title: Comparison of platelet-derived and plasma factor VIII efficacy using a novel native whole blood thrombin generation assay.
abstract: BACKGROUND
  We have recently developed a successful gene therapy approach for hemophilia A in which factor VIII (FVIII) expression is targeted to platelets by the αIIb promoter. Levels of platelet-expressed FVIII (2bF8) achieved by gene therapy may vary between individuals due to differences in ex vivo transduction and gene expression efficiency. Accurate assays to evaluate 2bF8 efficacy are desirable.
  
  
  OBJECTIVE
  To compare the hemostatic efficacy of 2bF8 with replacement therapy over a wide therapeutic dose range.
  
  
  METHODS
  Efficacy of 2bF8 was assessed using a new transgenic mouse model expressing high 2bF8 levels (LV18(tg) ). Blood from LV18(tg) mice or FVIII(null) mice infused with recombinant FVIII was mixed with FVIII(null) blood at different ratios ex vivo to achieve several concentrations of 2bF8 or plasma FVIII. Samples were evaluated with a novel native whole blood thrombin generation assay that uses recalcified whole blood without the addition of tissue factor to initiate coagulation.
  
  
  RESULTS
  FVIII dose dependency was observed in all five thrombin generation parameters. While the total amount of thrombin generated was similar, 2bF8 significantly accelerated thrombin generation compared with plasma FVIII. Remarkably, a 10-fold lower dose of 2bF8 than plasma FVIII (0.2% vs. 2%) significantly shortened the onset and peak of thrombin generation compared with FVIII(null) blood.
  
  
  CONCLUSIONS
  Using a new transgenic mouse model, we showed that the novel native whole blood thrombin generation assay established here can be used to monitor platelet targeted FVIII gene therapy. The higher therapeutic efficacy of 2bF8 compared with factor replacement therapy seemed to be due to acceleration of thrombin generation.
authors.affiliation: Blood Research Institute, BloodCenter of Wisconsin, Milwaukee, WI, USA.
authors.forename: C K
authors.initials: CK
authors.lastname: Baumgartner
authors.affiliation: Blood Research Institute, BloodCenter of Wisconsin, Milwaukee, WI, USA.
authors.forename: G
authors.initials: G
authors.lastname: Zhang
authors.affiliation: Blood Research Institute, BloodCenter of Wisconsin, Milwaukee, WI, USA.|Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI, USA.
authors.forename: E L
authors.initials: EL
authors.lastname: Kuether
authors.affiliation: Blood Research Institute, BloodCenter of Wisconsin, Milwaukee, WI, USA.
authors.forename: H
authors.initials: H
authors.lastname: Weiler
authors.affiliation: Blood Research Institute, BloodCenter of Wisconsin, Milwaukee, WI, USA.|Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI, USA.|Children's Research Institute, Children's Hospital of Wisconsin, Milwaukee, WI, USA.
authors.forename: Q
authors.initials: Q
authors.lastname: Shi
authors.affiliation: Blood Research Institute, BloodCenter of Wisconsin, Milwaukee, WI, USA.|Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI, USA.|Children's Research Institute, Children's Hospital of Wisconsin, Milwaukee, WI, USA.
authors.forename: R R
authors.initials: RR
authors.lastname: Montgomery
mesh_terms: D000818:Animals; D001777:Blood Coagulation / Q000187:drug effects* / Q000235:genetics; D001780:Blood Coagulation Tests; D003029:Coagulants / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D005169:Factor VIII / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology*; D005838:Genotype; D006801:Humans; D007700:Kinetics; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008822:Mice, Transgenic; D010641:Phenotype; D040201:Platelet Membrane Glycoprotein IIb / Q000235:genetics; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000494:pharmacology; D013917:Thrombin / Q000378:metabolism
chemical_list: D003029:Coagulants; D040201:Platelet Membrane Glycoprotein IIb; D011994:Recombinant Proteins; C078147:F8 protein, human; D005169:Factor VIII; D013917:Thrombin
keywords: factor VIII; gene therapy; hemophilia A; platelets; thrombin