicates the geometric mean for each assay.
Figure 2
Figure 2
VWF multimers from VWF:IbCo ELISA. Serial VWF:IbCo ELISA measurements were performed and the supernatants analyzed for multimer distribution. A control plasma sample is shown in the first lane, followed by the supernatant from the initial ELISA well for the tGPIbα235Y;239V construct (mutant A sup) and the supernatant after 5 serial incubations with tGPIbα235Y;239V (mutant E sup) in the absence of ristocetin. The last wells show the supernatant from the initial ELISA well for the wild-type GPIbα construct (WT A sup) and the supernatant after 5 serial incubations with wild-type GPIbα (WT E sup) in the presence of 1 mg/mL ristocetin.
Figure 3
Figure 3
Effect of D1472H polymorphism on VWF:RCo and VWF:IbCo ELISA assays. VWF:RCo/VWF:Ag and VWF:IbCo ELISA/VWF:Ag are shown for wild-type A1 domain subjects (●) compared with subjects with the D1472H polymorphism (○). The line indicates the mean for each assay.
Figure 4
Figure 4
VWF assays in type 1 VWD subjects. Results for the VWF:Ag, VWF:RCo, and VWF:IbCo ELISA for 107 type 1 VWD subjects (all with VWF:Ag ≤ 50) enrolled in the ZPMCB-VWD. The line indicates the mean for each assay. All VWF:RCo values reported as less than 10 IU/dL are graphed at 10 IU/dL for visualization purposes.
Figure 5
Figure 5
VWF assays in type 2 VWD subjects. Results for the VWF:Ag, VWF:RCo, and VWF:IbCo ELISA for 18 type 2A (●) and 11 type 2B subjects (○) enrolled in the ZPMCB-VWD. The line indicates the mean for each assay. All VWF:RCo values reported as less than 10 IU/dL are graphed at 10 IU/dL for visualization purposes.
Figure 6
Figure 6
Comparison of VWF:RCo and the VWF:IbCo ELISA. VWF:RCo/VWF:Ag (●) and VWF:IbCo ELISA/VWF:Ag (○) are shown for healthy controls, type 1, type 2A, type 2B, type 2M, and type 2N VWD subjects. The line indicates the mean for each assay.
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