BACKGROUND: von Willebrand factor (VWF) serves a critical role as a carrier of factor (F)VIII in circulation. While it is generally believed that FVIII and VWF assemble in circulation after secretion from different cells, an alternative view is that cells should exist that co-express FVIII and VWF. OBJECTIVES: In this study, intracellular co-expression of FVIII and VWF was studied, with particular reference to complex assembly and high-affinity interaction. METHODS: Using yellow fluorescent protein-tagged FVIII (FVIII-YFP) and cyan fluorescent protein-tagged VWF (VWF-CFP), we studied intracellular trafficking in human embryonic kidney (HEK293) cells and human umbilical vein endothelial cells (HUVEC). The role of the high-affinity interaction between FVIII and VWF was assessed using a FVIII-YFP variant carrying a Tyr1680Phe substitution, which abolishes high-affinity binding to VWF. Cellular trafficking studies were complemented by binding studies employing purified proteins. RESULTS: Solid phase binding assays employing FVIII-YFP demonstrated that the presence of the fluorescent moiety did not compromise high-affinity binding (K(d) = 0.065 +/- 0.008 nm) whereas the binding of the Tyr1680Phe FVIII-YFP variant was significantly reduced. Co-expression studies in HEK293 cells revealed intracellular co-storage of both FVIII-YFP and Tyr1680Phe FVIII-YFP within VWF-containing storage organelles. In addition, expression of FVIII-YFP and Tyr1680Phe FVIII-YFP in HUVEC demonstrated co-trafficking with endogenous VWF to authentic Weibel-Palade bodies (WPBs). CONCLUSIONS: Our findings demonstrate that FVIII trafficking to WPBs is independent of Tyr1680 and high-affinity binding to VWF. We therefore conclude that the structural requirements that determine intracellular co-trafficking differ from those that determine complex assembly in circulation.