A REGION OF VITAMIN-K-DEPENDENT PROTEIN-S THAT BINDS TO C4B BINDING-PROTEIN (C4BP) IDENTIFIED USING BACTERIOPHAGE PEPTIDE DISPLAY LIBRARIES

Vitamin K-dependent protein S, a blood coagulation inhibitor, interact s with the C4b-binding protein (C4BP) in human plasma with high affini ty (K-D = 0.1 nM). Identification of a portion of protein S that binds to C4BP has been approached using random libraries of 6- and 15-mer p eptides displayed on bacteriophage surfaces. Bacteriophage binding to the beta-chain of C4BP were selected in several rounds of affinity pur ification with intervening amplification in E. coli. Homology searches of the affinity purified peptide sequences against protein S led to t he identification of four regions in protein S that were similar to se veral of the selected peptides. These regions were synthesized as line ar peptides and tested in inhibition experiments. Only one distinct pe ak (around position 450) was observed when the homology scores versus human protein S sequence were averaged over all affinity purified pept ides. A synthetic peptide comprising residues 439-460 in human protein S was found to inhibit protein S binding to C4BP. The same result was found with two overlapping peptides (residues 447-468 and 435-468, re spectively) in a second set of synthetic peptides. Direct binding of t he peptides to C4BP was inferred from titrations monitored by recordin g the near UV circular dichroism spectra or the polarization of trypto phan fluorescence. The results suggest that residues 447-460 constitut e a portion of protein S that is important for the interaction with C4 BP. These findings may have implications for patients suffering from t hrombosis, due to the lack of free protein S, by directing the design of drugs that disrupt protein S binding to C4BP.