Selection of Phages that Inhibit vWF Interaction with Collagen under both Static and Flow Conditions

Abstract

SummaryPhages from a pentadecamer phage display library were selected for binding to vWF by affinity panning. Bound phages were selectively eluted with human collagen type I. After the third round of panning 95% of individual phage clones bound to vWF. The B8-phage inhibited the binding of collagen to vWF with an IC50 of 0.6 × 1010 phages/ml, and of vWF to collagen with an IC50 of 1.0 × 1010 phages/ml at 0.5 μg/ml vWF. Under flow conditions, 1.5 × 1011 B8-phage/ml nearly completely inhibited platelet deposition on a human collagen type I coated surface at a shear rate of 1200 s-1, while phages without an insert had no effect. The peptide corresponding to the one displayed on the B8-phage competed with the phage for binding to vWF with an IC50 of 30 μg/ml (16 μM). The peptide furthermore inhibited vWF-binding to collagen with a maximum of 40% at a concentration of 1.25 mg/ml (650 μM), higher concentrations of peptide could not improve this. We thus have selected phages that are potent vWF-binders and that can be used as tools to detect vWF, to inhibit vWF-collagen interaction and to further analyse the role of vWF-collagen binding.