Structure-Resolved Mechanistic Phenotyping of Von Willebrand Disease

项目摘要：主要止血剂血管性血友病因子 (vWF) 隔离血小板以阻止出血- ing。受到血流的流变剪切作用，vWF 的多聚纤维解开，暴露出 A1 结构域钩 它通过结合血小板 GPIb↵ 受体捕获血小板。 A1域内发生突变 在冯·维勒布兰德病 (vWD) 的所有临床分类中，最常见的遗传性人类出血性疾病 顺序，导致血浆中 vWF 的定量不足和血小板粘附的功能缺陷。中央 vWF 在疾病中的功能范式是突变改变了 vWF 对血液流变学效应的反应 流，但这些突变如何改变血小板粘附 vWF 机制的结构基础并不明确 明白了。 A1 中的 vWD 突变诱导构象变化，从而展开 A1 结构域的局部区域 所提出的 2B 型（功能获得）和 2M 型（功能丧失）vWD 表型的结构 改变两个先前未识别的 A1-GPIB↵ 结合位点 该应用程序的总体目标是破译 A1 构象紊乱在 GPIb↵ 亲和力识别中的作用。中心假设是该机制 vWD 的功能障碍不是剪切依赖性的，而是由内在的构象动力学决定的 这些假定的结合位点。为了实现我们的目标，我们将 1）确定结构性决定因素 vWF-血小板功能的获得和丧失，2) 破译结合机制，3) 开发新技术 用于检测血浆中vWF的病理构象。我们的方法是创新的，因为它利用了 氢-氘交换和交联质谱法以获得结构的高分辨率图 天然疾病预先决定了 GPIb↵ 亲和力，它采用了新的 RNA 适体分子探针，专门 结合并抑制 vWD 患者血浆 vWF 中 A1 结构域的紊乱构象。本研究项目 满足 NHLBI 指出的明确需求，以增强对 vWD 机制的了解，改进 vWD 诊断 它建立了 vWD 表型分析的新方法。拟议的研究预计将 增强对 vWD 如何影响 vWF 折叠和功能之间联系的基本科学理解 并改进对当前诊断的解释，从而提供更明智的治疗建议和 加强患者护理。

PROJECT SUMMARY: The primary hemostatic Von Willebrand Factor (vWF) sequesters platelets to arrest bleed- ing. Subject to the rheological shear of blood flow, multimeric fibers of vWF unravel exposing A1 domain hooks which capture platelets through the binding of platelet GPIb↵ receptors. Mutations within the A1 domain occur in all clinical classifications of von Willebrand disease (vWD), the most common inherited human bleeding dis- order, causing quantitative deficiencies of vWF in plasma and functional flaws in platelet adhesion. The central paradigm of vWF function in disease is that mutations alter vWF's response to the rheological effects of blood flow, but the the structural basis for how these mutations alter the mechanics of platelet adhesion to vWF is not understood. vWD mutations in A1 induce conformational changes that unfold local regions of the A1 domain structure in both type 2B (gain-of-function) and type 2M (loss-of-function) vWD phenotypes which are proposed to alter two previously unidentified A1-GPIB↵ binding sites The overall objective of this application is to decipher the role of A1 conformational disorder in GPIb↵ affinity recognition. The central hypothesis is that the mechanism of dysfunction in vWD is not shear dependent, but rather, determined by the intrinsic conformational dynamics of these putative binding sites. To accomplish our objective, we will 1) identify the structural determinants for gain and loss of vWF-platelet function, 2) decipher the binding mechanism, and 3) develop novel technologies for the detection of pathological conformations of vWF in plasma. Our approach is innovative because it utilizes hydrogen-deuterium exchange and cross-linking mass spectrometry to attain high-resolution map of how struc- tural disorder predetermines GPIb↵ affinity and it employs new RNA aptamer molecular probes that specifically bind and inhibit disordered conformations of the A1 domain within vWD patient plasma vWF. This research project addresses explicit needs, stated by the NHLBI, to enhance knowledge of vWD mechanisms, improve vWD diag- nostics, and it establishes novel methods for the phenotyping of vWD. The proposed studies are expected to enhance a basic scientific understanding of how vWD affects the linkage between folding and function of vWF and to improve interpretation of current diagnostics leading to better informed treatment recommendations and enhanced patient care.