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

摘要: von Willebrand factor (VWF) is a complex plasma glycoprotein that modulates platelet adhesion at the site of vascular injury, and it also serves as carrier protein for (F)VIII. As megakaryocytes are only hematopoietic lineage to naturally synthesize store VWF within alpha-granules, this study was performed determine if expression FVIII transgene in could lead trafficking storage with alpha-granules. Isolex selected CD34+ cells from human G-CSF mobilized peripheral blood (PBC) murine bone marrow were transduced retrovirus encoding B-domain deleted form (BDD-FVIII). Cells then induced cytokines population multiple lineages including megakaryocytes. Chromogenic analysis culture supernatant FVIII-transduced demonstrated synthesis functional FVIII. Treatment agonists activation (ADP, epinephrine, thrombin receptor-activating peptide) resulted release antigen active into cells. Immunofluorescence cultured revealed punctate pattern staining consistent VWF. Electron microscopy using immunogold-conjugated antibodies colocalized retained its association platelets isolated NOD/SCID mice 2-6 weeks post-transplant PBC. These results suggest feasibility development locally inducible secretory pool patients hemophilia A.