title: Noncovalent stabilization of the factor VIII A2 domain enhances efficacy in hemophilia A mouse vascular injury models.
abstract: An important negative regulator of factor VIIIa (FVIIIa) cofactor activity is A2 subunit dissociation. FVIII molecules with stabilized activity have been generated by elimination of charged residues at the A1-A2 and A2-A3 interfaces. These molecules exhibited reduced decay rates as part of the enzymatic factor Xa generation complex and retained their activities under thermal and chemical denaturing conditions. We describe here the potency and efficacy of 1 such stability variant, D519V/E665V, derived from B domain-deleted FVIII (BDD-FVIII). The major effect of A2 stabilization was on cofactor activity. D519V/E665V potency was increased twofold by the 2-stage chromogenic assay relative to BDD-FVIII. D519V/E665V demonstrated enhanced thrombin generation responses (fivefold by peak thrombin) relative to BDD-FVIII. In vivo consequences of enhanced cofactor activity of D519V/E665V included >fourfold increased maximal platelet-fibrin deposition after laser injury and twofold increased protection from bleeding in acute and prolonged vascular injury model in hemophilia A mice. These results demonstrate that noncovalent stabilization of the FVIII A2 subunit can prolong its cofactor activity, leading to differential enhancement in clot formation over protection from blood loss in hemophilia. The FVIII molecule described here is the first molecule with clear efficacy enhancement resulting from noncovalent stabilization of the A2 domain.
authors.affiliation: Bayer HealthCare Pharmaceuticals, US Innovation Center, San Francisco, CA; and.
authors.forename: Lilley
authors.initials: L
authors.lastname: Leong
authors.affiliation: Bayer HealthCare Pharmaceuticals, US Innovation Center, San Francisco, CA; and.
authors.forename: Derek
authors.initials: D
authors.lastname: Sim
authors.affiliation: Bayer HealthCare Pharmaceuticals, US Innovation Center, San Francisco, CA; and.
authors.forename: Chandra
authors.initials: C
authors.lastname: Patel
authors.affiliation: Bayer HealthCare Pharmaceuticals, US Innovation Center, San Francisco, CA; and.
authors.forename: Katherine
authors.initials: K
authors.lastname: Tran
authors.affiliation: Bayer HealthCare Pharmaceuticals, US Innovation Center, San Francisco, CA; and.
authors.forename: Perry
authors.initials: P
authors.lastname: Liu
authors.affiliation: Bayer HealthCare Pharmaceuticals, US Innovation Center, San Francisco, CA; and.
authors.forename: Elena
authors.initials: E
authors.lastname: Ho
authors.affiliation: Bayer HealthCare Pharmaceuticals, US Innovation Center, San Francisco, CA; and.
authors.forename: Thomas
authors.initials: T
authors.lastname: Thompson
authors.affiliation: Bayer HealthCare Pharmaceuticals, US Innovation Center, San Francisco, CA; and.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Kretschmer
authors.affiliation: Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, NY.
authors.forename: Hironao
authors.initials: H
authors.lastname: Wakabayashi
authors.affiliation: Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, NY.
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Fay
authors.affiliation: Bayer HealthCare Pharmaceuticals, US Innovation Center, San Francisco, CA; and.
authors.forename: John E
authors.initials: JE
authors.lastname: Murphy
mesh_terms: D000818:Animals; D001160:Arterioles / Q000293:injuries; D004195:Disease Models, Animal; D005169:Factor VIII / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology*; D005260:Female; D006467:Hemophilia A / Q000235:genetics*; D051379:Mice; D018345:Mice, Knockout; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology
chemical_list: D011994:Recombinant Proteins; D005169:Factor VIII