Division of Plasma Protein Therapeutics, Office of Tissues and Advanced Therapies, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, Maryland, USA.
Confocal Imaging Facility, National Institute on Aging, National Institutes of Health, Baltimore, Maryland, USA.
J Thromb Haemost. 2022 Sep;20(9):2098-2108. doi: 10.1111/jth.15804. Epub 2022 Jul 12.
Von Willebrand factor (VWF) is elevated in sickle cell disease (SCD) and contributes to vaso-occlusion through its thrombogenic properties. VWF is regulated by ADAMTS13, a plasma protease that cleaves VWF into less bioactive multimers. Independent investigations have shown VWF to be elevated in SCD, whereas measurements of ADAMTS13 have been variable.
We assessed ADAMTS13 activity using multiple activity assays and measured levels of alternative VWF-cleaving proteases in SCD.
METHODS/ PATIENTS: Plasma samples were collected from adult patients with SCD (n = 20) at a single institution when presenting for routine red cell exchange transfusion therapy. ADAMTS13 activity was measured by FRETS-VWF73, Technozym ADAMTS-13 Activity ELISA kit and a full-length VWF digestion reaction. Alternative VWF-cleaving proteases were identified by ELISA. A cell culture model was used to study the impact of SCD stimuli on endothelial ADAMTS13 and alternative VWF-cleaving proteases.
ADAMTS13 activity was found to be moderately deficient across the SCD cohort as assessed by activity assays using a VWF A2 domain peptide substrate. However, SCD plasma showed preserved ability to digest full-length VWF, suggesting assay-discrepant results. Neutrophil and endothelial-derived proteases were found to be elevated in SCD plasma. Matrix metalloproteinase 9 specifically showed preferential cleavage of full-length VWF. Upregulation of alternative VWF-cleaving proteases occurred in endothelial cells exposed to SCD stimuli such as heme and hypoxia.
This is the first demonstration of accessory plasma enzymes contributing to the regulation of VWF in a specific disease state and may have implications for assessing the VWF/ADAMTS13 axis in other settings.
血管性血友病因子（VWF）在镰状细胞病（SCD）中升高，并通过其血栓形成特性导致血管阻塞。VWF 受 ADAMTS13 调节，ADAMTS13 是一种血浆蛋白酶，可将 VWF 切割成活性较低的多聚体。独立研究表明 VWF 在 SCD 中升高，而 ADAMTS13 的测量结果则有所不同。
我们使用多种活性测定法评估 ADAMTS13 活性，并测量 SCD 中替代 VWF 切割蛋白酶的水平。
方法/患者：在单个机构收集了 20 名成年 SCD 患者的血浆样本，这些患者在接受常规红细胞交换输血治疗时出现。通过 FRETS-VWF73、Technozym ADAMTS-13 活性 ELISA 试剂盒和全长 VWF 消化反应测定 ADAMTS13 活性。通过 ELISA 鉴定替代 VWF 切割蛋白酶。使用细胞培养模型研究 SCD 刺激对内皮 ADAMTS13 和替代 VWF 切割蛋白酶的影响。
使用 VWF A2 结构域肽底物的活性测定法评估 SCD 队列的 ADAMTS13 活性发现其为中度缺乏。然而，SCD 血浆显示出消化全长 VWF 的能力得到保留，这表明测定结果不一致。SCD 血浆中发现中性粒细胞和内皮衍生的蛋白酶升高。基质金属蛋白酶 9 特异性地显示出全长 VWF 的优先切割。暴露于 SCD 刺激（如血红素和缺氧）的内皮细胞中，替代 VWF 切割蛋白酶的上调发生。
这是首次证明辅助血浆酶在特定疾病状态下有助于 VWF 的调节，这可能对评估其他情况下的 VWF/ADAMTS13 轴具有重要意义。
