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

Coagulation factor VII bound to its cofactor tissue factor is the physiolog ical 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 ana logues derived from the first EGF-like domain of FVII comprising the sequen ce 45-83 are reported on. The six cysteine residues found in the native pro tein were replaced by Abu. The peptides were isolated from a multicomponent mixture following standard Fmoc solid phase synthesis. Purification and ch aracterisation of the heterogeneous product showed that aspartimide formati on was a major side-reaction, occurring predominantly at the Asp(46)-G1y(47 ) and Asn(57)-Gly(58) dipeptides. Although relatively common in peptide syn thesis, the extent to which this side-reaction had taken place was consider ed surprising. Reported herein are the analytical methods used to isolate a nd characterise several of the modified products. Also, the inhibitory effe ct of these peptides on the formation and enzymatic activity of the factor VIIa/tissue factor complex have been compared. Surprisingly, the peptide co ntaining an iso-Asp residue at position 57 possessed 66-fold higher inhibit ory activity compared with the original target peptide. A possible explanat ion for this increase in observed activity is presented. Copyright (C) 1999 European Peptide Society and John Wiley & Sons, Ltd.