OC 53.4 - O-linked glycosylation of VWF modulates Weibel-Palade body formation and VWF secretion

Background: Von Willebrand factor (VWF) is a key hemostatic glycoprotein that is predominantly 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-glycosylation on intracellular VWF trafficking, WPB formation and VWF secretory pathways.

Methods: HUVECs were treated with the O-glycosylation inhibitor BG (Benzyl-α-GalNAc). Site-directed mutagenesis was used to generate VWF mutants lacking specific O-glycan chains. WPB morphology was studied using confocal microscopy. VWF secretion was assessed by ELISA.

Results: BG-induced inhibition of O-glycosylation in HUVECs dramatically affected WPB formation, without altering the morphology of other intracellular compartments like the trans-Golgi network. WPBs were significantly shorter and more circular in BG-treated cells compared to controls (P < 0.0001). BG treatment also impacted VWF and angiopoietin-2 (Ang-2) secretion in HUVECs. In particular, histamine-stimulated VWF secretion was significantly reduced (P < 0.002) in BG-treated cells whereas unstimulated VWF secretion was increased (P < 0.001) (Figure 1). In addition, BG-treatment also resulted in significantly decreased length of VWF strings released from HUVECs in response to histamine-induced activation (P= 0.045). Interestingly and in keeping with the BG data, site-directed mutagenesis of the two O-linked glycan clusters which flank the A1 domain, led to a decrease in pseudo-WPB length (P < 0.0001) with WPB morphology mimicking that observed in BG-treated HUVECs.

Conclusion(s): Our novel data suggest that specific O-linked glycan determinants on VWF critically regulate intracellular trafficking and thereby influence WPB morphology and secretion of VWF strings.