VWF D1区突变导致的血管性血友病发病机制及治疗策略的研究

Von Willebrand disease (VWD) is mainly caused by gene mutations of von Willebrand factor (VWF). VWF propeptide (VWFpp) plays a key role in the intracellular biosynthesis, transport and secretion of VWF. As an indispensable component of VWFpp, D1 domain takes part in the function of VWFpp. However, the pathogenic nature of VWF D1 mutations in VWD patients remains largely unknown. Our previous studies have observed that four VWF mutations in the D1 domain, found in three unrelated VWD family, decreased the multimerization of VWF, and retained in the endoplasmic reticulum (ER). Therefore, we present that the mutations in D1 domain of VWF gene decrease the activity of VWFpp and VWF multimerization, fail to transport from ER to Golgi apparatus, and cause the decline of mature VWF. By using Western blot, confocal fluorescence immunoassay and VWF multimer analysis, we will further clarify the precise mechanism of the abnormalities of VWF multimerization and of intracellular transport in the VWD with D1 domain mutation. In order to restore the VWF defect in the VWD patients with D1 domain mutation, we will cultivate the blood outgrowth endothelial cells from the patients, which wil lbe further transfected with wild-type VWFpp plasmid and then be detected the level and multimerization of the mature VWF. This study will provide us new ideas and theoretical basis to improve the precision medicine of VWD patients with these mutations.

血管性血友病（VWD）主要是血管性血友病因子（VWF）基因突变所致。VWF前导肽（VWFpp）在VWF的细胞内合成、转运过程中至关重要。VWF D1区作为VWFpp的重要组成部分，其突变导致VWD的发病机制不明。本课题组前期研究在VWD患者中发现了4个位于VWF D1区的突变，这些突变降低了VWF多聚化水平，导致VWF滞留于内质网。我们提出假说，D1区突变降低了VWFpp的酶活性，影响VWF多聚化，导致VWF无法从内质网转运至高尔基体，成熟VWF分泌减少，最终导致VWD。为了验证该假说，我们将采用Western blot、荧光免疫共聚焦、VWF多聚体分析等技术方法，系统地研究D1区突变如何影响VWF多聚化及细胞内转运。此外，我们将野生型VWFpp质粒转染VWD患者来源的过度生长内皮细胞，以纠正D1区突变所致的VWF异常，探讨新的治疗策略。这将为D1区突变的VWD患者的精准治疗提供新思路。

血管性血友病（VWD）主要是血管性血友病因子（VWF）基因突变所致。VWF前导肽（VWFpp）在VWF的细胞内合成、转运过程中至关重要。VWF D1区作为VWFpp的重要组成部分，其突变导致VWD的发病机制不明。本课题组在VWD患者中发现了4个位于VWF D1区的突变。本项目中我们采用Western blot、荧光免疫共聚焦、VWF多聚体分析等技术方法，进一步证实了：D1区突变降低了VWFpp作为自身内源性氧化还原酶的活性酶活性，影响VWF多聚化，导致VWF无法从内质网转运至高尔基体，成熟VWF的基础分泌和刺激分泌均减少，最终导致VWD发生。针对上述致病机制，我们采用野生型VWF前导肽与4个突变体共转染细胞，发现野生型VWF前导肽可以部分纠正突变体VWF的多聚化障碍，减少VWF突变体在ER内的滞留，促进其细胞内转运，从而增加VWF的分泌。这说明转染前导肽可纠正VWD D1区突变所致的功能障碍，为D1区突变的VWD患者的精准治疗提供新策略。