title: A soluble tissue factor mutant is a selective anticoagulant and antithrombotic agent.
abstract: One approach to developing safer and more efficacious agents for the treatment of thrombotic disease involves the design and testing of inhibitors that block specific steps in the coagulation cascade. We describe here the development of a mutant of human tissue factor (TF) as a specific antagonist of the extrinsic pathway of blood coagulation and the testing of this mutant in a rabbit model of arterial thrombosis. Alanine substitutions of Lys residues 165 and 166 in human TF have been shown previously to diminish the cofactor function of TF in support of factor X (FX) activation catalyzed by factor VIIa (FVIIa). The K165A:K166A mutations have been incorporated into soluble TF (sTF; residues 1-219) to generate the molecule "hTFAA." hTFAA binds FVIIa with kinetics and affinity equivalent to wild-type sTF, but the hTFAA x FVIIa complex shows a 34-fold reduction in catalytic efficiency for FX activation relative to the activity measured for sTF x FVIIa. hTFAA inhibits the activation of FX catalyzed by the complex formed between FVIIa and relipidated TF(1-243). hTFAA prolongs prothrombin time (PT) determined with human plasma and relipidated TF(1-243) or membrane bound TF, and has no effect on activated partial thromboplastin time, but is 70-fold less potent as an inhibitor of PT with rabbit plasma. The rabbit homologue of this mutant ("rTFAA") was produced and shown to have greater potency with rabbit plasma. Both hTFAA and rTFAA display an antithrombotic effect in a rabbit model of arterial thrombosis with rTFAA giving full efficacy at a lower dose than hTFAA. Compared to heparin doses of equal antithrombotic potential, hTFAA and rTFAA cause less bleeding as judged by measurements of the cuticle bleeding time. These results indicate that TF x FVIIa is a good target for the development of new anticoagulant drugs for the treatment of thrombotic disease.
authors.affiliation: Department of Protein Engineering, Genentech, Inc., South San Francisco, CA 94080, USA.
authors.forename: R F
authors.initials: RF
authors.lastname: Kelley
authors.forename: C J
authors.initials: CJ
authors.lastname: Refino
authors.forename: M P
authors.initials: MP
authors.lastname: O'Connell
authors.forename: N
authors.initials: N
authors.lastname: Modi
authors.forename: P
authors.initials: P
authors.lastname: Sehl
authors.forename: D
authors.initials: D
authors.lastname: Lowe
authors.forename: C
authors.initials: C
authors.lastname: Pater
authors.forename: S
authors.initials: S
authors.lastname: Bunting
mesh_terms: D000818:Animals; D000925:Anticoagulants / Q000494:pharmacology*; D001921:Brain / Q000378:metabolism; D002339:Carotid Arteries / Q000187:drug effects / Q000473:pathology; D002460:Cell Line; D003001:Cloning, Molecular; D003029:Coagulants / Q000494:pharmacology; D017931:DNA Primers; D004926:Escherichia coli; D015942:Factor VIIa / Q000378:metabolism; D005343:Fibrinolytic Agents / Q000494:pharmacology*; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000473:pathology; D016297:Mutagenesis, Site-Directed; D010314:Partial Thromboplastin Time; D016133:Polymerase Chain Reaction; D011517:Prothrombin Time; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology; D012995:Solubility; D013925:Thromboplastin / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology*
chemical_list: D000925:Anticoagulants; D003029:Coagulants; D017931:DNA Primers; D005343:Fibrinolytic Agents; D011994:Recombinant Proteins; D006493:Heparin; D013925:Thromboplastin; D015942:Factor VIIa