New assay for quantifying functional domain-specific inhibitors against human VWF in plasma
Abstract number: PP-WE-619
Varadi1 K., Schrenk1 G., Langlet2 S., Martineau2 N., Arnaud2 E., Rottensteiner1 H., Turecek1 P.L., Ehrlich1 H.J., Schwarz1 H.P.
11Baxter BioScience, Baxter Innovations GmbH, Vienna, Austria 22Research and Development, Diagnostica Stago SAS, Gennevilliers, France
How-to-cite Varadi K, Schrenk G, Langlet S, Martineau N, Arnaud E, Rottensteiner H, Turecek PL, Ehrlich HJ, Schwarz HP. New assay for quantifying functional domain-specific inhibitors against human VWF in plasma. Journal of Thrombosis and Haemostasis 2009; Volume 7, Supplement 2: Abstract PP-WE-619
Although substitution therapy of patients with von Willebrand's disease rarely causes inhibitor development, it is important in preclinical and clinical studies to assess if the administration of VWF concentrates might induce inhibitor formation. von Willebrand factor (VWF) stabilizes FVIII and mediates platelet adhesion involving different epitopes of VWF. The binding site for FVIII is located at the N-terminal domain. The A1 domain interacts with the glycoprotein Ib receptor of platelets, and the A1 and A3 domains both bind to collagen. We established assay systems for determining inhibitors against the different functional epitopes of VWF, based on the Bethesda assay originally described for measuring FVIII inhibitors in plasma. Plasma samples to be tested were mixed with an equivalent amount of human normal plasma and the residual VWF:RCo (platelet binding) and VWF:CB (collagen binding) activities were measured by commercially available assays. The VWF:FVIII binding was measured with a new prototype assay kit from Stago (France). The assays were validated for human plasma samples and for plasma samples from cynomolgus monkeys treated with human rVWF. All assays showed a precision with a coefficient of variation < 25% and clearly allowed the differentiation between low and high titer anti-human VWF inhibitors. The assays were also able to exclude the presence of inhibitors. Analyses of plasma samples from 3 VWD patients with high (> 50 Bethesda-like U/mL), medium (10–20 BU/mL), and low titers (< 3 BU/mL) of inhibitors resulted in similar inhibitor titers for all 3 assays suggesting a polyclonal nature of the anti-VWF inhibitors.
Disclosure of interest: K Varadi, Baxter, Employee; G Schrenk, Baxter, Employee; S Langlet, Stago, Employee; N Martineau, Stago, Employee; E Arnaud, Stago, Employee; Hp Rottensteiner, Baxter, Employee; PL Turecek, Baxter, Employee; HJ Ehrlich, Baxter, Employee; HP Schwarz, Baxter, Employee.