Effective Factor VIII Endocytosis Is under Conformational Control of von Willebrand Factor.

Von Willebrand Factor (VWF) protects Factor VIII (FVIII) from rapid clearance of the cofactor from the circulations. This notion may agree with the observation that VWF blocks FVIII binding to LDL receptor (LDLR) and LDL receptor-related protein (LRP) in vitro. In spite of the presence of VWF, however, we have previously demonstrated that LRP and LDLR are involved in the catabolism of FVIII in vivo. This suggests that dissociation of the FVIII-VWF complex in plasma or at the cell surface drives the LRP/LDLR dependent clearance of FVIII. We therefore first assessed the effect of VWF on the binding of FVIII to the cell surface employing confocal microscopy. To this end, a functional FVIII derivative containing the yellow fluorescent protein (FVIIIYFP) was incubated with LRP-expressing or LDLR-expressing CHO cells at 4 °C in the presence and absence of VWF. The result showed that there was a distinct yellow fluorescent staining of the cell surface irrespective of the presence of VWF. When the same experiment was performed at 37 °C, however, yellow fluorescent focal spots rapidly appeared inside the cells but only in the absence of VWF. Co-localization studies demonstrated that these spots originated from FVIIIYFP present in early endosomes. These findings suggest that VWF blocks the transfer of FVIII to its endocytic receptors but not the binding of FVIII to the cell surface. Intriguingly, in the presence of ristocetin, FVIIIYFP was localized inside the cells not only in the absence of VWF but also in its presence. In agreement with this finding, flow cytometric analysis confirmed that VWF was no longer able to prevent endocytosis of FVIIIYFP in the presence of ristocetin. These results suggest that switching VWF in the VWF-FVIIIYFP complex into its active conformation triggers the endocytic uptake of FVIII. The restored endocytosis of FVIII was not the consequence of a reduced affinity of “active-VWF” for FVIII. The latter was concluded from solid phase binding studies that showed that FVIII-VWF complex formation is indistinguishable in the presence and absence of ristocetin. We propose that switching VWF in the VWF-FVIII complex into its active conformation initiates at the cell surface a sequence of molecular events, which ultimately lead to the endocytic uptake of FVIII by cells expressing LRP or LDLR.