前肽TIL-1结构域介导血管性血友病因子多聚体组装的机制及血管性血友病基因治疗新策略的研究

血管性血友病因子（VWF）多聚体形成是其发挥正常凝血功能的结构基础，其在高尔基体中的多聚体组装机制不明。我们前期研究发现VWF前肽TIL-1结构域内293-333位氨基酸缺失突变体虽能以二聚体形式正常合成和分泌，却无法组装成多聚体。高尔基体内缺乏分子间二硫键形成所需二硫键异构酶及伴侣蛋白，提示TIL-1结构域可能具备二硫键异构酶活性或促进二聚体间彼此靠近与相互作用而介导VWF多聚体组装。本研究拟通过生物化学和生物物理手段分析 TIL-1结构域二硫键异构酶活性及其与自身和D’D3区域的相互作用，并解析TIL-1结构域及二聚体三维结构。由于前期研究发现外源性前肽区分子可与突变前肽竞争结合成熟VWF分子，促进后者多聚体形成，本研究将在此基础上尝试蛋白水平恢复前肽区突变导致的VWF多聚体形成的能力并挽救其生物活性为目标的新颖VWD治疗策略，为多聚体组装障碍型VWD患者的基因治疗研发开辟新的策略。

Only multimeric von Willebrand Factor (VWF) is fully functional in blood coagulation reactions, however, it still unclear how multimerization of VWF takes place in Golgi apparatus. In the pilot study of patients with von Willebrand Disease (VWD), we found that the deletion of residues 293-333 in TIL-1 domain of the VWF propeptide led to significant loss of the high-molecular-weight VWF multimers with normal synthesis, secretion and dimerization. In the absence of oxidoreductases and chaperones for disulfide bonds formation, it indicates that TIL-1 domain of the VWF propeptide might facilitate aggregation and interaction among VWF dimers and mediate multimerization of VWF through the possible isomerase activity it retains in Golgi apparatus. We thus propose to resolve the structure of TIL-1 domain and investigate its structural and functional properties involved in multimerization of VWF by biochemical and biophysical analysis. In the preliminary study, we found that external wild type propeptide could compete with its mutant counterpart to bind with mature VWF and restored multimerization of VWF. Taking advantage of these exceptional experimental findings, the proposed study will also explore the novel gene therapy strategy of VWD by supplementing only wild type propeptide, instead of full-length VWF gene, to rescue the multimerization and procoagulation function of VWF. The proposed study is anticipated to elucidate the molecular mechanism of TIL-1 domain mediated VWF multimerization facilitate the development and clinical application of gene therapy regimens for von Willebrand Disease.