Structure-Resolved Mechanistic Phenotyping of Von Willebrand Disease

冯维勒布兰德病的结构解析机制表型

• 批准号: 10188620
• 负责人: Matthew Auton
• 金额: $ 42.99万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10188620
• 关键词: Address; Adhesions; Adhesives; Affect; Affinity; Behavior; Binding; Binding Sites; Biological Assay; Blood; Blood Coagulation Disorders; Blood Platelets; Blood flow; Classification; Clinical; Data; Detection; Deuterium; Diagnosis; Diagnostic; Disease; Elements; Fiber; Functional disorder; Genotype; Goals; Heart Diseases; Hematological Disease; Hemorrhage; Hemostatic Agents; Human; Hydrogen; Inherited; Knowledge; Label; Lead; Lung diseases; Maps; Mass Spectrum Analysis; Measures; Mechanics; Methods; Modeling; Molecular Conformation; Molecular Probes; Mutation; National Heart, Lung, and Blood Institute; Pathologic; Pathology; Patient Care; Patients; Phenotype; Physicians; Plasma; Prevalence; Proteins; Public Health; Publishing; Pyrimidine; RNA; Recombinants; Recommendation; Reporting; Research; Research Project Grants; Resistance; Resolution; Rheology; Role; Severities; Site; Sleep Disorders; Structure; Testing; Therapeutic; Variant; Vision; aptamer; base; blood rheology; clinical diagnostics; crosslink; dimer; disease phenotype; disease-causing mutation; gain of function; improved; innovation; loss of function; new technology; novel; novel strategies; nuclease; platelet function; predictive modeling; prevent; receptor; response; stoichiometry; vascular injury; von Willebrand Disease; von Willebrand Factor

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.

项目总结：主要止血血管性血友病因子（vWF）隔离血小板以阻止出血- ing.受血液流变学剪切的影响，vWF的多聚体纤维解开，暴露A1结构域钩 其通过结合血小板GPIb β受体捕获血小板。A1结构域内的突变发生 在血管性血友病（vWD）的所有临床分类中，最常见的遗传性人类出血性疾病是 顺序，导致血浆中vWF的定量缺陷和血小板粘附的功能缺陷。中央 vWF在疾病中的功能范例是突变改变vWF对血液流变学效应的反应 然而，这些突变如何改变血小板与vWF粘附机制的结构基础并不清楚。 明白A1中的vWD突变诱导构象变化，使A1结构域的局部区域展开 2B型（功能获得）和2 M型（功能丧失）vWD表型中的结构， 改变两个以前未鉴定的艾德A1-GPIB结合位点 A1构象紊乱在GPIb亲和力识别中的作用。核心假设是， vWD中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，以获得高分辨率的地图， 先天性疾病预先决定了GPIb的亲和力，它采用了新的RNA适体分子探针， 结合并抑制vWD患者血浆vWF内A1结构域的无序构象。本研究项目 解决了明确的需求，由NHLBI，以提高知识的vWD机制，改善vWD诊断， nostics，并建立了vWD表型分型的新方法。预计拟议的研究将 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 并改善对当前诊断的解释，从而更好地提供知情的治疗建议， 加强病人护理。