Linear analogues derived from the first EGF-like domain of human blood coagulation factor VII: enhanced inhibition of FVIIa/TF complex activity by backbone modification through aspartimide formation

Abstract

Coagulation factor VII bound to its cofactor tissue factor is the physiological initiator of blood coagulation. The interaction between factor VII and tissue factor involves all four of the structural modules found in factor VII, with the most significant contribution coming from the first EGF-like domain. In this study, the synthesis and biological activity of several analogues derived from the first EGF-like domain of FVII comprising the sequence 45-83 are reported on. The six cysteine residues found in the native protein were replaced by Abu. The peptides were isolated from a multicomponent mixture following standard Fmoc solid phase synthesis. Purification and characterisation of the heterogeneous product showed that aspartimide formation was a major side-reaction, occurring predominantly at the Asp46-Gly47 and Asn57-Gly58 dipeptides. Although relatively common in peptide synthesis, the extent to which this side-reaction had taken place was considered surprising. Reported herein are the analytical methods used to isolate and characterise several of the modified products. Also, the inhibitory effect of these peptides on the formation and enzymatic activity of the factor VIIa/tissue factor complex have been compared. Surprisingly, the peptide containing an iso-Asp residue at position 57 possessed 66-fold higher inhibitory activity compared with the original target peptide. A possible explanation for this increase in observed activity is presented.

A novel approach to the synthesis of EGF‐like domains: a method for the one‐pot regioselective formation of the three disulfide bonds of a human blood coagulation Factor VII EGF‐1 analogue

“…One recurrent problem we have observed during the assembly of EGF‐like domains was the formation, often in high yield, of aspartimide ring byproducts (23). Analysis of the amino acid sequence revealed several potentially sensitive areas where an asparagine or aspartic acid residue was positioned at the N ‐terminal side of a glycine residue.…”

“…Analysis of the amino acid sequence revealed several potentially sensitive areas where an asparagine or aspartic acid residue was positioned at the N ‐terminal side of a glycine residue. In vitro data of the EGF‐like analogues indicated that one of the ring‐opened β‐aspartic acid peptides, isolated as an impurity during the synthesis, possessed increased biological activity compared with the parent sequence (23). We were therefore interested in synthesizing analogues where the β‐Asp residue was introduced deliberately during the assembly of the peptide in order to compare and contrast the structure and biological activity of this product.…”

“…This module also provides a major part of the contact area between the two proteins (5,27). We have previously evaluated peptides from both the EGF‐1 and the EGF‐2 domain of FVII as inhibitors of the FVIIa/TF enzymatic activity (23,25). The isolated impurity Asn57β–Asp had an IC 50 of 1 µ m , which was 66 times better than the native linear EGF‐1 sequence (23).…”

“…We have previously evaluated peptides from both the EGF‐1 and the EGF‐2 domain of FVII as inhibitors of the FVIIa/TF enzymatic activity (23,25). The isolated impurity Asn57β–Asp had an IC 50 of 1 µ m , which was 66 times better than the native linear EGF‐1 sequence (23). Constraining this molecule by reintroducing in a stepwise manner two and three of the disulfide bridges was anticipated to increase the activity of these peptides.…”

Synthesis, biological activity, and solution structures of a cyclic dodecapeptide from the EGF‐2 domain of blood coagulation factor VII

“…We have previously shown the functional importance of both EGF domains of FVII in the induction of coagulation via the extrinsic pathway (13,14). Furthermore, we reported short peptides derived from the EGF‐2 domain, which possess anticoagulant activity (15).…”