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    Oral Communication

    Platelet Disorders, von Willebrand Disease and Thrombotic Microangiopathies

    von Willebrand Factor Biology

    OC 13.3 - O-glycan and sialic acid residues differentially regulate von Willebrand factor trafficking, storage and secretion.

    Sunday, June 25, 2023
    11:15 – 11:30 ET
    Room: 710B
    Session Available As: On-Demand

      Presenting Author(s)

    • Ellie Karampini, PhD (she/her/hers)

      Post-Doctoral Researcher
      Irish Centre for Vascular Biology, School of Pharmacy and Biomolecular Sciences, Royal College of Surgeons in Ireland, Dublin, Ireland
      Dublin, Dublin, Ireland

      • Co-Author(s)

    • MG

      Massimiliano Garre

      Imaging Specialist
      Super-Resolution Imaging Consortium, Royal College of Surgeons in Ireland
      Dublin, Dublin, Ireland

    • Dearbhla Doherty, MD (she/her/hers)

      Clinical Research Fellow
      Irish Centre for Vascular Biology, School of Pharmacy and Biomolecular Sciences, Royal College of Surgeons in Ireland, Dublin, Ireland
      Dublin 2, Dublin, Ireland

    • SE

      Stephanie Elliott

      Research assistant
      Irish Centre for Vascular Biology, School of Pharmacy and Biomolecular Sciences, Royal College of Surgeons in Ireland, Dublin, Ireland
      Dublin, Dublin, Ireland

    • BC

      Brenton Cavanagh

      Research Engineer
      Cellular and Molecular Imaging Core, Royal College of Surgeons in Ireland, Dublin, Ireland
      Dublin, Dublin, Ireland

    • Jamie O'Sullivan, PhD (she/her/hers)

      Principal Investigator
      Royal College of Surgeons in Ireland, Irish Centre for Vascular Biology, Dublin, Ireland

    • Ingmar Schoen, PhD (he/him/his)

      Lecturer
      RCSI University of Medicine and Health Sciences
      Dublin, Dublin, Ireland

    • JO

      James S. O'Donnell, MB, PhD

      Professor of Haematology
      Irish Centre for Vascular Biology, Royal College of Surgeons in Ireland,Dublin, Ireland
      Dublin, Dublin, Ireland

    Background: Von Willebrand factor (VWF) is a key hemostatic glycoprotein synthesized and secreted by endothelial cells. During biosynthesis, VWF undergoes extensive N- and O-linked glycosylation, before finally being stored in Weibel-Palade bodies (WPBs). WPB morphology influences VWF string formation upon activation. Although glycan alterations have been implicated in von Willebrand disease pathophysiology, the role of glycosylation in regulating VWF intracellular trafficking remains poorly understood.

    Aims: In this study, we investigated the role of O-glycans and terminal sialylation on intracellular VWF trafficking, WPB formation and VWF secretory pathways.

    Methods: HUVECs were treated with the O-glycosylation inhibitor BG (Benzyl-α-GalNAc) or sialylation inhibitor FAX (P-3FAX-Neu5Ac). α2-3 sialylation was reduced by shRNA knockdown (KD) of ST3Gal4. WPB morphology and string formation was studied using confocal microscopy. VWF secretion was assessed by ELISA.

    Results: Inhibition of VWF O-glycosylation by BG significantly increased basal VWF secretion (p < 0.01). In contrast, inhibition of VWF sialylation by FAX significantly attenuated VWF release into the supernatant (p < 0.0001). Similarly, KD of ST3Gal4 resulted in a significant decrease of VWF basal release (p < 0.01). BG-induced inhibition of O-glycosylation in HUVECs markedly affected WPB formation, such that WPBs were significantly shorter and more circular in BG-treated cells compared to controls (p < 0.0001). Additionally, BG-treatment resulted in the activation of A1 domain intracellularly and negatively affected the cells’ ability to produce histamine-induced VWF strings under shear stress (1.5 ml/min) with both fluorescent-labelled and platelet-decorated VWF strings being significantly reduced in length (p < 0.0001). In FAX and shST3Gal4 treated ECs, WPB morphology appeared normal without A1 activation, but a significant increase in the WPB number per cell was observed in FAX-treated ECs (p < 0.01).

    Conclusion(s): Our novel data suggest that VWF O-linked glycosylation and terminal sialylation regulate intracellular trafficking and storage of VWF in EC. Moreover, these glycan effects impact VWF biosynthesis through a variety of different mechanisms.

    Learning Objectives:

    • upon completion, participants will be able to describe the intracellular VWF trafficking
    • upon completion, participants will be able to define the role of O-glycosylation in VWF storage and secretion
    • upon completion, participants will be able to differentiate between O-linked glycan forms and their role in VWF biology