Submitted May 29, 2008
Accepted November 25, 2008
Intracellular co-trafficking of factor VIII and von Willebrand factor type 2N variants to storage organelles
Maartje van den Biggelaar, Alexander B Meijer, Jan Voorberg, and Koen Mertens*
Department of Plasma Proteins, Sanquin Research, Amsterdam, Netherlands
Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, Netherlands
Landsteiner Laboratory of AMC and Sanquin, University of Amsterdam, Amsterdam, Netherlands
* Corresponding author; email: k.mertens{at}sanquin.nl.
Weibel-Palade bodies (WPBs) are the endothelial storage organelles that are formed upon von Willebrand factor (VWF) expression. Apart from VWF, WPBs contain a variety of hemostatic and inflammatory proteins. Some of these are thought to be targeted to WPBs by directly interacting with VWF in the secretory pathway. Previous studies have demonstrated that co-expression of factor VIII (FVIII) with VWF results in co-storage of both proteins. However, whether co-trafficking is driven by intracellular FVIII-VWF assembly has remained unclear. We now have addressed this issue using recombinant VWF type 2N variants that are known to display reduced FVIII binding in the circulation. Binding studies using purified fluorescent FVIII and VWF type 2N variants revealed FVIII binding defects varying from moderate (Arg854Gln, Cys1060Arg) to severe (Arg763Gly, Thr791Met, Arg816Trp). Upon expression in HEK293 cells, all VWF variants induced formation of WPB-like organelles that were able to recruit P-selectin, as well as FVIII. WPBs containing FVIII did not display their typical elongated shape, suggesting that FVIII affects the organization of VWF tubules therein. The finding that VWF type 2N variants are still capable of co-targeting FVIII to storage granules implies that trafficking of WPB cargo proteins does not necessarily require high-affinity assembly with VWF.
