title: Selection and characterization of a new class of peptide exosite inhibitors of coagulation factor VIIa.
abstract: A new series of peptide inhibitors of human Factor VIIa (FVIIa) has been identified and affinity matured from naive and partially randomized peptide phage libraries selected against the immobilized tissue factor x Factor VIIa (TF x FVIIa) complex. These "A-series" peptides contain a single disulfide bond and a 13-residue minimal core required for maximal affinity. They are exemplified by peptide A-183 (EEWEVLCWTWETCER), which binds at a newly identified exosite on the FVIIa protease domain, described in the accompanying report [Roberge, M., Santell, L., Dennis, M. S., Eigenbrot, C., Dwyer, M. A., and Lazarus, R. A. (2001) Biochemistry 40, 9522-9531]. A-183 was obtained from a trypsin digest of A-100-Z, a recombinant protein comprising A-183 and the Z domain of protein A. Surprisingly, A-183 was a very potent inhibitor of TF x FVIIa, inhibiting activation of Factor X (FX) and Factor IX and amidolytic activity of Chromozym t-PA with IC50 values of 1.6 +/- 1.2, 3.5 +/- 0.3, and 8.5 +/- 3.5 nM, respectively. Kinetic analysis revealed that A-183 was a partial (hyperbolic) mixed-type inhibitor of FX activation having a Ki of 200 pM as well as a partial competitive inhibitor of amidolytic activity. The A-series peptides were also specific and potent inhibitors of TF-dependent clotting as measured in a prothrombin time (PT) clotting assay and had no effect on the TF-independent activated partial thromboplastin time. At saturating concentrations of peptide, the maximal extent by which A-183 and A-100-Z inhibited the rate of FX activation was 78 +/- 3 and 89 +/- 6%, respectively. The degree of inhibition of the rate of FX activation correlated with a maximum fold prolongation in the PT assay of 1.8-fold for A- 183 and 3.3-fold for A-100-Z. The A-series peptides represent a new class of peptide exosite inhibitors that are capable of attenuating, rather than completely inhibiting, the activity of TF x FVIIa, potentially leading to anticoagulants with an increased therapeutic window.
authors.affiliation: Department of Protein Engineering, Genentech, Inc., South San Francisco, California 94080, USA. dennis.mark@gene.com
authors.forename: M S
authors.initials: MS
authors.lastname: Dennis
authors.forename: M
authors.initials: M
authors.lastname: Roberge
authors.forename: C
authors.initials: C
authors.lastname: Quan
authors.forename: R A
authors.initials: RA
authors.lastname: Lazarus
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D001777:Blood Coagulation / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D015942:Factor VIIa / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism / Q000494:pharmacology*; D019151:Peptide Library; D010455:Peptides / Q000378:metabolism / Q000494:pharmacology*; D011480:Protease Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D010446:Peptide Fragments; D019151:Peptide Library; D010455:Peptides; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D015942:Factor VIIa