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: 10421216
• 负责人: Zhao Wang
• 金额: $ 41.16万
• 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/10421216
• 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; Diffusion; 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; Integrin alphaXbeta2; Integrins; Investigation; Ions; Kinetics; Laboratories; Length; 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 Glycoprotein GPIIb-IIIa Complex; 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; base; 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; 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是血小板活化的中心分子，通过高度复杂的分子发生 相互作用和结构变化。最终，αIIbβ3对纤维蛋白原和von Willebrand的亲和力成熟 因素。αIIbβ3亲和成熟事件的失调导致格兰兹曼的血栓减少或 血小板减少症。因此，通过αIIbβ3受体微调这些相互作用是被证明有效的 治疗性血栓病理靶向和免疫治疗的策略。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引起严重的 血小板减少症。我们提出的研究的总体目标是了解αIIbβ3的分子基础 变形和小分子激活和抑制αIIbβ3的机械力化学，包括临床- 毒品。我们的指导性假设是多种形式的αIIbβ3存在于动态构象平衡中 在时间和空间上受细胞信号、与细胞骨架的关联以及与细胞骨架的相互作用的调节 胞外配体。我们将首次确定完整的αIIbβ3的单粒子低温EM结构， 在原子水平上表征与生理配体(例如纤维蛋白原)有关的结构转变，以及 临床相关拮抗剂对原位和原位αIIbβ3构象平衡的影响 体外培养。所有特定的目标都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αIIbβ3在配体或临床应用中引起的变形结构变化。 相关药品。我们将研究纯化的全长αIIbβ3的构象动力学 使用单颗粒低温EM方法和广泛的生物化学技术对溶液中的蛋白质进行检测。 目的2实验将通过探测完整的αIIbβ3的构象状态来研究其结构变化 使用构象报告单抗和使用尖端直接求解原位结构的细胞表面 先进的低温冷凝器成像技术。总的来说，我们提议的研究将提供独特的分子洞察力 深入到αIIbβ3的结构和双向信号调节中，将有助于我们对整合素的理解 生物学，并可能找到新的拮抗剂来调节αIIbβ3的功能。