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Updated: Oct 26, 2026
Investigating von Willebrand Factor Pathophysiology Using a Flow Chamber Model of von Willebrand Factor-platelet String Formation
Published on: August 14, 2017
A screening assay for type 2B von Willebrand factor variants using an enzyme-linked immunosorbent assay based on
Maria Teresa Pagliari1, Luciano Baronciani1, Paola Colpani1
1Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Milan, Italy.
Background:
Type 2B von Willebrand disease (VWD) is caused by gain-of-function variants in the A1 domain of von Willebrand factor (VWF), leading to increased platelet binding, loss of high-molecular-weight multimers, and sometimes thrombocytopenia. Conventional diagnosis relies on ristocetin-induced platelet agglutination or molecular testing.
Aim:
To evaluate an in-house enzyme-linked immunosorbent assay (ELISA) based on a recombinant glycoprotein Ib wild-type (rGPIb-WT) fragment and ristocetin, as a screening tool to identify first-time referral patients requiring further evaluation for possible type 2B VWD.
Materials And Methods:
Plasma from 131 healthy controls and 31 type 2B patients was analyzed using rGPIb-WT ELISA at ristocetin concentrations of 0.06, 0.09, and 0.12 mg/mL. Optical density ratios of plasma sample to normal pooled plasma were used to generate receiver operating characteristic curves and define screening cut-offs. Plasma from 254 first-time referrals was tested. Positive samples were evaluated for platelet-dependent VWF activity over VWF antigen ratio (VWF:GPIbR/VWF:Ag) and VWF multimers. Candidate patients underwent VWF exon 28 Sanger sequencing.
Results:
Twenty-six of 254 patients tested positive at least at 1 ristocetin concentration. Sixteen were considered candidates for type 2B VWD evaluation based on a VWF:GPIbR/VWF:Ag ratio <0.7 and/or an abnormal multimeric pattern. Type 2B variants were found in 12 of 16 sequenced patients. The remaining 10 patients showed ultra-large multimers or an increased proportion of high-molecular-weight multimers; 8 of these had a normal VWF:GPIbR/VWF:Ag ratio (>0.7).
Discussion:
The rGPIb-WT ELISA positivity may reflect type 2B variants or the presence of larger multimers. Integrated with conventional assays, positivity may guide type 2B-directed evaluation.
