期刊
JOURNAL OF THROMBOSIS AND HAEMOSTASIS
卷 1, 期 12, 页码 2477-2489出版社
BLACKWELL PUBL LTD
DOI: 10.1111/j.1538-7836.2003.00534.x
关键词
FVIII transgene expression; hemophilia A; platelet alpha-granules
资金
- NATIONAL HEART, LUNG, AND BLOOD INSTITUTE [P01HL044612, R01HL033721, R01HL068138] Funding Source: NIH RePORTER
- NHLBI NIH HHS [HL-33721, HL-44612, HL-68138] Funding Source: Medline
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(R) 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.
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