Hordijk Sophie, Groten Stijn A, Bürgisser Petra E, Laan Sebastiaan N J, Korenke Georg-Christoph, Beysen Diane, Leebeek Frank W G, Skehel Paul A, van den Biggelaar Maartje, Carter Tom, Bierings Ruben
Erasmus University Medical Center, Rotterdam, Netherlands.
Sanquin Research, Amsterdam, Netherlands.
Blood. 2025 Jul 15. doi: 10.1182/blood.2024027935.
The regulated secretion of von Willebrand factor (VWF) from Weibel-Palade bodies (WPB) in endothelial cells is fundamental to hemostasis. This process relies on recruiting Rab GTPases and their effectors to the WPB membrane, with the guanine nucleotide exchange factor (GEF) MAP-kinase activating death domain (MADD) playing a central role. Biallelic variants in MADD lead to a pleiotropic neurological and developmental disorder that can include bleeding abnormalities. This study investigates the impact of pathogenic MADD variants on VWF secretion using patient-derived endothelial cells. We isolated endothelial colony forming cells (ECFCs) from three pediatric patients with biallelic MADD variants and unaffected heterozygous family members. All patients exhibited low VWF plasma levels (22-30 IU/dL). Proteomic analysis of patient-derived ECFCs revealed an absence of MADD peptides, reduced VWF, and downregulation of proteins involved in the exocytotic machinery, including Rab3D and the Rab3/27 effector Slp4-a. Functional assays demonstrated diminished Rab27A and Rab3D activity and their failure to localize to WPBs in patient cells. Biochemical and live-imaging studies showed that histamine-induced VWF and VWFpp secretion were significantly reduced in patient cells due to delayed and reduced degranulation of WPBs. Our findings demonstrate the critical role of MADD in maintaining the secretion competence of WPBs and the magnitude of VWF secretion by regulating the recruitment of the endothelial exocytotic machinery. This study highlights the in vivo significance of WPB exocytosis in maintaining plasma VWF levels and establishes MADD as the first causal gene for quantitative von Willebrand Disease (VWD) in patients without pathogenic VWF variants.
内皮细胞中血管性血友病因子（VWF）从魏-帕小体（WPB）的调节性分泌是止血的基础。这一过程依赖于将Rab GTP酶及其效应器招募到WPB膜上，鸟嘌呤核苷酸交换因子（GEF）丝裂原活化蛋白激酶激活死亡结构域（MADD）发挥核心作用。MADD的双等位基因变异导致一种多效性神经和发育障碍，可能包括出血异常。本研究使用患者来源的内皮细胞研究致病性MADD变异对VWF分泌的影响。我们从三名患有双等位基因MADD变异的儿科患者及其未受影响的杂合子家庭成员中分离出内皮集落形成细胞（ECFC）。所有患者的VWF血浆水平均较低（22 - 30 IU/dL）。对患者来源的ECFC进行蛋白质组学分析发现，MADD肽缺失、VWF减少，以及参与胞吐机制的蛋白质下调，包括Rab3D和Rab3/27效应器Slp4-a。功能试验表明，患者细胞中Rab27A和Rab3D活性降低，且无法定位于WPB。生化和实时成像研究表明，由于WPB脱颗粒延迟和减少，组胺诱导的患者细胞中VWF和VWF前体蛋白分泌显著减少。我们的研究结果表明，MADD在维持WPB的分泌能力以及通过调节内皮胞吐机制的募集来控制VWF分泌量方面起着关键作用。本研究强调了WPB胞吐作用在维持血浆VWF水平方面的体内重要性，并将MADD确立为无致病性VWF变异患者定量血管性血友病（VWD）的首个致病基因。
