Plantier J-L, Enjolras N, Rodriguez M-H E, Massé J-M, Cramer E M, Négrier C
INSERM U331, Laboratoire d'Hémobiologie-Faculté de Médecine RTH, Laennec, Lyon, France.
J Thromb Haemost. 2003 Feb;1(2):292-9. doi: 10.1046/j.1538-7836.2003.00071.x.
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.
血友病B被认为是基因治疗的一个良好候选对象。已经尝试了几种策略，在血友病动物模型中取得了有希望的结果，但在人类中未能达到纠正水平。为了克服这一不便，我们旨在在参与止血反应的细胞（如内皮细胞和血小板）中产生细胞内的因子IX（F IX）池。受到刺激时，这些细胞会释放其颗粒内容物，在这种情况下会导致局部FIX浓度增加，并可在局部产生有效的止血作用。为了产生可释放的凝血FIX细胞内池，将P-选择素（pselCT）分子的细胞质结构域与人FIX蛋白的羧基末端融合。在具有储存颗粒的AtT20细胞系中研究了这种嵌合分子（FIX-pselCT）的特性。如先前对跨膜分子而非可溶性蛋白如FIX的研究所示，pselCT片段可诱导FIX-pselCT的储存。添加pselCT尾部不影响FIX-pselCT的凝血活性。用不同抑制剂处理AtT20细胞表明，FIX-pselCT不会被细胞内降解，且嵌合分子的半衰期至少比野生型FIX长两倍。免疫电子显微镜分析表明FIX-pselCT特异性定位于含促肾上腺皮质激素的颗粒内。使用佛波酯肉豆蔻酸酯乙酸盐（PMA）、离子载体A-23187或8-溴环磷酸腺苷（8-Br-cAMP）刺激细胞可有效释放活性FIX-pselCT。这些数据表明，将P-选择素的细胞质结构域添加到FIX中，可通过将重组蛋白导向调节性分泌颗粒来改变FIX分子的细胞命运，而不会改变其凝血活性。
