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 monoclo nal antibody (MoAb) 82D6A3 (an anti-von Willebrand Factor [VWF] antibo dy 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 s pecific and dose-dependent manner. The two phage clones were further u sed in a two-direction competition experiment with VWF. VWF was able t o displace phages from collagen in a dose-dependent manner with an IC5 0 of 35 mu g/mL and phages were able to inhibit VWF binding to collage n. With the use of specific primers, the sequence of the cysteine-flan ked hexapeptide inserts could be deduced. The two phage crones carried an almost identical sequence, CVWLWEQC and CVWLWENC, with a substitut ion of an hi for a Q at position 6 of the hexapeptide. Sequence compar ison with the known VWF sequence showed the presence of a comparable s equence at position 1129-1136 (VWTLPDQC), located between the collagen -binding A(3)-domain and the D-4-domain. The two cyclic peptides, the putative corresponding VWF peptide, and a peptide with a scrambled cyc lic sequence were synthesized. The two cyclic peptides inhibited VWF b inding 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 mu mol/L and 34.8 +/- 8.59 mu mol/L (mean +/- SEM, n = 3) of the N- and the Q-peptide, respectiv ely, were needed. The two cyclic peptides were also able to inhibit VW F binding to calfskin and human collagen type I, but effective concent rations were some 5 to 10 times higher. (C) 1998 by The American Socie ty of Hematology.