Abstract Number: OC 13.1
Meeting: ISTH 2020 Congress
Background: Von Willebrand Factor (VWF) can be activated by shear force, but the underlying molecular mechanism has remained elusive. A discontinuous autoinhibitory module (AIM) composed of both flanking regions around the A1 domain of VWF can impede its binding to platelets.
Aims: To test if tensile force releases the AIM from A1 and if the nanobody VHH81 that comprises FDA-approved caplacizumab could inhibit this process.
Methods: Single-molecule optical tweezers were used to apply tensile force on recombinant VWF-A1 fragments, and bio-layer interferometry to assess binding of VWF-A1 with platelet GPIbα. Platelet aggregometry and laminar flow chamber assays were utilized to measure VWF-A1-induced platelet activation/aggregation.
Results: A fragment containing the AIM and A1 (residues 1238-1493) exhibits a single unfolding event around 15 pN that is attributable to the unfolding of the AIM (Fig. A,B,C). The contour length is 29.9 ± 0.5 nm, consistent with ~60 residues in the AIM. VWF constructs lacking either N-terminal part of the AIM (1268-1493) or C-terminal AIM (1238-1461) show significantly altered unfolding events at lower forces (Fig A,C,D). VHH81 binds to 1238-1493, 1238-1461 but not 1268-1493 with high affinity, and greatly increases the unfolding force of the AIM (Fig. E,F,G). Consistently, VHH81 inhibits platelet accumulation to collagen surface at 2500/s shear rate, but the inhibition greatly diminishes at 10,000/s. In comparison, DNA aptamer ARC1172 that directly blocks the GPIbα-binding site in A1 abolishes platelet accumulation under both shear rates (Fig. H).
Conclusions: We provide the first direct evidence that tensile force unfolds the AIM from A1, supporting a model in which the folded AIM binds to A1 and critically masks its binding to platelets. Furthermore, VHH81 impedes VWF binding to platelets by binding to residues in the N-AIM and increasing the mechanical stability of the AIM. Thus, it is the first shear-reversible antagonist of the VWF-GPIbα interaction.
To cite this abstract in AMA style:Arce N, Cao W, Brown A, Legan E, Wilson M, Emsley J, Zhang F, Li R. Force-induced Unfolding of the Autoinhibitory Module Activates VWF [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/force-induced-unfolding-of-the-autoinhibitory-module-activates-vwf/. Accessed November 29, 2023.
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