title: Fusion of two distinct peptide exosite inhibitors of Factor VIIa.
abstract: Highly potent bifunctional inhibitors of Factor VIIa (FVIIa) were generated by linking two distinct peptides, recently shown to bind to two discrete exosites on the FVIIa protease domain [Dennis, Eigenbrot, Skelton, Ultsch, Santell, Dwyer, O'Connell and Lazarus (2000) Nature (London) 404, 465-470; Dennis, Roberge, Quan and Lazarus (2001) Biochemistry 40, 9513-9521; Roberge, Santell, Dennis, Eigenbrot, Dwyer and Lazarus (2001) Biochemistry 40, 9522-9531]. Fusion peptides consisting of an N-terminal A-series peptide followed by flexible linkers, an E-series peptide, and the Z-domain of protein A were expressed in Escherichia coli and purified using IgG-Sepharose affinity chromatography. The fusion peptides were potent anticoagulants and had steep concentration dependence curves in tissue factor-dependent prothrombin time (PT) assays in comparison to the individual peptides or their noncovalent combination. This phenomenon was dependent on the length of the linker joining the A- and E-peptides. The fusion of the peptides increased the extent of inhibition of Factor X (FX) activation to 100% at saturating peptide concentrations, but did not improve the binding affinity for Factor VIIa (FVIIa) at the A- and E- binding sites or the IC(50) for the inhibition of FX activation. Differences between the peptides in the PT fold prolongation in normal and FVII-deficient plasma, in conjunction with the inhibition of (125)I-FVII activation, suggest that the enhanced effects of the fusion peptides involve the inhibition of FVII autoactivation.
authors.affiliation: Department of Protein Engineering, Genentech Inc., 1 DNA Way, South San Francisco, California 94080, U.S.A.
authors.forename: Martin
authors.initials: M
authors.lastname: Roberge
authors.forename: Mark
authors.initials: M
authors.lastname: Peek
authors.forename: Daniel
authors.initials: D
authors.lastname: Kirchhofer
authors.forename: Mark S
authors.initials: MS
authors.lastname: Dennis
authors.forename: Robert A
authors.initials: RA
authors.lastname: Lazarus
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D015942:Factor VIIa / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D005170:Factor X / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology*; D011487:Protein Conformation; D011516:Prothrombin / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000494:pharmacology; D013925:Thromboplastin / Q000378:metabolism
chemical_list: D010455:Peptides; D011993:Recombinant Fusion Proteins; D011516:Prothrombin; D005170:Factor X; D013925:Thromboplastin; D015942:Factor VIIa