Soluble Siglec-5 associates to PSGL-1 and displays anti-inflammatory activity.

Interactions between endothelial selectins and the leukocyte counter-receptor PSGL1 mediates leukocyte recruitment to inflammation sites. PSGL1 is highly sialylated, making it a potential ligand for Siglec-5, a leukocyte-receptor that recognizes sialic acid structures. Binding assays using soluble Siglec-5 variants (sSiglec-5/C4BP and sSiglec-5/Fc) revealed a dose- and calcium-dependent binding to PSGL1. Pre-treatment of PSGL1 with sialidase reduced Siglec-5 binding by 79 ± 4%. In confocal immune-fluorescence assays, we observed that 50% of Peripheral Blood Mononuclear Cells (PBMCs) simultaneously express PSGL1 and Siglec-5. Duolink-proximity ligation analysis demonstrated that PSGL1 and Siglec-5 are in close proximity (<40 nm) in 31 ± 4% of PBMCs. In vitro perfusion assays revealed that leukocyte-rolling over E- and P-selectin was inhibited by sSiglec-5/Fc or sSiglec-5/C4BP, while adhesion onto VCAM1 was unaffected. When applied to healthy mice (0.8 mg/kg), sSiglec-5/C4BP significantly reduced the number of rolling leukocytes under basal conditions (10.9 ± 3.7 versus 23.5 ± 9.3 leukocytes/field/min for sSiglec-5/C4BP-treated and control mice, respectively; p = 0.0093). Moreover, leukocyte recruitment was inhibited over a 5-h observation period in an in vivo model of TNFalpha-induced inflammation following injection sSiglec-5/C4BP (0.8 mg/kg). Our data identify PSGL1 as a ligand for Siglec-5, and soluble Siglec-5 variants appear efficient in blocking PSGL1-mediated leukocyte rolling and the inflammatory response in general.

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). DESIGN AND 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.

Macrophage LRP1 contributes to the clearance of von Willebrand factor.

The relationship between low-density lipoprotein receptor-related protein-1 (LRP1) and von Willebrand factor (VWF) has remained elusive for years. Indeed, despite a reported absence of interaction between both proteins, liver-specific deletion of LRP1 results in increased VWF levels. To investigate this discrepancy, we used mice with a macrophage-specific deficiency of LRP1 (macLRP1(-)) because we previously found that macrophages dominate VWF clearance. Basal VWF levels were increased in macLRP1(-) mice compared with control mice (1.6 ± 0.4 vs 1.0 ± 0.4 U/mL). Clearance experiments revealed that half-life of human VWF was significantly increased in macLRP1(-) mice. Ubiquitous blocking of LRP1 or additional lipoprotein receptors by overexpressing receptor-associated protein in macLRP1(-) mice did not result in further rise of VWF levels (0.1 ± 0.2 U/mL), in contrast to macLRP1(+) mice (rise in VWF, 0.8 ± 0.4 U/mL). This points to macLRP1 being the only lipoprotein receptor regulating VWF levels. When testing the mechanism(s) involved, we observed that VWF-coated beads adhered efficiently to LRP1 but only when exposed to shear forces exceeding 2.5 dyne/cm(2), implying the existence of shear stress-dependent interactions. Furthermore, a mechanism involving ß2-integrins that binds both VWF and LRP1 also is implicated because inhibition of ß2-integrins led to increased VWF levels in control (rise, 0.19 ± 0.16 U/mL) but not in macLRP1(-) mice (0.08 ± 0.15 U/mL).

Identification of galectin-1 and galectin-3 as novel partners for von Willebrand factor.

OBJECTIVE: Although von Willebrand factor (VWF) is a heavily glycosylated protein, its potential to associate with glycan-binding proteins is poorly investigated. Here, we explored its interaction with the glycan-binding proteins galectin-1 and galectin-3. METHODS AND RESULTS: Immunofluorescence analysis using Duolink proximity ligation assays revealed that VWF colocalizes with galectin-1 and galectin-3 in endothelial cells, both before and after stimulation of endothelial cells. Moreover, galectin-1 was found along the typical VWF bundles that are released by endothelial cells. Galectin-1 and galectin-3 could be coprecipitated with VWF from plasma in immunoprecipitation assays, whereas plasma levels of galectin-1 and galectin-3 were significantly reduced in VWF-deficient mice. Binding studies using purified proteins confirmed that VWF could directly interact with both galectins, predominantly via its N-linked glycans. In search of the physiological relevance of the VWF-galectin interaction, we found that inhibition of galectins in in vitro perfusion assays was associated with increased VWF-platelet string formation, a phenomenon that was reproduced in galectin-1/galectin-3 double-deficient mice. These mice were also characterized by a more rapid formation of initial thrombi following ferric chloride-induced injury. CONCLUSIONS: We have identified galectin-1 and galectin-3 as novel partners for VWF, and these proteins may modulate VWF-mediated thrombus formation.

Regulation of von Willebrand factor-platelet interactions.

The formation of thrombi is a multistep process involving several components, including von Willebrand factor (VWF). VWF is an adhesive multimeric protein, which acts as a molecular bridge between the subendothelial matrix and the glycoprotein Ib/IX/V receptor complex. Furthermore, VWF promotes the expansion of the platelet plug by cross-linking platelets via binding to integrin alphaIIbbeta3. In terms of thrombus formation, it is essential that VWF-platelet interactions occur timely, that is: it should happen not too early or too late. Given the co-existence of VWF and platelets in the circulation, this implies that there must be regulatory mechanisms that prevent premature formation of VWF-rich platelet aggregates that could occlude the vasculature. Indeed, several mechanisms have been identified at the level of VWF, which are dedicated to the prevention of excessive VWF-platelet interactions following endothelial release of VWF (which may include limited exposure to shear stress, the presence of Mg2+ ions, inhibition of VWF-platelet interactions by endothelial proteins, ADAMTS13-mediated proteolysis) and of circulating VWF-platelet aggregates during normal circulation (shielding of the platelet-binding A1 domain by other regions of the VWF molecule, inhibition of VWF-platelet interactions by beta2-glycoprotein I). In the present review an overview of these mechanisms will be discussed.