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 variant sequences of the human factor VII gamma-carboxyglutamic acid (Gla)-containing domain have been prepared by direct peptide synthesis using the Fmoc-based protection strategy. Peptides were purified by ion-exchange and reversed-phase chromatography and characterized as the correct products. A peptide comprising residues 1-49 (GP 1-49) inhibited the activation of factor X (FX) by soluble tissue factor (sTF) and recombinant activated factor VII (rFVIIa). In the absence of phospholipid, no inhibition by this peptide was observed. GP 1-49 did not inhibit the hydrolysis of a peptidyl substrate by rFVIIa in the presence of either sTF or relipidated TF apoprotein in the presence or absence of phospholipid. A similar peptide (residues 1-38, GP 1-38) that did not contain the aromatic stack region was also inhibitory. Two variant peptides, one identical to GP 1-49 but lacking the N-terminal alanine residue (GP 2-49) and one identical to GP 1-38 but with an arginine to alanine substitution at position 9 (GP 1-38 R9A), showed substantially reduced inhibitory activity. Kinetic analysis of the inhibition of Xa generation by GP 1-49 revealed a noncompetitive mode of inhibition, probably via a substrate-depletion mechanism. GP 1-49 does not inhibit by preventing FX binding to phospholipid surfaces. This indicates that the N-terminal residues of the FVII Gla domain are important for the structural integrity of the peptide, and implicates the Gla domain per se in a direct interaction with phospholipid- bound FX.[1]References
- Synthesis and characterization of wild-type and variant gamma-carboxyglutamic acid-containing domains of factor VII. Martin, D.M., O'Brien, D.P., Tuddenham, E.G., Byfield, P.G. Biochemistry (1993) [Pubmed]
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