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.

摘要 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机制发生， 相互作用和结构改变。最终，αIIbβ3成熟其对纤维蛋白原和血管性血友病的亲和力 因子αIIbβ3亲和力成熟事件中的失调导致Glanzmann血栓无力症或 血小板减少症因此，通过αIIbβ3受体微调这些相互作用是一种已被证明有效的方法。 治疗性血栓形成病理靶向和免疫治疗策略。目前，没有αIIbβ3 长期临床使用的抑制剂，而目前短期使用的抑制剂可能会导致严重的 血小板减少症。我们研究的总体目标是了解αIIbβ3的分子基础。 变形和机械化学的αIIbβ3激活和抑制的小分子，包括临床- 毒品我们的指导假设是，多种形式的αIIbβ3存在于动态构象平衡中， 在时间和空间上受细胞信号传导、与细胞骨架的结合以及与 细胞外配体我们将首次确定完整αIIbβ3的单粒子cryoEM结构， 在原子水平上表征与生理配体相关的结构转变（例如，纤维蛋白原），和 确定临床相关拮抗剂对αIIbβ3构象平衡的影响，无论是在原位还是在体外， 体外所有具体目标将在分子水平上阐述与这些目标相关的整合素αIIbβ3事件。 目的1实验将表征由配体或临床- 相关药物。我们将研究纯化的全长αIIbβ3的构象动力学 使用单粒子cryoEM方法和广泛的生化技术的蛋白质溶液。 目的2实验将通过探测完整αIIbβ3的构象状态来研究其结构变化。 细胞表面使用构象报告单克隆抗体，并通过直接解决原位结构使用尖端 先进的冷冻ET成像技术。总的来说，我们提出的研究将提供独特的分子见解 αIIbβ3的结构和双向信号调节，这将促进我们对整合素的理解 生物学，并可能确定新的拮抗剂调节αIIbβ3功能。