title: Potent bifunctional anticoagulants: Kunitz domain-tissue factor fusion proteins.
abstract: A strategy to design potent antagonists of human coagulation factor VIIa (FVIIa) by linking two proteins that independently inhibit activity and bind at separate, nonoverlapping sites is presented. A bifunctional inhibitor (KDTF5), comprising a Kunitz-type domain engineered to inhibit the FVIIa active site and a soluble tissue factor (TF) variant that is defective as a cofactor for factor X (FX) activation, was developed from structure-based modeling of a ternary FVIIa-Kunitz domain-TF complex. KDTF5 inhibited FVIIa-dependent FX activation with a Ki* of 235 +/- 45 pM, a 193-fold and 398-fold increase in potency compared to the TF variant and Kunitz domain individually. Similarly, KDTF5 was a more potent anticoagulant in vitro compared to either inhibitory domain alone. The results demonstrate the harnessing of a macromolecular chelate effect by fusing two inhibitory ligands that bind a target at spatially distinct sites.
authors.affiliation: Department of Protein Engineering, Genentech, Inc., South San Francisco, California 94080, USA.
authors.forename: G F
authors.initials: GF
authors.lastname: Lee
authors.forename: R A
authors.initials: RA
authors.lastname: Lazarus
authors.forename: R F
authors.initials: RF
authors.lastname: Kelley
mesh_terms: D000925:Anticoagulants / Q000737:chemistry* / Q000494:pharmacology*; D015195:Drug Design; D015942:Factor VIIa / Q000037:antagonists & inhibitors; D006801:Humans; D006868:Hydrolysis; D046911:Macromolecular Substances; D008958:Models, Molecular; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000502:physiology*; D013925:Thromboplastin / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D014360:Trypsin Inhibitor, Kunitz Soybean / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology*
chemical_list: D000925:Anticoagulants; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D013925:Thromboplastin; D014360:Trypsin Inhibitor, Kunitz Soybean; D015942:Factor VIIa