OC 58.1 - The molecular basis underpinning the enhanced clearance of VWF Vicenza (R1205H)

Tuesday, June 25, 2024

09:30 – 09:45 ICT

Room: 211 A-D

Presenting Author(s)

Ferdows Atiq, MD, MSc, PhD

Senior Research Fellow
Royal College of Surgeons in Ireland
Dublin, Dublin, Ireland

Co-Author(s)

OR

Orla Rawley

Post-Doctoral Fellow
Molecular Hemostasis Research Group, Department of Pathology and Molecular Medicine, Queen's University, Kingston, ON, Canada.
Kingston, Ontario, Canada
Jamie O'Sullivan

Lecturer/StAR Investigator in Vascular Biology
Irish Centre for Vascular Biology, School of Pharmacy and Biomolecular Sciences, Royal College of Surgeons in Ireland, Dublin, Ireland.
Dublin, Dublin, Ireland
Dearbhla Doherty

PhD Candidate
Irish Centre for Vascular Biology, School of Pharmacy and Biomolecular Sciences, Royal College of Surgeons in Ireland, Dublin, Ireland.
Dublin, Dublin, Ireland
SW

Soracha Ward

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
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
Ross Baker, MB BS BMedSc FRACP, FRCPA

Professor
Western Australia Centre for Thrombosis and Haemostasis, Perth Blood Institute, Murdoch University, Perth, Australia
Perth, Western Australia, Australia
Roger J.S. Preston

Senior Lecturer
Irish Centre for Vascular Biology, School of Pharmacy and Biomolecular Sciences, Royal College of Surgeons in Ireland, Dublin, Ireland.
Dublin, Dublin, Ireland
David Lillicrap, MD

Professor Department of Pathology and Molecular Medicine
Molecular Hemostasis Research Group, Department of Pathology and Molecular Medicine, Queen's University, Kingston, ON, Canada.
Kingston, Ontario, Canada
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: The R1205H Vicenza mutation causes type 1C VWD due to accelerated macrophage mediated VWF clearance. However, the biological mechanisms underlying this enhanced clearance remain poorly understood.

Aims: We hypothesized that the R1205H substitution triggers pathological enhanced clearance by (i) enhancing D’D3-domain interaction with specific macrophage receptors or (ii) by removing normal inhibitory effect of D’D3-region on A1-domain mediated clearance pathways.

Methods: In vivo clearance of VWF variants with specific R1205 substitutions was investigated in VWF-/- mice. Flow cytometry was employed to analyze VWF variant binding to THP-1 macrophages and plate-binding assays to characterize VWF binding to purified LRP1 cluster II or cluster IV receptors, MGL or SR-A1.

Results: In full-length rVWF missing the A1 domain (VWF-ΔA1), introduction of R1205H led to significantly enhanced clearance in VWF-/- mice compared to WT VWF-ΔA1 (Fig. 1A, p< 0.001). Importantly, R1205H in a truncated VWF-D’D3 fragment also triggered increased in vivo clearance compared to WT VWF-D’D3 (Fig.1B, p< 0.001). Furthermore, positive charge at 1205 is conserved amongst species (Fig. 1C). In contrast to VWF-R1205H, we observed that VWF-R1205K (preserving the positive charge) exhibited normal clearance in VWF-/- mice (Fig. 1D). Conversely, VWF-R1205E (resulting in loss of positive charge) resulted in significantly increased clearance, similar to VWF-R1205H. In vitro studies demonstrated increased macrophage-binding for VWF-R1205H compared to WT VWF. This binding was attenuated in the presence of RAP. Finally, plate-binding studies confirmed increased VWF-R1205H interaction with purified SR-AI compared to WT VWF (Fig. 2A, p< 0.001). Furthermore, consistent with the RAP data, significantly enhanced VWF-R1205H binding to LRP1 cluster II (p=0.034) and IV (p < 0.001) was also observed (Fig. 2B-C). In contrast, VWF-R1205H binding to the MGL clearance receptor was similar to WT (Fig 2D).

Conclusion(s): Disruption of the positive charge at VWF amino acid 1205 results in VWF-D’D3 mediated enhanced clearance regulated by macrophage LRP1 and SR-AI receptors.