A nanobody against the von Willebrand factor A3-domain detects ADAMTS13-induced proteolysis in congenital & acquired VWD.

Von Willebrand factor (VWF) is a multimeric protein, the size of which is regulated via ADAMTS13-mediated proteolysis within the A2-domain. We aimed to isolate nanobodies distinguishing between proteolyzed and non-proteolyzed VWF, leading to the identification of a nanobody (designated KB-VWF-D3.1) targeting the A3-domain, the epitope of which overlaps the collagen-binding site. While KB-VWF-D3.1 binds with similar efficiency to dimeric and multimeric derivatives of VWF, binding to VWF was lost upon proteolysis by ADAMTS13, suggesting that proteolysis in the A2-domain modulates exposure of its epitope in the A3-domain. We therefore used KB-VWF-D3.1 to monitor VWF degradation in plasma samples. Spiking experiments showed that a loss of 10% intact-VWF could be detected using this nanobody. By comparing plasma from volunteers to that of congenital VWD-patients, intact-VWF levels were significantly reduced for all VWD-types, and most severely in VWD-type 2A-group 2 in which mutations promote ADAMTS13-mediated proteolysis. Unexpectedly, we also observed increased proteolysis in some patients with VWD-type 1 and VWD-type 2M. A significant correlation (r=0.51, p<0.0001) between the relative amount of high molecular weight-multimers and levels of intact-VWF was observed. Reduced levels of intact-VWF were further found in plasmas from patients with severe aortic stenosis and patients receiving mechanical circulatory support. KB-VWF-D3.1 is thus a nanobody that detects changes in the exposure of its epitope within the collagen-binding site of the A3-domain. In view of its unique characteristics, it has the potential to be used as a diagnostic tool to investigate whether a loss of larger multimers is due to ADAMTS13-mediated proteolysis.