Investigation of the Cellular and Molecular Mechanisms of Thrombocyte Integrin Signaling

血小板整合素信号传导的细胞和分子机制研究

• 批准号: 10616738
• 负责人: Zhao Wang
• 金额: $ 39.66万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-05 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10616738
• 关键词: 3-Dimensional; Adaptor Signaling Protein; Affinity; Agonist; Architecture; Binding; Biochemical; Biological Assay; Biology; Blood Coagulation Disorders; Blood Platelets; Cell Surface Receptors; Cell surface; Cells; Cessation of life; Cholesterol; Classification; Clinical; Complex; Cryo-electron tomography; Cryoelectron Microscopy; Cytoskeleton; Defect; Detergents; Development; Disease; Dose; Drug Design; Drug Targeting; Drug usage; EGF gene; Equilibrium; Event; Family; Feasibility Studies; Fibrinogen; Future; Goals; Hematological Disease; Hemorrhage; Hour; Human; Hybrids; Imaging Techniques; Immunotherapy; In Situ; In Vitro; Inflammation; Integral Membrane Protein; Integrins; Investigation; Ions; Kinetics; Laboratories; Length; Licensing; Ligands; Link; Macrophage-1 Antigen; Malignant Neoplasms; Maps; Membrane; Modeling; Molecular; Molecular Conformation; Molecular Probes; Monoclonal Antibodies; Myocardial Infarction; Pathology; Pharmaceutical Preparations; Physiological; Platelet Activation; Platelet aggregation; Process; Property; Protein Structure Initiative; Proteins; Regulation; Reporting; Research; Resolution; Roentgen Rays; Role; Shapes; Signal Transduction; Specimen; Stroke; Structure; Surface; Techniques; Therapeutic; Thermodynamics; Thick; Thigh structure; Thrombasthenia; Thrombocytopenia; Thrombosis; Time; Vesicle; Visualization; antagonist; biophysical techniques; clinical effect; clinically relevant; effective therapy; experimental study; improved; in situ imaging; inhibitor; insight; microvesicles; nanodisk; nanometer resolution; novel; particle; platelet function; pre-clinical; receptor; reconstitution; skills; small molecule; structural biology; success; targeted treatment; therapeutic target; thrombotic; tomography; von Willebrand Factor

Abstract Platelet integrin αIIbβ3 is central to platelet activation, which occurs through highly complex molecular interactions and structural alteration. Ultimately, αIIbβ3 matures its affinity for fibrinogen and von Willebrand factor. Dysregulation in αIIbβ3 affinity maturation events causes Glanzmann's thrombasthenia or thrombocytopenia. Therefore, fine-tuning these interactions through the αIIbβ3 receptor is a proven and effective strategy for both therapeutic thrombotic pathology targeting and immunotherapy. Currently, there are no αIIbβ3 inhibitors for long-term clinical use, while current inhibitors for short-term use may cause serious thrombocytopenia. The overall goal of our proposed research is to understand the molecular basis of the αIIbβ3 shapeshifting and the mechanochemistry of αIIbβ3 activation and inhibition by small molecules, including clinical- drugs. Our guiding hypothesis is that multiple forms of αIIbβ3 exist in a dynamic conformational equilibrium that is temporally and spatially regulated by cell signaling, association with the cytoskeleton, and interactions with exocellular ligands. We will determine, for the first time, the single-particle cryoEM structure of intact αIIbβ3, characterize structural transitions at the atomic level as they relate to physiological ligands (e.g., fibrinogen), and determine the effects of clinically relevant antagonists on αIIbβ3 conformational equilibrium both in situ and in vitro. All specific aims will elaborate integrin αIIbβ3 events at the molecular level pertaining to these objectives. Aim 1 experiments will characterize shapeshifting structural changes of αIIbβ3 induced by ligands or clinical- relevant drugs. We will investigate the kinetics of the conformational dynamics of the purified full-length αIIbβ3 using a single-particle cryoEM approach and wide-ranging biochemical techniques for the protein in solution. Aim 2 experiments will study architectural changes in intact αIIbβ3 by probing its conformational states on the cell surfaces using conformation-reporting mAbs and by directly solving in situ structures using cutting-edge advanced cryoET imaging techniques. Collectively, our proposed studies will provide unique molecular insights into structural and bidirectional signaling regulation of αIIbβ3, which will advance our understanding of integrin biology and may identify new antagonists for modulating αIIbβ3 function.

抽象的 血小板整合素 αIIbβ3 是血小板活化的核心，血小板活化通过高度复杂的分子进行 最终，αIIbβ3 成熟其对纤维蛋白原和血管性血友病的亲和力。 αIIbβ3 亲和力成熟事件的失调会导致格兰兹曼血小板无力症或。 因此，通过 αIIbβ3 受体微调这些相互作用是一种行之有效的方法。 目前，还没有针对血栓病理学靶向和免疫治疗的策略。 临床上长期使用抑制剂，而目前的抑制剂短期使用可能会导致严重的 我们提出的研究的总体目标是了解 αIIbβ3 的分子基础。 小分子对 αIIbβ3 激活和抑制的变形和机械化学，包括临床- 我们的指导性假设是药物多种形式的 αIIbβ3 存在于动态构象平衡中， 在时间和空间上受到细胞信号传导、与细胞骨架的关联以及与 我们将首次确定完整 αIIbβ3 的单颗粒冷冻电镜结构， 在原子水平上表征与生理配体（例如纤维蛋白原）相关的结构转变，以及 确定临床相关拮抗剂对原位和原位 αIIbβ3 构象平衡的影响 所有具体目标都将在与这些目标相关的分子水平上阐述整合素 αIIbβ3 事件。 目标 1 实验将表征由配体或临床-诱导的 αIIbβ3 的变形结构变化。 我们将研究纯化的全长αIIbβ3的构象动力学。 使用单颗粒冷冻电镜方法和广泛的生化技术来分析溶液中的蛋白质。 目标 2 实验将通过探测完整 αIIbβ3 的构象状态来研究其结构变化。 使用构象报告单克隆抗体并使用尖端技术直接求解原位结构 总的来说，我们提出的研究将提供独特的分子见解。 深入研究αIIbβ3的结构和双向信号调节，这将增进我们对整合素的理解 生物学并可能鉴定出调节 αIIbβ3 功能的新拮抗剂。

项目成果

Systematic mapping of the conformational landscape and dynamism of soluble fibrinogen.

可溶性纤维蛋白原的构象景观和动态的系统绘图。

• 发表时间: 2023-06
• 作者: Pinelo, Jose E E;Manandhar, Pragya;Popovic, Grega;Ray, Katherine;Tasdelen, Mehmet F;Nguyen, Quoc;Iavarone, Anthony T;Offenbacher, Adam R;Hudson, Nathan E;Sen, Mehmet
• 通讯作者: Sen, Mehmet