Mechanical and biochemical regulation of von Willebrand Factor adhesion in flow

血管性血友病因子（VWF）是一种长链、多聚的血液蛋白。它粘附在损伤部位的血小板上， 形成栓塞止血。VWF功能的缺乏或不足导致血管性血友病， 最常见的遗传性出血性疾病，影响1%的美国人口。另一方面如果 血小板栓的形成发生在错误的时间或地点，它可以导致血栓形成，这可以切断 血液循环，导致中风或心脏病发作。只有在损伤严重时才能精确地打开VWF-血小板粘附。 检测到VWF，自然界已经编程VWF来感知血流的变化，并通过激活其 粘附于血小板表面的受体蛋白GPIb β。 我建议将新型微流控系统、单分子方法和特殊荧光联合收割机 技术，以提高我们对调节VWF功能的生化和机械因素的理解 通过流动。使用我开发的先进技术，我们通过流动拉伸单个VWF多聚体， 并首次直接可视化了它们的力激活构象转变和激活。这些 测量结果显示，VWF多聚体首先伸长，然后激活其GPIb结合位点， 在流动下的力量。在此基础上，我将回答与VWF的机械调节相关的三个问题。 首先，我将测量VWF中的流动诱导力如何调节ADAMTS 13蛋白酶对其的降解。这 降解过程限制了VWF的大小以降低VWF的凝血潜力。我将测试一个“分子拉链” 结合模型，其中ADAMT 13的一端几乎不与球状VWF的D4-CK结构域结合， 一旦A2结构域解折叠，进一步结合传播到蛋白水解位点。我也会决定 在GPIb κ B或血小板存在下ADAMTS 13裂解的量。第二，我将检验一个假设， 活性氧（ROS）和无细胞血红蛋白直接与 VWF调节其功能。我将测量在以下情况下流动诱导的VWF粘附的增强： 这些化学信号第三，我将揭示如何在破裂或狭窄的血管中发现伸长流， 激活自由循环的VWF和GPIb之间的粘附，以增加血栓形成。我会完成 这是通过结合高流速交叉槽微流体系统，共聚焦显微镜和特殊的 荧光技术来测量VWF对伸长流动变化的时间依赖性响应。 这项工作将提高对VWF的生化和机械调控的理解， 血管中的线索我的定量方法和先进的仪器将带来的见解， 聚合物物理学和先进的单分子和荧光方法，进入血液学领域。这 学习还将使我获得生物化学、分子生物学和血液学方面的第一手经验，使我能够 今后能更好地将我的定量和物理训练运用到更多的生物学研究课题中去。

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.