Identification of peptides, selected by phage display technology, that inhibit von Willebrand factor binding to collagen.
A repeated selection of phages from a cyclic hexapeptide phage display library resulted in an enrichment of phages that bound to the monoclonal antibody (MoAb) 82D6A3 (an anti-von Willebrand Factor [vWF] antibody that inhibits binding of vWF to collagen). Two clones were selected that bound both to MoAb 82D6A3 and to rat tail collagen type I in a specific and dose-dependent manner. The two phage clones were further used in a two-direction competition experiment with vWF. vWF was able to displace phages from collagen in a dose-dependent manner with an IC50 of 35 micrograms/mL and phages were able to inhibit vWF binding to collagen. With the use of specific primers, the sequence of the cysteine-flanked hexapeptide inserts could be deduced. The two phage clones carried an almost identical sequence, CVWLWEQC and CVWLWENC, with a substitution of an N for a Q at position 6 of the hexapeptide. Sequence comparison with the known vWF sequence showed the presence of a comparable sequence at position 1129-1136 (VWTLPDQC), located between the collagen-binding A3-domain and the D4-domain. The two cyclic peptides, the putative corresponding vWF peptide, and a peptide with a scrambled cyclic sequence were synthesized. The two cyclic peptides inhibited vWF binding to rat tail collagen type I in a dose-dependent manner, whereas the linear vWF peptide and the scrambled cyclic peptide were inactive. For half maximal inhibition, 100 +/- 12.7 micromol/L and 34.8 +/- 8.59 micromol/L (mean +/- SEM, n = 3) of the N- and the Q-peptide, respectively, were needed. The two cyclic peptides were also able to inhibit vWF binding to calfskin and human collagen type I, but effective concentrations were some 5 to 10 times higher.
Depraetere
H
H
Laboratory for Thrombosis Research, Interdisciplinary Research Center, KU Leuven Campus Kortrijk, Belgium. Hilde.Depraetere@kulak.ac.be
Viaene
A
A
Deroo
S
S
Vauterin
S
S
Deckmyn
H
H
D000818:Animals; D001435:Bacteriophages; D003094:Collagen / Q000378:metabolism*; D006801:Humans; D019151:Peptide Library; D010455:Peptides / Q000235:genetics* / Q000494:pharmacology*; D011485:Protein Binding / Q000187:drug effects; D051381:Rats; D014841:von Willebrand Factor / Q000378:metabolism*
D019151:Peptide Library; D010455:Peptides; D014841:von Willebrand Factor; D003094:Collagen