PB0345 - The novel variable heavy chain antibody Syn-VWFA1 inhibits platelet von Willebrand factor interaction in solution

Sunday, June 23, 2024

13:45 – 14:45 ICT

Room: Exhibition Hall

Presenting Author(s)

Dana Huskens, PhD

PhD, Senior Research Project Leader
Synapse Research Institute
Maastricht, Limburg, Netherlands

Co-Author(s)

Mark Roest

PhD, Clinical Director
Synapse Research Institute
Maastricht, Limburg, Netherlands
Lv

Lisa N. van der Vorm

PhD
Department of Clinical Chemistry and Hematology, Gelre Ziekenhuizen, Apeldoorn
Apeldoorn, Gelderland, Netherlands
Steven J. Humphreys, MPhil

PhD student
Aberdeen Cardiovascular & Diabetes Centre, Institute of Medical Sciences, School of Medicine, Medical Sciences & Nutrition, University of Aberdeen
Aberdeen, Scotland, United Kingdom
Joke Konings, PhD

Senior Scientist
Synapse Research Institute
Maastricht, Limburg, Netherlands
NA

Nicholas A. Arce

PhD
Aflac Cancer and Blood Disorders Center, Children’s Healthcare of Atlanta, Department of Pediatrics, Emory University School of Medicine
Atlanta, Georgia, United States
Claire S. Whyte, PhD

PhD, Advanced Research Fellow
Aberdeen Cardiovascular & Diabetes Centre, Institute of Medical Sciences, School of Medicine, Medical Sciences & Nutrition, University of Aberdeen
Aberdeen, Scotland, United Kingdom
Pd

Philip G. de Groot

Professor Emeritus, advisor
Synapse Research Institute
Maastricht, Limburg, Netherlands
Nicola J. Mutch, MSc Hons, PhD

Professor
Aberdeen Cardiovascular & Diabetes Centre, Institute of Medical Sciences, School of Medicine, Medical Sciences & Nutrition, University of Aberdeen
Aberdeen, Scotland, United Kingdom
Renhao Li, PhD

Professor
First Affiliated Hospital, Zhejiang University School of Medicine
Hangzhou, Zhejiang, China (People's Republic)
JR

Jasper A. Remijn

PhD, Clinical Chemist
Department of Clinical Chemistry and Hematology, Gelre Ziekenhuizen
Apeldoorn, Gelderland, Netherlands
Bd

Bas de Laat

General Director
Synapse Research Institute
Maastricht, Limburg, Netherlands

Background: Blocking the interaction between GP1bα and von Willebrand Factor (VWF) is an attractive strategy for treatment and prevention of platelet-VWF agglutinate formation in von Willebrand disease type 2B and thrombotic thrombocytopenic purpura.

Aims: Characterize the effect of a llama-derived variable heavy chain antibody (VHH) targeting the A1 domain of active VWF (Syn-VWFA1), on platelet VWF interactions.

Methods: Flow cytometric analysis of platelet-VWF binding in whole blood using FITC-conjugated anti-VWF and PE-conjugated anti-CD41a. Light transmission aggregometry in platelet-rich plasma using ristocetin. Whole blood microfluidic analysis at high shear (1600 s−1) using Vena8 Fluoro+ Biochips (Cellix) coated with Horm collagen type 1. VWF-collagen binding assay (Asserachrom, Stago) to study GPIbα binding. Beads on a string assay using histamine stimulated endothelial cells (HUVECs). Cleavage of Haemate® P VWF (HVWF) by recombinant ADAMTS13 was measured using semi-automated electrophoresis (Hydrasys 2 instrument). Hydrogen-deuterium exchange mass spectrometry (HDX-MS) of recombinant A1.

Results: Syn-VWFA1 abolished platelet binding to ristocetin-activated VWF (native and HVWF) and R1306W (VWF type 2B) (Figure 1) and inhibited ristocetin-activated VWF and R1306W VWF platelet agglutination. Syn-VWFA1 did not alter adhesion of platelets to collagen under flow or binding of GPIbα to immobilized VWF. Syn-VWFA1 reduced platelet string length and number on histamine stimulated endothelial cells and increased the cleavage of VWF by ADAMTS13. The binding site of Syn-VWFA1 is located in the β1 to α1 loop (1288-1293) of the A1 domain (Figure 2) and binding results in a conformational change exposing regions in the α2 and α3 helices.

Conclusion(s): Syn-VWFA1 blocks platelet-binding to activated VWF in solution in a dose-dependent manner, but does not interact with (collagen-) immobilized active VWF. Therefore, Syn-VWFA1 has potential as treatment for patients with increased circulating active VWF, to prevent 3-dimensional pathological platelet-VWF aggregate formation whilst maintaining hemostatic 2-dimensional thrombus formation.