title: Enhancing the anticoagulant potency of soluble tissue factor mutants by increasing their affinity to factor VIIa.
abstract: Complexation of factor VIIa (FVIIa) and tissue factor (TF) initiates the extrinsic pathway of blood coagulation. Inappropriate triggering of this pathway has been linked to thrombotic disorders. We have previously shown that a mutant form ofsoluble tissue factor (sTF, residues 1-219) having Lys165 and Lys166 replaced with alanine residues (hTFAA) gave an antithromboticeffect in a rabbit model of arterial thrombosis. Here we have improved the potency of hTFAA by incorporating amino acid substitutions thatincrease the affinity of sTF for FVIIa. Phage display has been used toselect consensus sequences at two FVIIa-contact regions on sTF, segments 44-50 and 130-140, that individually showed modestly increased (approximately 2-fold) FVIIa-affinity. These consensus sequences have been combined with other previously selected mutations to generate a series of variants (hTFAA-2, hTFAA-3, and hTFAA-4) having 4 to 10-fold increased FVIIa-binding affinity. Surface plasmon resonance measurementsindicate that the increased affinity results mostly from an increase in the association rate constant. All of these variants displayed increased inhibitor potency relative to hTFAA in an assay of Factor X activation catalyzed by the complex of FVIIa with membrane TF. In addition, these affinity improved hTFAA variants are more potent anticoagulants and have increased antithrombotic activity relative to hTFAA in an ex vivo flow chamber model of thrombosis. The potency trend parallels increases in FVIIa-binding affinity; however, the absolute fold increases in potencies are greater than increases in binding affinity, consistent with kinetic studies of the FVIIa-binding event. Through incorporation of 10 amino acid substitutions (hTFAA-3) we have increased the antithrombotic activity of hTFAA by 20-fold.
authors.affiliation: Department of Protein Engineering, Genentech, Inc., South San Francisco, CA 94080, USA.
authors.forename: Jihong
authors.initials: J
authors.lastname: Yang
authors.forename: Geoffrey F
authors.initials: GF
authors.lastname: Lee
authors.forename: Markus A
authors.initials: MA
authors.lastname: Riederer
authors.forename: Robert F
authors.initials: RF
authors.lastname: Kelley
mesh_terms: D000925:Anticoagulants / Q000493:pharmacokinetics* / Q000494:pharmacology; D015942:Factor VIIa / Q000037:antagonists & inhibitors* / Q000378:metabolism; D005343:Fibrinolytic Agents / Q000493:pharmacokinetics / Q000494:pharmacology; D006801:Humans; D009154:Mutation; D011485:Protein Binding / Q000235:genetics; D015202:Protein Engineering; D012995:Solubility; D020349:Surface Plasmon Resonance; D013925:Thromboplastin / Q000235:genetics* / Q000493:pharmacokinetics* / Q000494:pharmacology
chemical_list: D000925:Anticoagulants; D005343:Fibrinolytic Agents; D013925:Thromboplastin; D015942:Factor VIIa