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.
Martin
D M
DM
Haemostasis Research Group, MRC Clinical Research Centre, Harrow, Middlesex, U.K.
O'Brien
D P
DP
Tuddenham
E G
EG
Byfield
P G
PG
D015055:1-Carboxyglutamic Acid / Q000737:chemistry*; D000577:Amides / Q000737:chemistry; D000818:Animals; D002417:Cattle; D002852:Chromatography, Ion Exchange; D005167:Factor VII / Q000737:chemistry*; D005170:Factor X / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D010455:Peptides / Q000138:chemical synthesis; D010743:Phospholipids / Q000737:chemistry; D011487:Protein Conformation
D000577:Amides; D010455:Peptides; D010743:Phospholipids; D015055:1-Carboxyglutamic Acid; D005167:Factor VII; D005170:Factor X