Galectin-1 and Galectin-3 Constitute Novel-Binding Partners for Factor VIII

Abstract

Objective-Recent studies have demonstrated that galectin-1 (Gal-1) and galectin-3 (Gal-3) can bind von Willebrand factor and directly modulate von Willebrand factor-dependent early thrombus formation in vivo. Because the glycans expressed on human factor VIII (FVIII) are similar to those of von Willebrand factor, we investigated whether galectins might also bind and modulate the activity of FVIII. Approach and Results-Immunosorbant assays and surface plasmon resonance analysis confirmed that Gal-1 and Gal-3 bound purified FVIII with high affinity. Exoglycosidase removal of FVIII N-linked glycans significantly reduced binding to both Gal-1 and Gal-3. Moreover, combined removal of both the N-and O-glycans of FVIII further attenuated Gal-3 binding. Notably, specific digestion of FVIII high-mannose glycans at N239 and N2118 significantly impaired FVIII affinity for Gal-1. Importantly Gal-1, but not Gal-3, bound to free FVIII in the plasma milieu, and significantly inhibited FVIII functional activity. Interestingly, commercial recombinant FVIII (rFVIII) concentrates are manufactured in different cell lines and differ in their glycosylation profiles. Although the biological mechanism has not been defined, recent studies in previously untreated patients with severe hemophilia A reported significant differences in inhibitor development associated with different rFVIII products. Interestingly, Gal-1 and Gal-3 both displayed enhanced affinity for BHK-rFVIII compared with CHO-rFVIII. Furthermore, binding of Gal-1 and Gal-3 to BDD-FVIII was markedly reduced compared with full-length rFVIII. Conclusions-We have identified Gal-1 and Gal-3 as novel-binding partners for human FVIII and demonstrated that Gal-1 binding can influence the procoagulant activity of FVIII.

von Willebrand factor clearance – biological mechanisms and clinical significance

“…Plasma VWF:Ag levels were normal in Gal‐1/Gal‐3 double‐deficient mice, suggesting that although galectins can bind to VWF and may influence its functional properties, they may not play a direct role in modulating its clearance. In this context, it is interesting that Gal‐1 and Gal‐3 have also both recently been shown to bind to FVIII in a glycan‐dependent manner (O'Sullivan et al , ).…”

A novel role for the macrophage galactose-type lectin receptor in mediating von Willebrand factor clearance

“…As described in the supplemental Methods, surface plasmon resonance (SPR) was used to evaluate MGL binding to VWF. 13 Briefly, purified pd-VWF was immobilized on a CM5 chip, and binding to recombinant MGL (R&D Systems, United Kingdom) was determined. Furthermore, proximity ligation assay (Duolink-PLA; Sigma Aldrich, Ireland) was performed to evaluate colocalization of VWF and MGL on THP1 macrophages.…”

Early ovariectomy reveals the germline encoding of natural anti‐A‐ and Tn‐cross‐reactive immunoglobulin M (IgM) arising from developmental O‐GalNAc glycosylations. (Germline‐encoded natural anti‐A/Tn cross‐reactive IgM)

“…This occurs in human‐specific, epistatic cooperation with the fucosyltransferase 1 (FUT1) and 2 (FUT2), encoded by the H and Se genes on chromosome 19. The membrane‐located N ‐linked glycosylations are associated with soluble enzyme versions, which independently of the secretor status, are involved in identically specific N‐ and O ‐linked glycosylations on (muco) epithelial cells and plasma proteins , such as clotting factor VIII and vWF , carried by A2M . It is important to mention, that the dynamic, functional connection between the A2M structure and FVIII/vWF activity is based on both N ‐ and O ‐glycosylations , while the levels of A2M‐bound ABO(H) blood group reactivity correlate strictly with the phenotype expression on red cell surfaces .…”

“…The histochemistry of murine WAP‐T mammary cancer has revealed glycoconjugate changes similar to that in human breast cancer . In plasma, the major HPA‐binding proteins are blood group ABO(H)‐reactive glycoproteins, such as clotting factor VIII (FVIII) and von Willebrand factor (vWF) , carried by α 2‐macroglobulin (A2M) . This is an abundant polyfunctional protein occurring in plasma of mammals and considered an evolutionarily conserved arm of the innate immune system , while in the human is expressing the ABO(H) phenotype in plasma, strictly in accordance with the expression on red cell surfaces .…”