Differential targeting of von Willebrand factor depending on oxidizing conditions

项目摘要/摘要 血液蛋白von Willebrand因子(Vwf)是一种大的多聚体蛋白，当被激活时，它与 血小板将他们拴在血管损伤的部位，引发血液凝固。这一过程至关重要。 对于正常的止血，特别是在fl炎症条件下，它被认为是致病-死亡的主要参与者。 合理的血栓形成。因此，VWF一直是抗血栓药物开发的靶点 治疗学。然而，一个特别的挑战是如何防止病理性血栓的形成，同时仍然允许- 生理血凝正常。事实上，目前可用的抗血栓疗法是已知的 导致颅内出血，如E侧ff等。这里介绍的工作提出，通过识别Confor- VWF在fl中经历的分子变化将有可能设计出靶向VWF的分子 选择性地仅在fl的促血栓形成环境中进行。体外实验表明， 在fl合成过程中释放的氧化剂增加了vWF的血小板结合活性，这已经 与VWF内蛋氨酸残基的氧化有关。这项研究的目的是描述什么是抑制 在vWF中，通过蛋氨酸氧化和可以最佳靶向的identifiES位点来去除机制 在氧化条件下。我们假设氧化激活了A1结构域(VWF中的结构域 包含与血小板的结合部位)，通过去除其邻近区域的掩蔽功能。穿过 结合动态flow实验、平衡去折叠实验和结合实验，我们将鉴定 VWF的哪些自身抑制机制通过氧化转变为ff，哪些蛋氨酸残基是关键 氧化条件下的活化。这些知识随后将用于计算对接研究，以创建 A1结构域与其邻近结构域之间的络合物模型，并研究了其结构效应 蛋氨酸氧化在原子水平的细节。一种使用fl炎症条件下患者样本的检测方法 将被用来测试在fl炎症条件下和体内更高的vwf活性之间的联系。结构性的 在这里获得的见解对于基于结构的药物设计将是非常宝贵的，以便开发针对 VWF只有在其处于氧化状态时才能防止血栓形成，同时允许止血。

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.