SYNTHESIS AND CHARACTERIZATION OF WILD-TYPE AND VARIANT GAMMA-CARBOXYGLUTAMIC ACID-CONTAINING DOMAINS OF FACTOR-VII

Synthetic peptides corresponding to portions of the wild-type and vari ant sequences of the human factor VII gamma-carboxyglutamic acid (Gla) -containing domain have been prepared by direct peptide synthesis usin g the Fmoc-based protection strategy. Peptides were purified by ion-ex change and reversed-phase chromatography and characterized as the corr ect products. A peptide comprising residues 1-49 (GP 1-49) inhibited t he activation of factor X (FX) by soluble tissue factor (sTF) and reco mbinant activated factor VII (rFVIIa). In the absence of phospholipid, no inhibition by this peptide was observed. GP 1-49 did not inhibit t he hydrolysis of a peptidyl substrate by rFVIIa in the presence of eit her sTF or relipidated TF apoprotein in the presence or absence of pho spholipid. A similar peptide (residues 1-38, GP 1-38) that did not con tain the aromatic stack region was also inhibitory. Two variant peptid es, one identical to GP 1-49 but lacking the N-terminal alanine residu e (GP 2-49) and one identical to GP 1-38 but with an arginine to alani ne substitution at position 9 (GP 1-38 R9A), showed substantially redu ced inhibitory activity. Kinetic analysis of the inhibition of Xa gene ration by GP 1-49 revealed a noncompetitive mode of inhibition, probab ly via a substrate-depletion mechanism. GP 1-49 does not inhibit by pr eventing FX binding to phospholipid surfaces. This indicates that the N-terminal residues of the FVII Gla domain are important for the struc tural integrity of the peptide, and implicates the Gla domain per se i n a direct interaction with phospholipid-bound FX.