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1193 The Variable Heavy Chain Antibody Syn-VWFA1 Inhibits the Interaction of Platelets to Von Willebrand Factor in Solution but Not to Immobilized Von Willebrand Factor Thereby Inhibiting 3-Dimensional but Not 2-Dimensional Thrombus Formation

Program: Oral and Poster Abstracts
Session: 301. Vasculature, Endothelium, Thrombosis and Platelets: Basic and Translational: Poster I
Hematology Disease Topics & Pathways:
Research, Bleeding and Clotting, Fundamental Science, thrombocytopenias, Diseases, thrombotic disorders, VWD
Saturday, December 9, 2023, 5:30 PM-7:30 PM

Dana Huskens, PhD1*, Lisa Nynke Van Der Vorm, PhD2*, Joke Konings, PhD2*, Nicholas Arce, PhD3, Mark Roest1*, Philip G. De Groot, PhD2*, Renhao Li, PhD3, Jasper A. Remijn, PhD4* and Bas De Laat, MD, PhD2

1Department of Platelet Pathophysiology, Synapse Research Institute, Maastricht, Netherlands
2Synapse Research Institute, Maastricht, Netherlands
3Aflac Cancer and Blood Disorders Center, Children’s Healthcare of Atlanta, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA
4Department of Clinical Chemistry, Meander Medical Center, Amersfoort, Netherlands

Neutralizing 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.

The aim of this study was to characterize a llama-derived variable heavy chain antibody (VHH) targeting the A1 domain of active VWF (Syn-VWFA1), in terms of its interference with platelet VWF interaction.

Syn-VWFA1 (1.5 µM) abolished platelet binding to ristocetin-activated VWF (native and Haemate® P VWF) and R1306W VWF (VWF type 2B) in a flow-cytometric platelet function assay (Figure 1). Moreover, Syn-VWFA1 dose-dependently inhibited ristocetin-activated VWF and R1306W VWF platelet agglutination, but not collagen- and ADP-induced platelet aggregation. Under flow conditions, Syn-VWFA1 (1.8 µM) did not affect adhesion of platelets to collagen at high shear. In the shear- and collagen-dependent PFA-200, Syn-VWFA1 prolonged the closure time, but only at high concentrations (>3 µM), while GPIba binding to immobilized VWF remained unaffected by Syn-VWFA1. Syn-VWFA1 increased the cleavage of VWF multimers by ADAMTS13. Furthermore, by applying deuterium exchange, we found a small region in the β1 to α1 loop (1288-1293) in the A1 domain of VWF, which is likely to constitute the nanobody binding site (Figure 2) and binding of Syn-VWFA1 to the A1 domain results in a conformational change exposing regions in the α2 and α3 helices.

In conclusion, Syn-VWFA1 dose-dependently blocks platelet-binding to activated VWF in solution, but not to (collagen-) immobilized active VWF. This implies that immobilization of VWF to collagen affects VWF conformation in such a way (1) that access of Syn-VWFA1 to its epitope in the A1 domain is hindered or (2) that conformational changes following Syn-VWFA1 binding are blocked, while allowing platelet binding. This implies that Syn-VWFA1 has potential treatment opportunities for patients with increased circulating active VWF, to prevent 3-dimensional thrombus formation (pathological platelet-VWF aggregate formation) but not 2-dimensional thrombus formation (leaving physiological hemostasis at sites of vascular injury intact).

Disclosures: Huskens: Synapse Research Institute: Current Employment, Other: Synapse Research Institute is part of the Diagnostica Stago group. Konings: Synapse Research Institute: Current Employment. Arce: Loxo Oncology at Lilly: Current Employment, Current equity holder in publicly-traded company. Roest: Synapse Research Institute: Current Employment, Other: Synapse Research Institute is part of the Diagnostica Stago group. De Groot: Synapse Research Institute: Consultancy, Honoraria. De Laat: Diagnostica Stago: Other: Synapse Research Institute is part of the Diagnostica Stago group..

*signifies non-member of ASH