Mechanical and biochemical regulation of von Willebrand Factor adhesion in flow

血管性血友病因子(VWF)是一种长的、聚合性的血液蛋白。它附着在受伤部位的血小板上，以 形成止血的塞子。VWF功能缺失或功能不全导致von Willebrand病， 最常见的遗传性出血性疾病，影响美国1%的人口。另一方面，如果 在错误的时间或地点形成血小板堵塞，可能会导致血栓形成，从而切断血栓的形成 循环，导致中风或心脏病发作。仅当受伤时才精确打开VWF-血小板黏附 检测到，大自然已经编程VWF来感知血流的变化，并通过激活其 与血小板表面受体蛋白GPIB的黏附。 我建议将新的微流控系统、单分子方法和特殊荧光相结合 提高我们对调节VWF功能的生化和机械因素的理解的技术 按流量。使用我开发的先进技术，我们已经通过流动拉伸了单个VWF倍增器 并首次直接可视化了它们的力激活构象转变和激活。这些 测量表明，vwf多聚体首先拉长并激活其与GPIB的结合部位 流中的力。在此基础上，我将回答三个与VWF的机械调节有关的问题。 首先，我将测量VWF中的流动诱导力如何调节ADAMTS13蛋白酶对其降解的影响。这 降解过程限制了VWF的大小，从而降低了VWF的凝血潜力。我要测试一种“分子拉链” 结合模型，其中ADAMT13的一端几乎不与球状VWF的D4-CK结构域结合，具有 一旦A2结构域展开，进一步结合传播到蛋白水解点。我还将决定 在GPIB或血小板存在的情况下，ADAMTS13的裂解量。第二，我将检验这一假设 活性氧物种(ROS)和无细胞血红蛋白直接与流量传感能力相结合 VWF调节其功能。我将测量在存在的情况下流动诱导的VWF粘附性的增强 这些化学线索。第三，我将揭示在破裂或狭窄的血管中如何发现延长的血流 激活自由循环血管内皮细胞因子和GPIB之间的黏附，以增强血栓形成。我会完成的 这是通过结合高流量交叉缝隙微流控系统、共聚焦显微镜和特殊的 荧光技术测量VWF对伸长流变化的依赖时间的响应。 这项工作将提高对生物化学和机械调节VWF的理解 血管中的线索。我的量化方法和先进的工具将带来来自 聚合物物理学，以及先进的单分子和荧光方法，进入血液学领域。这 学习还将使我在生物化学、分子生物学和血液学方面获得第一手经验，使我能够 更好地将我的量化和体能训练应用到未来更多的生物学研究课题中。

Von Willebrand factor (VWF) is a long, polymeric blood protein. It adheres to platelets at sites of injury to form plugs that stops bleeding. The lack or inadequacy of VWF function causes von Willebrand disease, the most common hereditary bleeding disorder, which affects 1% of the US population. On the other hand, if platelet plug formation occurs at the wrong time or place, it can lead to thrombosis, which can cut off circulation, causing a stroke or heart attack. To precisely turn on VWF-platelet adhesion only when injuries are detected, nature has programmed VWF to sense changes in blood flow and to respond by activating its adhesion to GPIb, the receptor protein on platelet surface. I propose to combine novel microfluidic systems, single-molecule methods and special fluorescence techniques to improve our understanding of the biochemical and mechanical factors regulating VWF function by flow. Using advanced techniques that I have developed, we have stretched single VWF multimers by flow and directly visualized their force-activated conformational transitions and activation for the first time. These measurements revealed that VWF multimers first elongate and then activate their binding sites for GPIb with force under flow. Building on this, I will answer three questions related to the mechanical regulation of VWF. First, I will measure how flow-induced force in VWF regulates its degradation by ADAMTS13 protease. This degradation process limits the size of VWF to lower the clotting potential of VWF. I will test a “molecular zipper” binding model, in which one end of the ADAMT13 scarcely binds to the D4-CK domains of globular VWF, with further binding propagating to the proteolysis site once the A2 domain is unfolded. I will also determine the amount of ADAMTS13 cleavage in the presence of GPIb or platelets. Second, I will test the hypothesis that reactive oxygen species (ROS) and cell-free hemoglobin directly integrate with the flow sensing capability of VWF to regulate its function. I will measure the enhancement of flow-induced VWF adhesion in the presence of these chemical cues. Third, I will reveal how elongational flow found in ruptured or narrowed blood vessels activates the adhesion between freely circulating VWF and GPIb to augment thrombosis. I will accomplish this by combining a high flow-rate cross-slot microfluidic system, a confocal microscope and special fluorescence techniques to measure the time-dependent response of VWF to changes in elongational flow. This proposed work will improve the understanding of VWF regulation by biochemical and mechanical cues in blood vessels. My quantitative approaches and advanced instrumentation will bring insights from polymer physics, and advanced single-molecule and fluorescence methods, into the field of hematology. This study will also give me firsthand experience in biochemistry, molecular biology and hematology, enabling me to better apply my quantitative and physical training to more topics in biology research in the future.