PEPTIDES CORRESPONDING TO THE 2ND EPIDERMAL GROWTH FACTOR-LIKE DOMAINOF HUMAN BLOOD-COAGULATION FACTOR-VII - SYNTHESIS, FOLDING AND BIOLOGICAL-ACTIVITY

Factor VIIa (FVIIa) is the enzymatically active constituent of the FVI Ia/tissue factor (TF) complex, the initiator of the extrinsic pathway of blood coagulation. The zymogen FVII and FVIIa are composed of discr ete domains, two of which are homologous to the epidermal growth facto r (EGF). This investigation examined the significance of the FVII EGF- 2 domain in the processes leading to activation of factor X (FX). Pept ides 47 residues in length and corresponding to the amino acid sequenc e of the EGF-2 domain of human FVII were prepared by solid-phase synth esis methods. Peptide variants with all six Cys residues replaced by L -2-aminobutyryl residues (1), or containing one (2a-c), two (3a,b) or three (4) disulfide bonds, were obtained by application of various S-p rotecting groups and oxidation methods. Peptide 4, containing the cyst ine bridge arrangement corresponding to that found in the native prote in, was prepared by a two-step regioselective disulfide bond formation method. An evaluation of the anti-coagulant properties of peptides 1- 4 revealed that all peptides, with the exception of the two-cystine is omer containing non-native disulfide pairings (3b), were potent inhibi tors of TF/FVIIa-mediated activation of FX. The fully constrained pept ide 4 was found to be twice as active as its completely non-constraine d counterpart 1, the two peptides showing ICS, values of 1.6 +/- 0.5 m u M (1) and 0.8 +/- 0.2 mu M (4) with respect to TF/FVIIa-dependent FX activation. The results of this study demonstrate the functional impo rtance of the EGF-2 domain of FVII in the induction of coagulation by the extrinsic pathway. (C) Munksgaard 1997.