Von Willebrand factor (VWF) is a multimeric protein whose size directly governs its hemostatic function: high-molecular-weight multimers are the most active in capturing platelets and adhering to collagen. This size is regulated by a protease, ADAMTS13, which cleaves VWF within its A2 domain. However, many pathological conditions are characterized by a loss of large multimers, without it being straightforward to determine whether this loss results from increased proteolysis by ADAMTS13 or from other mechanisms. A tool capable of distinguishing intact VWF from proteolyzed VWF would therefore address a genuine diagnostic need.
To this end, the authors sought to isolate nanobodies capable of discriminating cleaved from non-cleaved VWF. This screening strategy, carried out using recombinant VWF and degraded VWF, led to the identification of a nanobody, designated KB-VWF-D3.1, directed against the A3 domain and whose epitope overlaps the collagen-binding site. Notably, this nanobody binds with comparable efficiency to the dimeric and multimeric forms of VWF, but loses its binding once the protein is proteolyzed by ADAMTS13. This behavior suggests that cleavage within the A2 domain alters, at a distance, the exposure of the epitope located in the A3 domain. Controlled spiking experiments showed that a loss of as little as 10% of intact VWF could be detected using this approach.
Applied to plasma samples, KB-VWF-D3.1 revealed a significant reduction in intact VWF levels across all types of congenital von Willebrand disease, with the most pronounced decrease observed in type 2A–group 2, where the mutations specifically promote proteolysis by ADAMTS13. Unexpectedly, increased proteolysis was also observed in certain patients with type 1 and type 2M von Willebrand disease. The authors further report a significant correlation (r = 0.51; p < 0.0001) between the relative proportion of high-molecular-weight multimers and intact VWF levels. Reduced levels were also demonstrated in patients with severe aortic stenosis and in patients receiving mechanical circulatory support. KB-VWF-D3.1 thus detects variations in the exposure of its epitope within the collagen-binding site of the A3 domain. In light of its characteristics, the authors consider that it has the potential to serve as a diagnostic tool for determining whether a loss of large multimers is attributable to ADAMTS13-mediated proteolysis.