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Synthesis and characterization of wild-type and variant .gamma.-carboxyglutamic acid-containing domains of factor VII

Biochemistry1993Vol. 32(50), pp. 13949–13955

David Martin, Donogh P. O'Brien, Edward G. D. Tuddenham, PETER G. H. BYFIELD

Abstract

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.

Abstract

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