Siglecs as Novel Cellular Partners for Von Willebrand Factor
Authors: Pegon Julie N , Denis Cécile V , Odouard Soline , Christophe Olivier D , Lenting Peter J 
Published: October 2019

Abstract
Abstract 4306

Introduction:
Von Willebrand factor (VWF) is a multimeric protein that is pertinent to the haemostatic process by promoting platelet recruitment to the growing thrombus and acting as a carrier-protein for factor VIII. It is well established that VWF is able to interact with several cellular receptors present on the surface of platelets and leukocytes. In search for novel cellular partners for VWF present on these cells, we made use of the notion that VWF is heavily glycosylated, with the mature molecule containing 12 N-linked glycans and 10 O-linked glycans. Importantly, the vast majority of these glycans (>90%) are sialylated. The human proteome contains a family of sialic acid-binding receptors, known as Sialic acid-binding Immunoglobulin-like Lectins (Siglecs), which are expressed on cells of hematopoietic origin. The current study was designed to investigate the potential of Siglecs to interact with VWF.

Methods:
Stable human HEK293 cell lines were established that express human Siglec-5, Siglec-7 or Siglec-9. In addition, soluble variants of Siglecs were obtained: monomeric HPC4-tagged Siglec-5 and Siglec-7 (sSg5/HPC4 & sSg7/HPC4) and dimeric Fc-fusion proteins for human Siglec-3, -5, -7, -9, -10, -11 and murine Siglec-F (sSg-3/Fc etc). These Siglecs were used to study their interaction with purified plasma-derived (pdVWF) or recombinant BHK-cell derived VWF (rVWF). Protein-protein interactions were investigated via immuno-sorbent assays and via surface plasmon resonance (SPR) using Octet QK equipment. Binding of VWF to Siglec-expressing cells was assessed via immuno-fluorescence microscopy.

Results:
We observed consistent association of VWF (irrespective whether immuno-sorbent or SPR assays were used) to all of the immobilized Siglecs tested, with the exception of sSg-11/Fc that did not interact with pdVWF or with rVWF. Inversely, all soluble Siglecs but sSg-11/Fc efficiently bound to immobilized pdVWF or rVWF. Half-maximal binding in immuno-sorbent assays was between 97 and 645 nM (binding of VWF to immobilized Siglecs) versus 166 and 270 nM (binding of Siglecs to immobilized VWF). More detailed studies on the determination of the kinetic parameters using SPR technology are in progress. Specificity of the interaction was confirmed by treating VWF with sialidase before testing Siglec binding, and such treatment resulted in a strongly reduced (up to 80%) ability of Siglecs to bind to immobilized VWF. VWF was further found to bind to Siglec-5, -7 or -9 expressing HEK293 cells as assessed via immuno-fluorescence microscopy. In general, 10–15 % of the Siglec-expressing cells were covered with VWF after incubation, with the fluorescent intensity increasing dose-dependently with the applied VWF concentration. No fluorescent signal was observed upon incubation of VWF with non-transfected cells or when VWF was omitted from the incubation. Since Siglecs may be masked by the presence of endogenous sialylated proteins at the cellular surface, we also tested VWF binding to these cells following sialidase treatment. Sialidase treatment of cells resulted in a marked increase in the number of VWF-binding cells (up to 80% of the Siglec-positive cells) as well as an increase in intensity of the fluorescent signal. Co-localization of VWF with Siglecs at the cellular surface was confirmed by DuoLink-analysis, which allows the detection of proteins that are in the vicinity of ≤40 nm. Emerging evidence demonstrates that endothelial.

Conclusions:
We identified one murine and six human Siglec-family members as novel partners for VWF. Interactions between VWF and Siglecs are mediated by sialic acid structures present on VWF. In addition, the cellular accessibility of Siglecs for VWF is modulated by cis-acting sialylated proteins at the cell surface. In conclusion, our data demonstrate that the VWF glycome allows the interaction with a novel family of cellular receptors, potentially opening previously unrecognized avenues in the biology of VWF.

Disclosures:
No relevant conflicts of interest to declare.