Von Willebrand Factor Protects Factor VIII from Rapid Endocytosis Via LDL Receptor-Related Protein By Blocking the Initial Rate-Limiting Cell Surface Binding Step of Factor VIII
Authors: Meems Henriet , van der Zwaan Carmen , Mertens Koen , Meijer Alexander 
Published: October 2019

Abstract
Blood coagulation factor VIII is internalized and degraded by cellular systems via an endocytic mechanism that involves low-density lipoprotein receptor-related protein (LRP). Factor VIII's chaperone protein von Willebrand factor (VWF) has a dual role in this uptake mechanism. Under conditions of low shear stress VWF is particularly effective in blocking LRP-dependent endocytosis of factor VIII, whereas VWF itself mediates the uptake of factor VIII under high shear stress conditions. This study addresses the poorly understood uptake mechanism of factor VIII and the role therein of VWF in the absence of shear stress.
LRP binds its ligands via ligand-binding domains that are clustered in four regions. Cluster II, III and IV have each been demonstrated to bind factor VIII. However, one of the poorly understood aspects of the LRP dependent endocytosis of factor VIII is that the affinity of LRP for factor VIII is an order of magnitude lower compared to the affinity of LRP for other ligands like, for instance, alpha-2-macroglobulin (α2M). We and others have previously hypothesized that, in contrast to α2M, factor VIII may first require pre-concentration on the cell surface before it is transferred to LRP for effective endocytosis. Direct evidence for the existence of this two-step mechanism of endocytosis is, however, still lacking. In the present study, we compared the endocytic uptake mechanism of fluorescent labeled α2M and a factor VIII GFP variant employing a confocal microscopy study. To this end, U87MG cells were employed, which are particularly effective in internalizing ligands via the LRP-dependent pathway. The role of vWF in the LRP-mediated endocytosis of factor VIII was assessed as well.
The confocal microscopy studies surprisingly showed that factor VIII traffics to early endosomes irrespective of the presence of α2M. Co-incubation of factor VIII and α2M, revealed that both proteins end up in the same endocytic compartment of the cells. This finding can be explained if LRP can simultaneously bind and endocytose both factor VIII and α2M. Taking maximal advantage of this phenomenon, factor VIII and α2M were together incubated with the cells to accurately assess whether or not there is any difference in the rate of endocytosis of these proteins. The results demonstrated that α2M was present at the cell surface after 2 minutes of incubation. After 5 minutes, α2M was distributed in a punctate pattern over the cells indicating that α2M has been internalized by the cells. Interestingly, a 15 minutes incubation of factor VIII with the cells was required before factor VIII could be detected at the cell surface and inside the cells. This finding implies that the uptake of factor VIII is delayed compared to the rate of endocytosis of α2M. This is in agreement with the hypothesis that factor VIII may first require a cell surface binding step prior to LRP mediated endocytosis. These observations also imply that the cell surface binding step is rate-limiting in the endocytic uptake mechanism of factor VIII. Compelling evidence of the two-step endocytic model was obtained when factor VIII was incubated with the cells in the presence of receptor-associated-protein (RAP), which prevents direct binding of ligands to LRP. The results of these studies showed a striated fluorescent pattern that was localized at the borders of the cells. Notably, in the presence of VWF, no factor VIII was observed on or within the cells at all. These findings imply that VWF blocks cell surface binding of factor VIII.
Based on our findings, we propose that the ligand-binding clusters of LRP can mediate the binding of more than one ligand to LRP. The combined results further convincingly show that factor VIII endocytosis includes an initial cell surface binding step which is rate limiting in the uptake mechanism of factor VIII. The observations also explain why VWF is particularly effective in blocking the endocytosis of factor VIII. VWF may not only block the direct binding of factor VIII to LRP but also the rate limiting cell surface binding step prior to LRP mediated endocytosis.

Disclosures
No relevant conflicts of interest to declare.