Factor VIII and von Willebrand factor co-delivery by endothelial cells

A defect in coagulation factor VIII (FVIII) results in the inherited bleeding disorder hemophilia A. Current treatment of hemophilia A is hampered by the need of frequent administration of costly FVIII products. Therefore gene therapy is an attractive alternative for protein replacement to treat hemophilia A patients. Recent insights have suggested that it may be beneficial to express FVIII in cells that also secrete its natural carrier protein von Willebrand factor (VWF), i.e. platelets and endothelial cells. In this thesis our aim was to explore the potential of endothelial cells as a cellular release-on-demand device for co-delivery of FVIII and VWF. We demonstrate that blood outgrowth endothelial cells (BOECs) transduced with a lentiviral vector encoding FVIII are capable of long-term production of high FVIII levels. A substantial part of FVIII synthesized by BOECs is stored with VWF in storages organelles called Weibel-Palade bodies (WPBs), and can be released upon stimulation of WPB exocytosis. Using confocal microscopy we observed that the appearance of FVIII-containing WPBs was round as opposed to the typical elongated shape of normal WPBs. We found that FVIII expression prohibits the formation of long VWF tubules that normally determine the elongated shape of WPBs. In addition, our data suggest that an intracellular interaction of FVIII with VWF impairs recruitment of platelets by VWF released from WPBs. The effect of FVIII expression on VWF stored in WPBs implies that FVIII and VWF associate with each other inside WPBs. Therefore we studied whether or not WPB-derived FVIII and VWF remain associated after release from WPBs. We found that FVIII co-stored with VWF in WPBs binds to secreted VWF strings without a need of the extracellular high-affinity VWF binding site Y1680. Our data further suggest that intracellular and extracellular FVIII-VWF complex assembly differ with regard to the contribution of the Y1680 residue in VWF binding. We also determined the role of VWF for storage of FVIII in WPBs by studying the sorting ability of FVIII variants displaying reduced VWF interaction. We found that FVIII sorting to WPBs is not affected by a reduced extracellular FVIII-VWF interaction. Moreover, using domain deletion mutants and FVIII-FV chimeras, we demonstrate that FVIII sorting to WPBs is driven by FVIII-specific structural elements in both C domains. In conclusion, BOECs comprise a promising cell type as these cells synthesize and store considerable quantities of FVIII with VWF in WPBs. Furthermore, FVIII is probably released from WPBs in complex with VWF as exocytosis of WPB content under flow results in strong association of FVIII with VWF strings.