Tischer Alexander, Moon-Tasson Laurie, Auton Matthew
Division of Hematology, Departments of Internal Medicine and Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA.
Thromb J. 2025 Jan 17;23(1):3. doi: 10.1186/s12959-025-00688-x.
The REAADS VWF activity assay is often assumed to be specific for the A1 domain, the portion of VWF that binds platelet GPIbα. We tested this assay on the A1A2A3 region of VWF with each domain expressed independently of one another and together in combination as a tri-domain. The monoclonal antibody used in this assay is found to be insensitive to the single A domains and does not recognize free A1 domains as it is often assumed. Rather, we find the assay to effectively recognize A1A2A3 with the domains together in their natural glycosylated sequence context. Furthermore, type 2M and 2B Von Willebrand Disease mutations differentially disrupt the sensitivity of the assay, indicating that mutational effects on the structure of A1 in the A1A2A3 context concomitantly disrupt the epitope of the antibody. The REAADS VWF activity assay therefore is conformationally sensitive to the native quaternary association of the A domains together and it is not specific to freely exposed A1 domains.
REAADS VWF活性测定通常被认为对A1结构域具有特异性，A1结构域是VWF中与血小板糖蛋白Ibα结合的部分。我们在VWF的A1A2A3区域对该测定进行了测试，其中每个结构域彼此独立表达，并作为一个三结构域组合在一起表达。发现该测定中使用的单克隆抗体对单个A结构域不敏感，并且不像通常所认为的那样识别游离的A1结构域。相反，我们发现该测定能够有效地识别处于天然糖基化序列背景下的A1A2A3三结构域。此外，2M型和2B型血管性血友病突变对该测定的敏感性有不同程度的破坏，这表明在A1A2A3背景下对A1结构的突变影响会同时破坏抗体的表位。因此，REAADS VWF活性测定对A结构域天然的四级缔合具有构象敏感性，并且对自由暴露的A1结构域不具有特异性。
