Structure-Resolved Mechanistic Phenotyping of Von Willebrand Disease

项目摘要：主要止血剂von Willebrand因子(VWF)隔离血小板以止血- 英。受血液fl流变性的影响，vWF的多聚体fi片段暴露出A1结构域挂钩 它通过与血小板GPIB↵受体结合来捕获血小板。A1结构域内发生突变 在所有von Willebrand病(VWD)的临床分类中，最常见的遗传性人类出血性疾病是fi。 导致血浆vWF和功能性fi在血小板黏附中的定量下降。中环 疾病中vWF功能的范例是突变改变vWF对血液流变性的反应 fl现在，但这些突变如何改变血小板与vWF黏附机制的结构基础并不是 明白了。A1中的vWD突变导致构象变化，从而展开A1结构域的局部区域 建议的2B型(功能增益)和2M型(功能丧失)vWD表型的结构 为了改变两个以前Unidentified的A1-GPIB↵结合位点，本申请的总体目标是解密 A1构象无序在GPIB↵affi识别中的作用中心假设是这种机制 VWD的功能障碍不是剪切相关的，而是由固有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)确定以下结构决定因素 VWF-血小板功能的得失，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-氚交换和交联质谱学获得高分辨率的结构图。 结构紊乱预先决定了GPIB的↵和fi的含量，它使用了新的rna适配子分子探针，特别是fi 结合和抑制vWD患者血浆vWF中A1结构域的无序构象。这项研究项目 满足NHLBI规定的明确需求，以增强对vWD机制的知识，改善vWD的诊断 并建立了vWD表型的新方法。拟议的研究预计将 加强对vWD如何影响vWF折叠和功能之间的联系的基础科学的理解(fic) 并改进对当前诊断的解释，从而获得更知情的治疗建议和 加强对病人的护理。

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.