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Induction of megakaryocytes to synthesize and store a releasable pool of human factor VIII. J Thromb Haemost 2003 Dec;1(12):2477-89

Date

12/17/2003

Pubmed ID

14675082

DOI

10.1111/j.1538-7836.2003.00534.x

Scopus ID

2-s2.0-1542544992   45 Citations

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.

Author List

Wilcox DA, Shi Q, Nurden P, Haberichter SL, Rosenberg JB, Johnson BD, Nurden AT, White GC 2nd, Montgomery RR

Authors

Bryon D. Johnson PhD Professor in the Medicine department at Medical College of Wisconsin
Robert R. Montgomery MD Clinical Professor in the Pediatrics department at Medical College of Wisconsin
Qizhen Shi MD, PhD Professor in the Pediatrics department at Medical College of Wisconsin
Gilbert C. White MD Professor in the Medicine department at Medical College of Wisconsin
David A. Wilcox PhD Associate Professor in the Pediatrics department at Medical College of Wisconsin




MESH terms used to index this publication - Major topics in bold

Animals
Cell Culture Techniques
Cell Lineage
Cytoplasmic Granules
Factor VIII
Genetic Therapy
Hematopoietic Stem Cell Transplantation
Hematopoietic Stem Cells
Hemophilia A
Humans
Megakaryocytes
Mice
Mice, SCID
Protein Transport
Transduction, Genetic
von Willebrand Factor
jenkins-FCD Prod-400 0f9a74600e4e79798f8fa6f545ea115f3dd948b2