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-covalent complex, being critical to hemostasis. Although structurally unrelated, both share the presence of sialylated glycan-structures, making them potential ligands for sialic-acid-binding-immunoglobulin-like-lectins (Siglecs).


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


RESULTS
In different systems using purified proteins, saturable, dose-dependent and reversible interactions between a soluble Siglec-5 fragment and both hemostatic proteins were found. Sialidase treatment of VWF resulted in a complete lack of Siglec-5 binding. In contrast, sialidase treatment left interactions between FVIII and Siglec-5 unaffected. FVIII and VWF also bound to cellsurface exposed Siglec-5, as was visualized by classical immunostaining as well as by Duolinkproximity ligation assays. Co-localization of FVIII and VWF with early endosomal markers further suggested that binding to Siglec-5 is followed by endocytosis of the proteins. Finally, overexpression of human Siglec-5 in murine hepatocytes following hydrodynamic gene transfer resulted in a significant decrease 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 may contribute to the regulation of plasma levels of the FVIII/VWF complex.
Inserm U770 Université Paris Sud, Le Kremlin-Bicêtre, France.
Julie N
JN
Pegon
Mohamad
M
Kurdi
Caterina
C
Casari
Soline
S
Odouard
Cécile V
CV
Denis
Olivier D
OD
Christophe
Peter J
PJ
Lenting
D000818:Animals; D015703:Antigens, CD / Q000378:metabolism*; D015214:Antigens, Differentiation, Myelomonocytic / Q000378:metabolism*; D002478:Cells, Cultured; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D005169:Factor VIII / Q000378:metabolism*; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism; D006801:Humans; D007124:Immunoenzyme Techniques; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D037102:Lectins / Q000378:metabolism*; D008264:Macrophages / Q000166:cytology / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D009000:Monocytes / Q000166:cytology / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism*; D014841:von Willebrand Factor / Q000378:metabolism*
D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D037102:Lectins; D011134:Polysaccharides; C114531:SIGLEC5 protein, human; D014841:von Willebrand Factor; D005169:Factor VIII