Induction of megakaryocytes to synthesize and store a releasable pool of human factor VIII.

Autor: Wilcox DA; Department of Pediatrics, Medical College of Wisconsin, and Children's Hospital of Wisconsin, Milwaukee, Wisconsin 53226, USA. dwilcox@mail.mcw.edu, Shi Q, Nurden P, Haberichter SL, Rosenberg JB, Johnson BD, Nurden AT, White GC 2nd, Montgomery RR
Jazyk: angličtina
Zdroj: Journal of thrombosis and haemostasis : JTH [J Thromb Haemost] 2003 Dec; Vol. 1 (12), pp. 2477-89.
DOI: 10.1111/j.1538-7836.2003.00534.x
Abstrakt: 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.
Databáze: MEDLINE
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