title: Improved hemostasis in hemophilia mice by means of an engineered factor Va mutant.
abstract: BACKGROUND
  Factor (F)VIIa-based bypassing not always provides sufficient hemostasis in hemophilia.
  
  
  OBJECTIVE
  To investigate the potential of engineered activated factor V (FVa) variants as bypassing agents in hemophilia A.
  
  
  METHODS
  Activity of FVa variants was studied in vitro using prothrombinase assays with purified components and in FV- and FVIII-deficient plasma using clotting and thrombin generation assays. In vivo bleed reduction after the tail clip was studied in hemophilia A mice.
  
  
  CONCLUSIONS
  FVa mutations included a disulfide bond connecting the A2 and A3 domains and ones that rendered FVa resistant to inactivation by activated protein C (APC). '(super) FVa,' a combination of the A2-A3 disulfide (A2-SS-A3) to stabilize FVa and of APC-cleavage site mutations (Arg506/306/679Gln), had enhanced specific activity and complete APC resistance compared with wild-type FVa, FVL eiden (Arg506Gln), or FVaL eiden (A2-SS-A3). Furthermore, (super) FVa potently increased thrombin generation in vitro in FVIII-deficient plasma. In vivo, (super) FVa reduced bleeding in FVIII-deficient mice more effectively than wild-type FVa. Low-dose (super) FVa, but not wild-type FVa, decreased early blood loss during the first 10 min by more than two-fold compared with saline and provided bleed protection for the majority of mice, similar to treatments with FVIII. During the second 10 min after tail cut, (super) FVa at high dose, but not wild-type FVa, effectively reduced bleeding. These findings suggest that (super) FVa enhances not only clot formation but also clot stabilization. Thus, (super) FVa efficiently improved hemostasis in hemophilia in vitro and in vivo and may have potential therapeutic benefits as a novel bypassing agent in hemophilia.
authors.affiliation: Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA, USA; Division of Hematology/Oncology, Department of Medicine, University California San Diego, San Diego, CA, USA.
authors.forename: A
authors.initials: A
authors.lastname: von Drygalski
authors.forename: T J
authors.initials: TJ
authors.lastname: Cramer
authors.forename: V
authors.initials: V
authors.lastname: Bhat
authors.forename: J H
authors.initials: JH
authors.lastname: Griffin
authors.forename: A J
authors.initials: AJ
authors.lastname: Gale
authors.forename: L O
authors.initials: LO
authors.lastname: Mosnier
mesh_terms: D000818:Animals; D001777:Blood Coagulation; D004220:Disulfides / Q000737:chemistry; D015943:Factor Va / Q000235:genetics* / Q000378:metabolism; D006467:Hemophilia A / Q000235:genetics; D006487:Hemostasis / Q000235:genetics*; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D009154:Mutation; D010314:Partial Thromboplastin Time; D011486:Protein C / Q000737:chemistry; D015202:Protein Engineering / Q000379:methods*; D011517:Prothrombin Time; D011994:Recombinant Proteins / Q000737:chemistry; D013917:Thrombin / Q000737:chemistry; D013925:Thromboplastin / Q000737:chemistry / Q000235:genetics
chemical_list: D004220:Disulfides; D011486:Protein C; D011994:Recombinant Proteins; D015943:Factor Va; D013925:Thromboplastin; D013917:Thrombin
keywords: animal experimentation; bleeding; factor V; hemophilia; hemostasis