Inhibition assay for the binding of biotinylated von Willebrand factor to platelet-bound microtiter wells in the presence of ristocetin or botrocetin.

We developed a solid-phase inhibition assay for the binding of biotinylated von Willebrand factor (vWF) to platelet glycoprotein (GP)Ib based on an enzyme-linked immunosorbent assay (ELISA) with platelet-bound microtiter wells. Washed platelets were immobilized onto the microplates via the anti-GPIIb/IIIa monoclonal antibody (mAb) VNR-5. In the presence of an antibiotic ristocetin (1 mg/ml, final) or the snake venom botrocetin (2 micrograms/ml, final), biotinylated vWF bound to the affixed platelets with half-maximal binding occurring at a vWF concentration of approximately 2 micrograms/ml. Several specific inhibitors of the binding of vWF to GPIb abolished the interaction of biotinylated vWF with GPIb in a dose-dependent fashion, with comparable protein concentrations to those seen in the liquid-phase inhibition assay using 125I-vWF. These inhibitors included mAb against vWF (NMC-4), mAb against GPIb (AP-1), and two vWF fragments containing vWF's putative GPIb-binding domain, namely a 39/34-kDa dispase-digested vWF fragment and a recombinant vWF fragment. Thus, this new assay is a convenient alternative to the conventional inhibition assay using 125I-vWF.