The Endothelial Lectin Receptor CLEC4M Internalizes Factor VIII and Von Willebrand Factor Via a Clathrin-Coated Pit-Dependent Mechanism
Authors: Swystun Laura L. , Notley Colleen , Georgescu Ilinca , James Paula D. , Lillicrap David 
Published: October 2019

Abstract
Regulation of plasma levels of the coagulation factors von Willebrand factor (VWF) and factor VIII (FVIII) involves a dynamic balance between biosynthesis, secretion, and clearance. Clearance of VWF and FVIII occurs through receptor-mediated endocytosis, with both LDL receptor gene family (eg. LRP1), and lectin receptors (eg. asialoglycoprotein receptor, siglec-5) contributing to this process. We have previously characterized the endothelial lectin CLEC4M as an endocytic receptor for FVIII and VWF. These proteins represent the first endogenous glycoprotein ligands indentified for CLEC4M.
Previous studies have characterized CLEC4M as an adhesive receptor capable of facilitating viral infection in trans. Thus, the mechanism by which CLEC4M internalizes VWF and FVIII, and the subsequent fate of these ligands, is uncharacterized. We hypothesize that CLEC4M is able to endocytose VWF and FVIII via a clathrin-coated pit-dependent mechanism. We further hypothesize that endocytosis of FVIII and VWF by CLEC4M-expressing cells targets FVIII and VWF to lysosomes for degradation.
As CLEC4M is expressed exclusively on the endothelial cells of the hepatic sinusoids and lymph nodes, and commercially prepared liver sinusoidal endothelial cells do not retain CLEC4M expression, we generated a HEK 293 cell line that stably expresses CLEC4M (>90% positive by flow cytometry). We first inhibited the CLEC4M-mediated endocytic pathways by preincubating cells with methyl-β-cyclodextrin to deplete cell membrane cholesterol, dynasore hydrate to inhibit dynamin GTPase activity, and pitstop-2 to block the N-terminus of clathrin. Cells were then incubated with recombinant human FVIII, or plasma derived VWF-FVIII complex for 1 hour and internalization was visualized by immunofluorescence. A quantitative analysis of VWF or FVIII-positive objects was performed using Image Pro software.
CLEC4M-expressing 293 cells internalized both FVIII as well as the VWF-FVIII complex. Binding and internalization of VWF was reduced by methyl-β-cyclodextran (65%, p=0.067), by dynasore hydrate (92%, p=0.055), and by pitstop-2 (83%, p=0.032). Binding and internalization of FVIII was similarly reduced by methyl-β-cyclodextran (50%, p=0.0050), by dynasore hydrate (60%, p=0.0225), and by pitstop-2 (90%, p=0.0086). This suggests that CLEC4M mediates endocytosis of VWF and FVIII via a clathrin-coated pit-dependent mechanism that involves lipid rafts.
To visualize the endocytic pathway of VWF and FVIII, CLEC4M-expressing 293 cells were incubated with FVIII or the VWF-FVIII complex for 15, 30, or 60 minutes. Colocalization of FVIII, VWF and/or CLEC4M with markers for early endosomes (early endosomal antigen-1), late endosomes (Rab9), and lysosomes (LAMP1) was observed using immunofluorescence. For all conditions, CLEC4M-negative and isotype controls were performed.
We have previously confirmed that FVIII and VWF are internalized by CLEC4M expressing cells, and that VWF colocalizes with a marker for early endosomes. When CLEC4M-expressing cells were exposed to the VWF-FVIII complex, we observed a partial colocalization of VWF and FVIII, confirming that CLEC4M is able to internalize the VWF-FVIII complex. When CLEC4M-expressing cells were exposed to FVIII, we observed colocalization between FVIII and early endosomal antigen 1 within 15 minutes, confirming that upon internalization by CLEC4M, VWF and FVIII are targeted to early endosomes.
We next observed the transport of VWF and FVIII to late endosomes and lysosomes. When CLEC4M-expressing cells were incubated with VWF and FVIII for 30 minutes, we observed colocalization of both proteins with Rab9, a marker for late endosomes. When CLEC4M-expressing cells were incubated with FVIII for 1 hour, we observed colocalization of FVIII with LAMP1, a lysosomal marker, suggesting that the internalization of FVIII by CLEC4M leads to lysosome-mediated degradation of FVIII. In contrast, incubation of CLEC4M-expressing cells with VWF for up to 2 hours did not result in co-localization with LAMP1.
These studies confirm that, in the context of the stable cell system used in these experiments, the cell surface lectin receptor CLEC4M mediates endocytosis of FVIII and VWF through a clathrin-coated pit-dependent pathway. Internalization of VWF and FVIII by CLEC4M targets these proteins to early and late endosomes; FVIII is subsequently targeted to lysosomes for degradation.

Disclosures:
James: CSL Behring: Honoraria, Research Funding; Octapharma: Honoraria, Research Funding; Baxter: Honoraria; Bayer: Honoraria.