The P-selectin cytoplasmic domain directs the cellular storage of a recombinant chimeric factor IX
| Autor: | M. H. Rodriguez, Claude Negrier, Massé Jm, Cramer Em, Nathalie Enjolras, Jean-Luc Plantier |
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| Rok vydání: | 2003 |
| Předmět: |
DNA
Complementary P-selectin Recombinant Fusion Proteins Genetic Vectors Biology In Vitro Techniques Transfection Hemophilia B law.invention Cell Line Factor IX chemistry.chemical_compound Mice Adrenocorticotropic Hormone law Animals Humans Base Sequence Hematology Transmembrane protein Peptide Fragments Cell biology Protein Structure Tertiary P-Selectin Biochemistry chemistry Cell culture Cytoplasm Phorbol Recombinant DNA Intracellular |
| Zdroj: | Journal of thrombosis and haemostasis : JTH. 1(2) |
| ISSN: | 1538-7933 |
| Popis: | Hemophilia B was recognized as a good candidate for gene therapy. Several strategies have been attempted and gave promising results in hemophilic animals but failed to achieve corrective levels in humans. To overcome this inconvenience we aimed to generate intracellular pools of factor (F)IX in cells that are implicated in the hemostatic response, e.g. endothelial cells and platelets. Upon stimulation, these cells release their granule content, which in this case would result in an increase in local FIX concentration, and could locally produce an effective hemostasis. In an attempt to produce an intracellular pool of releasable coagulation FIX, the cytoplasmic domain of the P-selectin (pselCT) molecule was fused to the carboxy-terminal extremity of the human FIX protein. The properties of this chimeric molecule (FIX-pselCT) were studied in AtT20, a cell line which possesses storage granules. As previously shown for transmembrane molecules but not for a soluble protein such as FIX, the pselCT fragment induces the storage of FIX-pselCT. The coagulant activity of FIX-pselCT was not affected by the addition of the pselCT tail. The treatment of AtT20 cells with different inhibitors revealed that FIX-pselCT was not submitted to intracellular degradation and that the half-life of the chimeric molecule was at least two times longer than that of FIX-WT. An immunoelectron microscopic analysis demonstrated a specific localization of FIX-pselCT within the ACTH-containing granules. Cell stimulation using Phorbol Myristrate Acetate (PMA), ionophore A-23187 or 8-Br-cAMP induced efficient release of an active FIX-pselCT. These data demonstrate that the addition of the cytoplasmic domain of P-selectin to FIX modifies the cellular fate of the FIX molecule by directing the recombinant protein toward regulated-secretory granules without altering its coagulant activity. |
| Databáze: | OpenAIRE |
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