title: Interspecies exchange mutagenesis of the first epidermal growth factor-like domain of human factor VII.
abstract: The first epidermal growth factor-like (EGF1) domain of human factor VII (FVII) is essential for binding to tissue factor (TF). We hypothesized that the previously observed increased coagulant activity of rabbit plasma (i.e. FVII) with human TF might be explained by the five non-conserved amino acids in the rabbit vs. the human FVII EGF1 domain. Accordingly, we 'rabbitized' the human FVII EGF1 domain either by exchanging the entire EGF1 domain creating human FVII(rabEGF1) or by the single amino acid substitutions S53N, K62E, P74A, A75D and T83K. After transient expression in HEK293 cells, the recombinant FVII (rFVII) mutant proteins were analyzed for biological activity and binding affinity to human TF by competitive enzyme-linked immunosorbent assay (ELISA). Biological activity of the unpurified rFVII mutant proteins was either depressed or statistically unchanged vs. rFVII(WT). However, three of six rFVII mutant proteins had increased affinity for human TF in the rank order rFVII(rabEGF1) (3.3-fold) > rFVII(K62E) (2.9-fold) > rFVII(A75D) (1.7-fold). The mutant protein rFVII(K62E) was then permanently expressed and purified. Fully activated, purified rFVIIa(K62E) had a twofold greater clotting activity and 2.8-fold greater direct FVIIa amidolytic activity when compared with rFVIIa(WT). Quantitation of the affinity of TF binding by surface plasmon resonance indicated that the KD of purified rFVII(K62E) for human soluble TF (sTF) was 1.5 nM compared with 7.5 nM for rFVII(WT), i.e. fivefold greater affinity. We conclude that substitution of selected amino acid residues of the FVII EGF1 domain facilitated the creation of human rFVII chimeric proteins with both enhanced biological activity and increased affinity for TF.
authors.affiliation: Department of Pathology & Molecular Medicine, McMaster University, Hamilton, Ontario, Canada.
authors.forename: V
authors.initials: V
authors.lastname: Williamson
authors.forename: A
authors.initials: A
authors.lastname: Pyke
authors.forename: S
authors.initials: S
authors.lastname: Sridhara
authors.forename: R F
authors.initials: RF
authors.lastname: Kelley
authors.forename: M A
authors.initials: MA
authors.lastname: Blajchman
authors.forename: B J
authors.initials: BJ
authors.lastname: Clarke
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001777:Blood Coagulation / Q000187:drug effects; D002460:Cell Line; D004815:Epidermal Growth Factor / Q000737:chemistry; D005167:Factor VII / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D040081:Protein Array Analysis; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D013925:Thromboplastin / Q000378:metabolism; D014162:Transfection
chemical_list: D011993:Recombinant Fusion Proteins; D004815:Epidermal Growth Factor; D005167:Factor VII; D013925:Thromboplastin