Differential targeting of von Willebrand factor depending on oxidizing conditions

根据氧化条件对冯维勒布兰德因子进行差异化靶向

• 批准号: 10474452
• 负责人: Gianluca Interlandi
• 金额: $ 40.67万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10474452
• 关键词: Acute; Agglutination; Binding; Binding Sites; Biological Assay; Blood Platelets; Blood Proteins; Blood coagulation; Blood flow; Blood specimen; C-terminal; Chronic; Clinical; Complement Factor D; Complex; Computing Methodologies; Dangerousness; Development; Disease; Docking; Drug Design; Endothelium; Environment; Equilibrium; Event; Hemorrhage; Hemostatic Agents; Hemostatic function; In Vitro; Inflammation; Inflammatory; Knowledge; Laboratories; Lead; Link; Masks; Mass Spectrum Analysis; Methionine; Methods; Modeling; Molecular Conformation; Mutagenesis; Mutation; N-terminal; Oxidants; Oxides; Pathologic; Patients; Physiological; Process; Proteins; Research; Risk; Ristocetin; Role; Sampling; Scanning; Sepsis; Site; Structure; Surface; Testing; Therapeutic; Thrombosis; Thrombus; Work; base; clinically relevant; design; experimental study; in vivo; insight; molecular dynamics; oxidation; prevent; receptor; response; risk minimization; shear stress; side effect; therapeutic target; therapy development; thrombotic; vascular injury; von Willebrand Factor

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.

项目摘要 /摘要 血蛋白von willebrand因子（VWF）是一种大型多聚体蛋白，激活后，与 血小板将它们绑在血管损伤部位上会引起血液凝血。这个过程至关重要 对于正常的止血，尤其是在炎症条件下，它被认为是病原体的主要参与者 逻辑血栓形成。因此，VWF一直是抗血栓形成的目标 疗法。但是，一个特殊的挑战是如何防止病理血栓形成，同时仍然允许 - 正常的生理血液凝血。实际上，当前可用的抗栓性疗法已知 引起颅内出血作为侧面作用。这里提出的作品通过确定同意 VWF在炎症中经历的隔离变化将有可能设计针对VWF的分子 仅在炎症性促进性环境中有选择地。在体外进行的实验表明 在炎症期间释放的氧化剂增加了VWF的血小板结合活性，这具有 与VWF内蛋氨酸残留物的氧化有关。这项研究旨在表征什么抑制作用 通过甲基氨酸氧化去除VWF中的机制，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域（VWF中的域 通过删除其相邻域的掩蔽函数，包含与血小板的结合位点。通过 动态流测定，平衡展开实验和结合测定的组合，我们将确定 VWF的哪种自动抑制机制通过氧化而变成旋转，哪种方法保留是关键的 在氧化条件下的激活。然后，这些知识将用于计算对接研究中以创建 A1域及其相邻域之间的复合物模型，并研究 在原子水平上的蛋氨酸氧化。使用来自炎症疾病患者的样品的测定 将用于测试炎性条件和体内VWF活性较高之间的联系。结构 这里获得的见解对于基于结构的药物设计将是无价的，以开发针对的治疗 VWF仅在处于氧化状态时才能预防血栓形成同时允许止血。