title: Induction of megakaryocytes to synthesize and store a releasable pool of human factor VIII.
abstract: von Willebrand factor (VWF) is a complex plasma glycoprotein that modulates platelet adhesion at the site of a vascular injury, and it also serves as a carrier protein for factor (F)VIII. As megakaryocytes are the only hematopoietic lineage to naturally synthesize and store VWF within alpha-granules, this study was performed to determine if expression of a FVIII transgene in megakaryocytes could lead to trafficking and storage of FVIII with VWF in platelet alpha-granules. Isolex selected CD34+ cells from human G-CSF mobilized peripheral blood cells (PBC) and murine bone marrow were transduced with a retrovirus encoding the B-domain deleted form of human FVIII (BDD-FVIII). Cells were then induced with cytokines to form a population of multiple lineages including megakaryocytes. Chromogenic analysis of culture supernatant from FVIII-transduced human cells demonstrated synthesis of functional FVIII. Treatment of cells with agonists of platelet activation (ADP, epinephrine, and thrombin receptor-activating peptide) resulted in the release of VWF antigen and active FVIII into the supernatant from transduced cells. Immunofluorescence analysis of cultured human and murine megakaryocytes revealed a punctate pattern of staining for FVIII that was consistent with staining for VWF. Electron microscopy of transduced megakaryocytes using immunogold-conjugated antibodies colocalized FVIII and VWF within the alpha-granules. FVIII retained its association with VWF in human platelets isolated from the peripheral blood of NOD/SCID mice at 2-6 weeks post-transplant of transduced human PBC. These results suggest feasibility for the development of a locally inducible secretory pool of FVIII in platelets of patients with hemophilia A.
authors.affiliation: Department of Pediatrics, Medical College of Wisconsin, and Children's Hospital of Wisconsin, Milwaukee, Wisconsin 53226, USA. dwilcox@mail.mcw.edu
authors.forename: D A
authors.initials: DA
authors.lastname: Wilcox
authors.forename: Q
authors.initials: Q
authors.lastname: Shi
authors.forename: P
authors.initials: P
authors.lastname: Nurden
authors.forename: S L
authors.initials: SL
authors.lastname: Haberichter
authors.forename: J B
authors.initials: JB
authors.lastname: Rosenberg
authors.forename: B D
authors.initials: BD
authors.lastname: Johnson
authors.forename: A T
authors.initials: AT
authors.lastname: Nurden
authors.forename: G C
authors.initials: GC
authors.lastname: White
authors.forename: R R
authors.initials: RR
authors.lastname: Montgomery
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D019070:Cell Lineage / Q000187:drug effects; D003594:Cytoplasmic Granules / Q000737:chemistry; D005169:Factor VIII / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism*; D015316:Genetic Therapy / Q000379:methods; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism; D006467:Hemophilia A / Q000188:drug therapy; D006801:Humans; D008533:Megakaryocytes / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D016513:Mice, SCID; D021381:Protein Transport / Q000187:drug effects; D014161:Transduction, Genetic; D014841:von Willebrand Factor / Q000378:metabolism
chemical_list: D014841:von Willebrand Factor; D005169:Factor VIII