The Effect of von Willebrand Factor Normandy Mutations on Intracellular Co-Trafficking of Factor VIII to Storage Organelles.
Authors: van den Biggelaar Maartje , Ruhdorfer Birgit , Voorberg Jan , Mertens Koen 
Published: October 2019

Abstract
Von Willebrand Factor (VWF) serves a critical role in factor (F)VIII life-cycle. It is generally believed that FVIII and VWF assemble in the circulation after secretion from vascular endothelial cells (VWF) and hepatocytes (FVIII). However, during the last few years evidence has accumulated in favor of significant extra-hepatic FVIII sources that might be compatible with in vivo biosynthesis and co-storage of FVIII and VWF within the same cell. In agreement with this view, in vitro co-expression of FVIII and VWF results in the storage of FVIII in VWF-containing organelles. We have recently demonstrated that this intracellular co-trafficking is independent on Tyr1680 of the FVIII molecule which is required for extracellular high-affinity interaction to VWF (van den Biggelaar et al. JTH, in press). In the present study, we studied the requirements for intracellular co-trafficking of VWF and FVIII in more detail by addressing VWF variants which have been established as being deficient in extracellular FVIII binding (Normandy variants). To facilitate intracellular visualization, we made use of fluorescently tagged FVIII (FVIII-YFP) and VWF (VWF-CFP). Four different VWF-CFP Normandy variants (Thr791Met, Arg816Trp, Arg854Gln, Cys1060Arg) were created as well as a VWF-CFP variant containing an Arg763Gly replacement which prevents cleavage of the VWF propeptide from mature VWF. (Dr. C. Mazurier (Lille, France) is acknowledged for kindly providing DNA constructs containing the Thr791Met and Arg816Trp replacement). Constructs were expressed in HEK293 cells and purified from conditioned medium using immuno-affinity chromatography. Multimer analysis revealed similar high-molecular weight multimers for all Normandy variants. As expected, the Arg763Gly replacement resulted in an increased size of all multimers. Solid phase binding assays in combination with Surface Plasmon Resonance binding studies showed that binding of FVIII-YFP was strongly reduced for the variants carrying an Arg763Gly, Thr791Met and Arg816Trp replacement and only mildly reduced for the variants carrying an Arg854Gln or Cys1060Arg replacement. Confocal Laser Scanning Microscopy analysis demonstrated that expression of all variants induced the formation of pseudo-Weibel-Palade bodies in HEK293 cells. In addition, all variants were able to redirect FVIII from localization at the trans-Golgi network to VWF-containing storage organelles despite their evident reduction in affinity. These data indicate that mutations in the FVIII binding region of VWF do not affect FVIII/VWF intracellular co-trafficking. We therefore conclude that the requirements that determine extracellular complex assembly differ from those that determine intracellular co-trafficking. We propose that FVIII/VWF co-trafficking may be a result of low-affinity interactions or even indirect passive targeting. We further conclude that co-trafficking is independent of post-translational propeptide cleavage. Together with the observation that co-trafficking is independent on Tyr1680, this suggests that the interactions that determine co-trafficking take place prior to and independent of post-translational processing in the late secretory pathway. Analysis of intracellular trafficking of FVIII in VWF producing cells could prove essential in unraveling the putative role of FVIII and VWF co-storage in vivo. Knowledge of FVIII/VWF co-storage may be particularly relevant to gene therapy approaches that target the FVIII gene to VWF-producing cells.