Selection of phages that inhibit vWF interaction with collagen under both static and flow conditions.
Phages 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 x 10(10) phages/ml, and of vWF to collagen with an IC50 of 1.0 x 10(10) phages/ml at 0.5 microg/ml vWF. Under flow conditions, 1.5 x 10(11) 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 microg/ml (16 microM). The peptide furthermore inhibited vWF-binding to collagen with a maximum of 40% at a concentration of 1.25 mg/ml (650 microM), 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.
Ulrichts
H
H
Laboratory for Thrombosis Research, IRC, Kortrijk, Belgium.
Depraetere
H
H
Harsfalvi
J
J
Deckmyn
H
H
D001435:Bacteriophages / Q000737:chemistry / Q000378:metabolism*; D024042:Collagen Type I / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004347:Drug Interactions / Q000502:physiology; D006801:Humans; D020128:Inhibitory Concentration 50; D010446:Peptide Fragments / Q000494:pharmacology; D010973:Platelet Adhesiveness; D011485:Protein Binding; D012212:Rheology; D013314:Stress, Mechanical; D014841:von Willebrand Factor / Q000037:antagonists & inhibitors / Q000378:metabolism*
D024042:Collagen Type I; D010446:Peptide Fragments; D014841:von Willebrand Factor