OC 15.2 - N-glycans located in the VWF-A2 domain regulate A1-domain mediated clearance through macrophage LRP1 and SR-A1 receptors

Sunday, June 23, 2024

09:45 – 10:00 ICT

Room: 110 A-C

Presenting Author(s)

Alain Chion, PhD

Post-Doctoral Fellow
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)

Sonia Aguila, PhD

Principal Investigator
Centro Regional de Hemodonación, IMIB-Pascual Parrilla, UCAM
Murcia, Murcia, Spain
JF

Judicael Fazavana

Postdoctoral
Royal College of Surgeons In Ireland
Dublin, Dublin, Ireland
Ciara Byrne, PhD

Postdoc
RCSI Dublin
Dublin, Dublin, Ireland
Patricia Lopes, MSc

PhD candidate
Irish Centre for Vascular Biology, School of Pharmacy and Biomolecular Sciences, Royal College of Surgeons in Ireland
Dublin, Dublin, Ireland
TM

Tom McKinnon

Principal Investigator
Imperial College London
London, England, United Kingdom
JO

James S. O'Donnell

Professor of Vascular Biology
Irish Centre for Vascular Biology, School of Pharmacy and Biomolecular Sciences, RCSI University of Medicine and Health Sciences, Dublin, Ireland.
Dublin, Dublin, Ireland

Background: Macrophage LRP1 & SR-A1 are major clearance receptors for VWF. The isolated VWF-A1 domain (but not -A2 nor -A3 domains) binds to LRP1 and SR-A1. We recently identified the lysine cluster K 1405-K1408 in VWF-A1 domain as a critical LRP1 binding site. Finally, loss of N-linked glycans in the VWF-A2 domain (N1515Q & N1574Q) markedly enhances macrophage-mediated VWF clearance

Aims: We hypothesized that the N-glycan determinants in the VWF-A2 domain influence macrophage-mediated clearance by affecting LRP1-mediated interaction with the K1405-K1408 cluster in the VWF-A1 domain

Methods: Binding of full-length VWF to HEK293 expressing LRP1 was assessed by flow cytometry. Plate-binding assays were used to characterize binding to purified LRP1-Cluster II or Cluster IV receptors, or SR-A1 respectively. Finally, in vivo clearance of VWF variants were investigated in VWF-/- mice

Results: Binding of both VWF-N1515Q and -N1574Q to purified LRP1 was significantly increased (p < 0.028). Similarly, binding to HEK293 cells expressing LRP1 (HEK293-LRP1) was enhanced (p < 0.05). LRP1 interaction was increased for the VWF truncations VWFA1A2A3-N1515Q and VWFA1A2A3-N1574Q (p < 0.004). Both rVWF-N1515Q and -N1574Q also displayed increased binding to purified SR-A1 and to HEK293-SR-A1 cells (p < 0.004). In line with our in vitro findings, markedly enhanced clearance of VWF-N1515Q and VWF-N1574Q was observed VWF-/- mice. Importantly, this rapid clearance was ablated in both cases by additional introduction of the K1408E mutation into the VWF-A1 domain lysine cluster. Consistently, introduction of K1408E also significantly reduced (p < 0.002) binding of VWFA1A2A3-N1515Q and VWFA1A2A3-N1574Q to LRP1 ex vivo. Finally, alanine mutagenesis of the K1405-K1408 cluster in VWF-A1 domain ablated LRP1 binding but did not significantly affect SR-A1 interaction

Conclusion(s): Collectively, our findings demonstrate that interaction between the VWF-A1 and VWF-A2 domains significantly impacts in vivo clearance pathways. In particular, the bulky glycans N1515 and N1574 in VWF-A2 domain critically regulate A1-domain mediated clearance through macrophage LRP1 and SR-A1