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    Oral Communication

    Platelet Disorders, von Willebrand Disease and Thrombotic Microangiopathies

    Von Willebrand Disease and Thrombotic Thrombocytopenic Purpura (TTP) Novel Treatments

    OC 08.4 - Aptamer BT200 prolongs VWF half-life by blocking interaction with macrophage scavenger receptor LRP1

    Saturday, June 24, 2023
    13:45 – 14:00 ET
    Room: 518
    Session Available As: On-Demand
    Background: The pegylated aptamer BT200 binds to the A1 domain of VWF and significantly prolongs VWF half-life in vivo. Although the mechanism(s) involved have not been defined, the LRP1 scavenger receptor binds to the VWF A1 domain and contributes to physiological VWF clearance.

    Aims: To determine the molecular mechanisms through which the BT200 pegylated aptamer extends VWF half-life.

    Methods: Binding of full-length and selected VWF truncations to bone marrow-derived macrophages (BMDM) was assessed by flow cytometry in the presence or absence of BT200. Immunosorbent plate-binding assays were used to characterize binding to purified LRP1-ClusterII or Cluster IV receptors respectively. Finally, clearance of HEK293T-expressed VWF variants were investigated in VWF-/- mice.

    Results: BT200 dose-dependently inhibited binding of recombinant VWF (Vonvendi) & plasma-derived VWF (Fandhi) to BMDMs (maximal binding inhibition: 88.55 ± 4.73 % at 10 μM BT200). In addition, BT200 significantly inhibited binding of full-length rVWF to purified LRP1-Cluster-IV (p = 0.0005). Plate binding assays confirmed that the truncated VWF-A1A2A3 fragment and the isolated VWF-A1 domain both bound to LRP1 Cluster-II and Cluster-IV. In both cases, binding was again inhibited by BT200 in a dose-dependent manner, recapitulating the BMDM results. Finally, interaction of VWF-A1A2A3 fragment with HEK-293T transfected with full length LRP1 cDNA was ablated by BT200. In contrast, BT200 had no inhibitory effects on VWF-A1A2A3 binding to either MGL or SR-A1 macrophage clearance receptors. Since BT200 binds close to Lys 1405 in VWF-A1, site-directed mutagenesis of Lys 1405 study was performed. VWFA1-K1405A did not bind BMDMs, nor to coated LRP1-Cluster-IV. Furthermore, full-length rVWF-K1405A demonstrated a significantly increased half-life in VWF-/- mice compared to wild type VWF (P=0.0049).

    Conclusion(s): Our novel findings demonstrate that Lys1405 in VWFA1 is critical for interaction with the LRP1 scavenger receptor. BT200 binds to VWFA1 close to this key Lys1405 residue, thereby preventing interaction with LRP1 and consequently extending VWF half-life.

    Learning Objectives:

    • Upon completion, participants will be able to understand how the pegylated aptamer BT200 prolongs the half-life of von Willebrand Factor
    • Upon completion, participants will be able to assess which VWF critical clearance receptor is affected by BT200
    • Upon completion, participants will be able to understand how macrophage scavenger receptor LRP1 binds VWF