OC 07.1 - The clearance receptor CLEC4M regulates plasma factor V levels through factor V binding and internalization

Background: Factor V (FV) has pro- and anticoagulant properties, and plasma FV levels are associated with both venous thrombosis and bleeding. A genome-wide association study in 4373 participants of European descent identified a significant association between the CLEC4M gene (lead SNP rs594793, p=2.22x10-8) and FV levels (Trégouët et al. ISTH 2022). CLEC4M is a pathogen-recognition receptor known to bind and internalize von Willebrand factor and factor VIII. Therefore, we hypothesized that CLEC4M may also clear FV and contribute to inter-individual variation of plasma FV- levels.

Aims: To test the hypothesis that CLEC4M binds FV and mediates its internalization by cells.

Methods: The interaction between FV and CLEC4M was probed by antigen-based solid-phase assays. HEK293 cells stably expressing CLEC4M were generated and cultured in the presence or absence of FV. FV binding to the cell surface and FV internalization were studied by flow-cytometry and immunoblot, respectively. FV endocytosis and degradation were investigated using inhibitors of clathrin-mediated endocytosis and lysosomal activity, respectively, followed by FV immunoblot and ELISA on cell lysates. HEK293 cells not transfected with CLEC4M served as a control.

Results: FV bound to immobilized CLEC4M in a calcium- and concentration-dependent manner (Kd ~1 nM). Activated FV, lacking the heavily glycosylated B-domain, also bound to CLEC4M, but with lower affinity than FV. CLEC4M-expressing HEK293 cells bound and internalized more FV than CLEC4M-negative cells. Inhibitors of clathrin-mediated endocytosis decreased FV detection in cell lysates, while lysosome inhibitors increased it, suggesting that FV is internalized through clathrin-coated pits and then degraded in the lysosomes.

Conclusion(s): FV binds to CLEC4M at physiological plasma concentration. CLEC4M expression in HEK293 cells enhances the binding and internalization of FV, which is trafficked through clathrin-coated pits and degraded by lysosomes. These findings support a role of CLEC4M in the clearance of FV.