Von Willebrand disease (VWD) is the most common inherited bleeding disorder. Patients mainly develop mucocutaneous bleeding, like bruises, epistaxis and menorrhagia. The more severely affected... Show moreVon Willebrand disease (VWD) is the most common inherited bleeding disorder. Patients mainly develop mucocutaneous bleeding, like bruises, epistaxis and menorrhagia. The more severely affected patients may also develop joint bleeding, or bleeding from the gastrointestinal tract. Also, trauma, surgery or dental procedures may lead to critical bleeding events. VWD-related bleeding are caused by defects in von Willebrand factor (VWF), a large multimeric protein that is produced by endothelial cells and megakaryocytes. Most VWD patients develop the disease because of dominant-negative mutations in VWF. In this thesis we investigated whether inhibition of production of mutant VWF with limited inhibition of wildtype VWF positively affects the function of VWF and improves VWD phenotypes. We used small interfering RNAs (siRNAs) to selectively inhibit production of mutant VWF. These siRNAs were tested in several models for VWD. We indeed prove that siRNAs can distinguish a mutant and wildtype VWF allele in vitro in heterologous cell systems, ex vivo in patient-derived endothelial cells, and in vivo in a VWD mouse model. We also show in these disease models that we can improve several VWD phenotypes. These results are promising for further development of allele-specific siRNAs as a new treatment strategy for VWD. Show less
Von Willebrand disease is the most common inherited bleeding disorder and is characterized by reduced plasma von Willebrand factor (VWF) levels or functionally abnormal VWF. VWF is best known for... Show moreVon Willebrand disease is the most common inherited bleeding disorder and is characterized by reduced plasma von Willebrand factor (VWF) levels or functionally abnormal VWF. VWF is best known for its three classical hemostatic functions: (i) as a carrier protein for coagulation factor VIII, (ii) binding to exposed collagen upon vascular damage and (iii) as a mediator of the recruitment of platelets to sites of vascular injury. However, in recent years it has become clear that VWF has a versatile function beyond hemostasis, and has been implicated in several pathophysiological processes, such as tumor metastasis, angiogenesis, cell proliferation and inflammatory processes. So, apart from its evident role in hemostasis, it has become clear that VWF is a much more complex multifaceted protein then initially thought. Several aspects __ from synthesis, secretion and clearance, to functions in the circulation __ of this protein have been studied and the results are described in this thesis. These results will advance our knowledge about the mechanism(s) of VWF biosynthesis, clearance and its role in angiogenesis, which might lead to a more personalized treatment for von Willebrand disease and its complications in the future. Show less
The studies presented in this thesis explored several pathogenic mechanisms underlying von Willebrand disease that is characterized by a quantitative or functional deficiency of von Willebrand... Show moreThe studies presented in this thesis explored several pathogenic mechanisms underlying von Willebrand disease that is characterized by a quantitative or functional deficiency of von Willebrand factor, in particular with respect to intracellular storage in Weibel-Palade body and regulated secretion of von Willebrand factor. By using molecular biology, confocal and electron microscopic techniques, storage and secretion of von Willebrand factor were analyzed for von Willebrand disease variants identified in the patients. These studies advanced our understanding of von Willebrand disease at the molecular and cellular levels. HEK293 cells and endothelial cells derived from patients__ peripheral blood were established as two useful model-systems for examining von Willebrand factor structure-function relationships in the context of von Willebrand disease. Using these model-systems we have demonstrated that von Willebrand factor mutations may impair its storage and secretion and thus lead to a quantitative deficiency of this factor in the patients. Furthermore, we demonstrated that alteration in the structure of von Willebrand factor, by natural mutations that occur in von Willebrand disease patients, modulates von Willebrand factor string formation and function. We propose that alteration in von Willebrand factor string formation and function may be another new mechanism that contributes to the bleeding tendency in von Willebrand disease. Show less
Von Willebrand factor (VWF) plays an important role in both primary and secondary hemostasis as a molecular glue between platelets and subendothelial structures, and as a carrier of FVIII.... Show moreVon Willebrand factor (VWF) plays an important role in both primary and secondary hemostasis as a molecular glue between platelets and subendothelial structures, and as a carrier of FVIII. Mutations in VWF may cause von Willebrand disease (VWD), which is the most common bleeding disorder. It is characterized by symptoms ranging from very mild to severe bleeding. Mutations may influence the level of VWF (quantitative defect; type 1 or type 3) or may affect the function of VWF. Especially important for the generation of functional VWF is the formation of disulfide linked bonds. Firstly, intrachain linking is essential for the monomer structure. Secondly, interchain linking is necessary for both dimerization and multimerization of VWF. All 169 cysteine residues in the mature VWF subunit (8.2%) participate in these intra- or interchain disulfide bonds. The interaction between proVWF dimers ultimately yields high-molecular weight VWF that is active in primary hemostasis in the bloodstream.The main aims of the studies reported in this thesis were to examine how loss of cysteines located in different domains of VWF results in quantitative and qualitative VWF defects; how these mutations interfere with dimerization and multimerization; and how they influence intracellular routing, secretion and clearance of VWF. Show less
