Characterization of a Nanobody That Binds Von Willebrand Factor and Prevents Von Willebrand Factor Cleavage By Plasmin

Introduction: Von Willebrand factor (vWF) is a multimeric glycoprotein that mediates platelet adherence to exposed subendothelial collagen at sites of vascular injury. To prevent spontaneous platelet aggregation, accumulation of high molecular weight vWF multimers is prevented by ADAMTS13-mediated cleavage in the vWF A2 domain. Moreover, emerging data demonstrates plasmin-mediated cleavage of vWF in its open conformation in the A1-A2 linker, suggesting a role for plasmin in regulation of vWF multimer composition as a back up for ADAMTS13.

Aim: We recently identified a llama-derived anti-vWF nanobody recognizing vWF in a conformation sensitive to plasmin cleavage. Our aim was to characterize this nanobody in terms of binding to- and interference with vWF functionality.

Methods and results: The nanobody was produced by immunization of llamas with wildtype vWF, followed by preparation of cDNA from isolated peripheral blood lymphocyte-derived RNA and selection of the vWF A1 domain specific nanobody via phage display. To study recognition of vWF by the nanobody and possible modulating factors we developed an ELISA assay based on a nanobody-coated microtiter plate. Plasmin pre-treatment of normal pooled plasma (NPP) significantly increased nanobody recognition of vWF, but only after subjecting the plasma to shear by vortexing. Ristocetin also increased recognition by the nanobody; however, addition of plasmin to ristocetin-opened vWF in NPP did not further enhance nanobody binding. This suggests that, in contrast to ristocetin, vortexing was not sufficient to fully expose all vWF A1 domains, but the remaining hidden A1 domains were exposed by plasmin cleavage, allowing additional vWF to bind to the nanobody.

Ellipsometry was used to assess competition of the nanobody and plasmin binding to vWF. Polyclonal anti-human vWF antibodies were coated to silicon slides, followed by addition of vWF concentrate (Haemate) or recombinant vWF R1306W, plasmin and the nanobody in varying order. We found that plasmin binds much more efficiently to R1306W vWF than to globular vWF. If the nanobody was bound to the R1306W vWF first, plasmin could not bind anymore. Conversely, after plasmin binding, only a small proportion of the nanobody still recognizes the plasmin-sensitive conformation of vWF, suggesting plasmin occupied the majority but not all binding sites and a possible difference in affinity for this conformation of vWF.

A flow-cytometry based platelet-activation assay was performed to study the effect of plasmin on the ability of vWF in its plasmin-sensitive conformation to bind to washed platelets. Fluorescent detection of vWF binding was mediated by FITC-labeled monoclonal anti-vWF antibodies or biotin-conjugated labeled nanobody in combination with streptavidin-FITC. Addition of plasmin increased ristocetin-modified vWF binding to platelets by 3.7-fold. This effect of plasmin was abrogated in the presence of heparin, consistent with work by Brophy et al. (2017) showing that heparin prevents cleavage of vWF by plasmin. Hence, it appears that plasmin-cleaved vWF has increased binding affinity to platelets. Of note, a similar pattern in ristocetin-activated vWF to platelet binding upon addition of plasmin (and heparin) was detected by the nanobody. This demonstrates that the nanobody does not interfere with platelet binding of vWF. Moreover, binding of plasmin-cleaved vWF resulted in reduced platelet activation, as determined from P-selectin expression.

Discussion: In conclusion, we have identified an anti-vWF nanobody capable of blocking plasmin binding to- and subsequent cleavage of vWF. This nanobody is unable to bind globular vWF in solution or vWF immobilized by polyclonal anti-vWF antibodies, but efficiently interacts with shear- and ristocetin-activated vWF and recombinant vWF comprising the type 2B R1306W mutation. The nanobody epitope on vWF is expected to overlap with- or lie in close proximity to the plasmin binding site in the A1-A2 linker, possibly resulting in steric hindrance. Current studies focus on providing more direct evidence of the vWF binding site of the nanobody. From a clinical perspective, the sensitivity of R1306W vWF to plasmin-mediated cleavage may explain the bleeding diathesis in VWD type 2B patients with this mutation.