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， 血小板将它们束缚在血管损伤部位，引发血液凝固。这个过程至关重要 对于正常止血，但特别是在炎症条件下，它被认为是病理性的主要参与者， 逻辑血栓形成。因此，VWF一直是抗血栓药物开发的靶点 治疗学然而，一个特别的挑战是如何防止病理性血栓形成，同时仍然允许- 正常的生理性血液凝固事实上，目前可用的抗血栓形成治疗剂是已知的， 副作用是引起颅内出血这里提出的工作建议，通过确定confor- 因此，设计靶向VWF的分子将是可能的 选择性地仅在炎症性促血栓形成环境中。体外实验表明 在复苏过程中释放的氧化剂增加了VWF的血小板结合活性， 与VWF内甲硫氨酸残基的氧化有关。这项研究旨在描述什么抑制 在VWF中通过蛋氨酸氧化去除机制，并确定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中的结构域， 包含血小板的结合位点）。通过 结合一个动态的流动分析，平衡解折叠实验和结合试验，我们将确定 VWF的哪些自身抑制机制是通过氧化而转变的，哪些甲硫氨酸残基是 在氧化条件下活化。这些知识将用于计算对接研究， A1结构域与其相邻结构域之间的复合物模型，并研究A1结构域的结构效应。 甲硫氨酸氧化在原子水平的细节。使用来自炎症性疾病患者的样品的测定 将用于测试炎症条件和体内VWF的较高活性之间的联系。结构性 在这里获得的见解将是非常宝贵的结构为基础的药物设计，以开发治疗的目标 VWF仅在其氧化状态时才能用于预防血栓形成，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.