title: Apoptotic effects of platelet factor VIII on megakaryopoiesis: implications for a modified human FVIII for platelet-based gene therapy.
abstract: BACKGROUND
  Ectopically expressed B-domainless factor VIII in megakaryocytes is stored in α-granules, is effective in a number of murine hemostatic models, and is protected from circulating inhibitors. However, this platelet (p) FVIII has different temporal-spatial availability from plasma FVIII, with limited efficacy in other murine hemostatic models.
  
  
  OBJECTIVE
  We sought to improve pFVIII hemostatic efficacy by expressing canine (c) FVIII, which has higher stability and activity than human (h) FVIII in FVIII(null) mice.
  
  
  CONCLUSIONS
  We found that pcFVIII was more effective than phFVIII at restoring hemostasis, but peak pcFVIII antigen levels were lower and were associated with greater megakaryocyte apoptosis than phFVIII. These new insights suggest that pFVIII gene therapy strategies should focus on enhancing activity rather than levels. We previously showed that modification of the PACE/furin cleavage site in hFVIII resulted in secretion of hFVIII primarily as a single-chain molecule with increased biological activity. In megakaryocytes, this variant was expressed at the same level as phFVIII with a lentiviral bone marrow transplant approach to reconstitute FVIII(null) mice, but was more effective, resulting in near-normal hemostasis in the cremaster laser injury model. These studies may have implications for pFVIII gene therapy in hemophilia A.
authors.affiliation: Division of Hematology, The Children's Hospital of Philadelphia, Philadelphia, PA, USA.
authors.forename: T K
authors.initials: TK
authors.lastname: Greene
authors.forename: R B
authors.initials: RB
authors.lastname: Lyde
authors.forename: S C
authors.initials: SC
authors.lastname: Bailey
authors.forename: M P
authors.initials: MP
authors.lastname: Lambert
authors.forename: L
authors.initials: L
authors.lastname: Zhai
authors.forename: D E
authors.initials: DE
authors.lastname: Sabatino
authors.forename: R M
authors.initials: RM
authors.lastname: Camire
authors.forename: V R
authors.initials: VR
authors.lastname: Arruda
authors.forename: M
authors.initials: M
authors.lastname: Poncz
mesh_terms: D000818:Animals; D017209:Apoptosis; D001792:Blood Platelets / Q000166:cytology*; D002339:Carotid Arteries / Q000473:pathology; D002460:Cell Line; D006224:Cricetinae; D004285:Dogs; D005169:Factor VIII / Q000737:chemistry* / Q000235:genetics*; D015316:Genetic Therapy / Q000379:methods*; D006467:Hemophilia A / Q000235:genetics; D006487:Hemostasis; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D008533:Megakaryocytes / Q000166:cytology*; D051379:Mice; D008822:Mice, Transgenic
chemical_list: D005169:Factor VIII
keywords: apoptosis; blood platelets; coagulation factor VIII; gene therapy; megakaryocytes