Unraveling the Mechano-Regulation of Von Willebrand Factor

血管性血友病因子(VWF)是一种与止血密切相关的大糖蛋白。VWF感觉血流中的剪切流异常：在血管损伤部位，流体动力力增加，VWF延伸并随后促进血小板黏附。由于血小板堵塞的形成是初次止血的关键，VWF的缺陷或缺乏可导致严重的出血性疾病，称为von Willebrand病。在血液中，VWF以线性多聚体的形式存在，包括数量可变的二聚体亚基，形成最小的重复亚基，并通过N端二硫键连接。在静态条件下，VWF采用坍塌构象；在流体动力升高的情况下，多聚体通过分子内和分子间相互作用的开放以及VWF A2结构域的展开而延长。由于分子内的流体动力峰力与其有效长度密切相关，加长可以在更高的力下触发进一步的相互作用。我们和其他人解释了单分子力谱在VWF力传感中的一些关键事件，特别是A2结构域在~15pN处展开和~50pN以上强单体间D4结构域相互作用的解离。尽管最近取得了这些进展，但仍有许多悬而未决的问题。模拟表明，初始VWF延伸率(~1Pn)的临界力要小得多。此外，间接的实验证据指出相互作用的损失，特别是在低于AFM力谱分辨率极限的力&lt；5pN的低pH下，单体间C-结构域和弱的D4结构域相互作用的损失。最后，由于缺乏高分辨率结构，目前还没有对D4结构域介导的单体间相互作用的机制了解。我们建议将新的力谱方法与高分辨率结构信息相结合来解开VWF的力诱导激活和调节。使用新的分子连接策略，我们希望探测VWF力诱导的转变，特别是使用磁钳，它可以将力分解到毫微微牛顿范围内，并能够在很长一段时间(~分钟到几小时)内进行稳定的测量，以研究复性和再结合动力学。野生型VWF和几个突变和缺失结构的力谱将与高分辨率和低分辨率结构确定相结合，特别是使用结晶学、小角X射线散射和AFM成像。结构知识将使我们能够解释力谱学的发现，并指导其他实验的设计。结构信息结合对VWF延长和调节的关键初始步骤的直接测量将提高我们对其在止血中的作用的机械理解，并有可能指导治疗方法。

Von Willebrand Factor (VWF) is a large glycoprotein critically involved in hemostasis. VWF senses shear flow irregularities in the blood stream: at sites of vascular injury, where hydrodynamic forces are increased, VWF extends and subsequently promotes platelet adhesion. Since formation of a platelet plug is essential for primary hemostasis, defects in or deficiency of VWF can lead to severe bleeding disorders, known as von Willebrand disease. In the blood, VWF exists in the form of linear multimers comprising a variable number of dimeric subunits, which form the smallest repeating subunits and are linked via N-terminal disulfide bonds. Under static conditions, VWF adopts collapsed conformations; under elevated hydrodynamic forces, the multimers lengthen through opening of intra- and intermolecular interactions and unfolding of the VWF A2 domain. As hydrodynamic peak forces within the molecule strongly correlate with its effective length, lengthening can trigger opening of further interactions at even higher forces. We and others have elucidated some of the critical events in VWF force sensing by single-molecule force spectroscopy, in particular A2 domain unfolding at ~15 pN and the dissociation of a strong intermonomer D4 domain interaction above ~50 pN. Despite these recent advances many open questions remain. Simulations suggest much lower critical forces for initial VWF elongation (~ 1 pN). Additionally, indirect experimental evidence points to loss of interactions, in particular loss of intermonomer C-domain and weak D4 domain interactions at low pH, at forces <5 pN, below the resolution limit of AFM force spectroscopy. Finally, there is currently no mechanistic understanding of the D4 domain mediated intermonomer interactions due to a lack of high-resolution structures.We propose to combine novel force spectroscopy approaches with high-resolution structural information to unravel the force-induced activation and regulation of VWF.Using novel molecular attachment strategies we want to probe VWF force-induced transitions in particular using magnetic tweezers, which can resolve forces down to the femto-Newton range and enable stable measurements for long periods of time (~min to hours) to investigate refolding and rebinding kinetics. Force spectroscopy of wildtype VWF and several mutant and deletion constructs will be combined with high- and low-resolution structure determination, in particular using crystallography, small-angle X-ray scattering, and AFM imaging. Structural knowledge will allow us to interpret the findings from force spectroscopy and guide the design of additional experiments. Structural information combined with direct measurements of the critical initial steps of VWF elongation and regulation will improve our mechanistic understanding of its role in hemostasis and have the potential to direct therapeutic approaches.