Differential targeting of von Willebrand factor depending on oxidizing conditions

项目概要/摘要 血液蛋白血管性血友病因子 (VWF) 是一种大的多聚体蛋白，当被激活时，它会结合到 血小板将它们束缚在血管损伤部位，引发血液凝固。这个过程很关键 对于正常止血，但特别是在炎症条件下，它被认为是病理过程中的主要参与者。 逻辑血栓形成。因此，VWF一直是抗血栓药物开发的目标 疗法。然而，一个特殊的挑战是如何预防病理性血栓形成，同时仍然允许- 正常生理凝血。事实上，目前可用的抗血栓治疗方法是已知的 导致颅内出血的副作用。这里介绍的工作建议通过确定符合 鉴于 VWF 在炎症中发生的变化，将有可能设计针对 VWF 的分子 仅在炎症促血栓环境中选择性地发挥作用。体外实验表明 炎症过程中释放的氧化剂会增加 VWF 的血小板结合活性，这已 与 VWF 内蛋氨酸残基的氧化有关。本研究旨在描述抑制作用的特征 VWF 中的机制通过蛋氨酸氧化被消除，并确定了可以最佳靶向的位点 在氧化条件下。我们假设氧化激活了 A1 结构域（VWF 中的该结构域 包含血小板的结合位点）通过去除其邻近结构域的掩蔽功能。通过 结合动态流测定、平衡展开实验和结合测定，我们将确定 VWF 的哪些自动抑制机制通过氧化而关闭，哪些蛋氨酸残基是其关键 氧化条件下活化。然后，这些知识将用于计算对接研究，以创建 A1域及其邻近域之间的复合体模型并研究结构效应 原子级详细的蛋氨酸氧化。使用炎症患者样本进行的检测 将用于测试体内炎症状况与 VWF 较高活性之间的联系。结构性 这里获得的见解对于基于结构的药物设计非常宝贵，以便开发靶向治疗药物 VWF仅在其处于氧化状态时才能防止血栓形成，同时实现止血。

Project Summary / Abstract The blood protein von Willebrand factor (VWF) is a large multimeric protein that, when activated, binds to blood platelets tethering them to the site of vascular injury initiating blood coagulation. This process is critical for normal haemostasis, but especially under inflammatory conditions it is thought to be a major player in patho- logical thrombus formation. For this reason, VWF has been the target for the development of anti-thrombotic therapeutics. However, a particular challenge is how to prevent pathological thrombus formation while still allow- ing normal physiological blood coagulation. In fact, currently available anti-thrombotic therapeutics are known to cause intracranial bleeding as side effect. The work presented here proposes that by identifying the confor- mational changes that VWF undergoes in inflammation it will be possible to design molecules that target VWF selectively only in an inflammatory pro-thrombotic environment. Experiments performed in vitro have indicated that oxidizing agents released during inflammation increase the platelet-binding activity of VWF, and this has been linked to the oxidation of methionine residues within VWF. This study aims to characterize what inhibitory mechanisms are removed in VWF through methionine oxidation and identifies sites that can be targeted optimally under oxidizing conditions. We hypothesize that oxidation activates the A1 domain (the domain in VWF that contains the binding site to platelets) by removing the masking function of its neighboring domains. Through a combination of a dynamic flow assay, equilibrium unfolding experiments and a binding assay, we will identify which auto-inhibitory mechanisms of VWF are turned off by oxidation and which methionine residues are key for activation under oxidizing conditions. This knowledge will then be used in computational docking studies to create a model of the complex between the A1 domain and its neighboring domains and study the structural effects of methionine oxidation at atomic level of detail. An assay using samples from patients with inflammatory conditions will be used to test the link between inflammatory conditions and higher activity of VWF in vivo. The structural insights gained here will be invaluable to structure-based drug design in order to develop therapeutics that target VWF only when it is in its oxidized state in order to prevent thrombosis while allowing haemostasis.