Factor VIII and von Willebrand factor are ligands for the carbohydrate-receptor Siglec-5

Background

Factor VIII (FVIII) and von Willebrand factor (VWF) circulate in plasma in a tight non-covalentcomplex, being critical to hemostasis. Although structurally unrelated, both share the presence ofsialylated glycan-structures, making them potential ligands forsialic-acid-binding-immunoglobulin-like-lectins (Siglecs).

Design and Methods

We explored the potential interaction between FVIII/VWF and Siglec-5, a receptor expressed inmacrophages using various experimental approaches, including binding experiments with purifiedproteins and cell-binding studies with Siglec-5 expressing cells. Finally, Siglec-5 wasoverexpressed in mice via hydrodynamic gene transfer.

Results

In different systems using purified proteins, saturable, dose-dependent and reversibleinteractions between a soluble Siglec-5 fragment and both hemostatic proteins were found. Sialidasetreatment of VWF resulted in a complete lack of Siglec-5 binding. In contrast, sialidase treatmentleft interactions between FVIII and Siglec-5 unaffected. FVIII and VWF also bound to cellsurfaceexposed Siglec-5, as was visualized by classical immunostaining as well as by Duolinkproximityligation assays. Co-localization of FVIII and VWF with early endosomal markers further suggestedthat binding to Siglec-5 is followed by endocytosis of the proteins. Finally, overexpression ofhuman Siglec-5 in murine hepatocytes following hydrodynamic gene transfer resulted in a significantdecrease in plasma levels of FVIII and VWF in these mice.

Conclusions

Our data indicate that FVIII and VWF may act as a ligand for Siglec-5, and that Siglec-5 maycontribute to the regulation of plasma levels of the FVIII/VWF complex.Key words: factor VIII, von Willebrand factor, ligand, Siglec-5, interaction