蛋白质水解
ADAMTS13号
血管性血友病因子
表位
化学
分子生物学
血管性血友病
抗体
血小板
免疫学
生物化学
生物
酶
作者
Claire Kizlik-Masson,Ivan Peyron,Stéphane Gangnard,Gaelle Claire Le Goff,Solen Lenoir,Sandra Damodaran,M. Clavel,Stéphanie Roullet,V. Régnault,Antoine Rauch,Flavien Vincent,Emmanuelle Jeanpierre,Annabelle Dupont,Catherine Ternisien,Thibault Donnet,Christophe Olivier,Éric Van Belle,Cécile V. Denis,Caterina Casari,Sophie Susen,Peter J. Lenting
出处
期刊:Blood
[American Society of Hematology]
日期:2023-03-23
卷期号:141 (12): 1457-1468
被引量:1
标识
DOI:10.1182/blood.2022017569
摘要
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. Although 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 from congenital von Willebrand disease (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 < .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.
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