Summary Factor (F)VIII is an essential cofactor in the enzymatic coagulation cascade. A defect in the gene encoding for FVIII results in the X-linked inherited bleeding disorder hemophilia A. In the circulation, FVIII is protected from proteolytic degradation and premature clearance by forming a complex with its carrier protein von Willebrand factor (VWF). While the complex of FVIII and VWF involves one of the strongest protein-protein interactions in the human body, it has remained unclear when and where FVIII and VWF first interact. The most widely accepted view is that FVIII and VWF are secreted from different cells and form a complex in the circulation, merely by virtue of their high-affinity interaction. During the last decade however, increasing evidence has been presented that suggests that the human body should also comprise a releasable storage pool of both proteins. In this thesis, we have studied the relationship between high-affinity interaction in the circulation and intracellular co-storage of FVIII and VWF. We have taken a new approach towards this issue by creating fluorescently tagged VWF and FVIII proteins that are established as being deficient in high-affinity interaction. We have demonstrated that FVIII is stored in VWF-containing storage organelles in absence of high-affinity interaction and that the extent of FVIII storage is independent of its affinity for VWF. In addition, we studied the therapeutic benefit of intracellular co-expression of FVIII and VWF in terms of potential treatment options for hemophilia A. We have shown that blood outgrowth endothelial cells can produce high levels of functional FVIII over a prolonged period of time. In addition, we have demonstrated that a substantial amount of FVIII can be stored in VWF-containing storage organelles whereas the majority of FVIII is released in a constitutive manner. From our data, we conclude that the structural requirements that determine high-affinity interaction between FVIII and VWF in the circulation differ from those that determine intracellular co-trafficking. Our data provide a molecular explanation for the fact that hemophilia A and von Willebrand’s disease patients that suffer from impaired complex assembly of FVIII and VWF in the circulation show concomitant increase of FVIII and VWF plasma levels following treatment with desmopressin. The mechanism that determines FVIII storage in VWF-containing granules as well as the therapeutic potential of FVIII expression in VWF producing cells now awaits further exploration
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