Platelet Integrin Structure and Function

血小板整合素结构和功能

• 批准号: 10656292
• 负责人: Joel S. Bennett
• 金额: $ 76.18万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-10 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10656292
• 关键词: Actins; Address; Adhesions; Agonist; Alanine; Alpha Granule; Behavior; Binding; Biochemical Reaction; Bioinformatics; Biological Process; Blood Platelets; Blood Vessels; Calpain; Cell-Matrix Junction; Cells; Chinese Hamster Ovary Cell; Clot retraction; Coagulation Process; Collaborations; Complex; Computing Methodologies; Connective Tissue; Contracts; Cryoelectron Microscopy; Cytoskeletal Proteins; Cytoskeleton; Data; Defect; Development; Extracellular Protein; Fibrin; Fibrinogen; Fibrinogen Receptors; Future Generations; Genetic; Goals; Hemostatic Agents; Hot Spot; Human; Integrin Binding; Integrin alphaVbeta3; Integrins; Kinetics; Length; Ligand Binding; Link; Mechanics; Mediating; Megakaryocytes; Membrane; Methods; Modeling; Molecular Conformation; Mus; Myocardial Infarction; Neoplasm Metastasis; Pathogenesis; Pathologic; Pharmacology; Physiological; Physiology; Polymers; Proteins; Regulation; Research Design; Resolution; Rest; Scanning; Site; Spectrum Analysis; Stroke; Structure; Syndrome; Talin; Testing; Therapeutic; Thrombosis; Thrombus; Traction; Transfection; Transmembrane Domain; Trauma; Vinculin; Work; cohesion; extracellular; human disease; in vivo evaluation; induced pluripotent stem cell; integrin alphavbeta8; interfacial; intermolecular interaction; mutant; novel; optic trap; optical traps; protein data bank; protein protein interaction; prototype; single molecule; transmission process; two-dimensional

Project 2: Summary Platelet αIIbβ3 has been considered the prototypic integrin whose quintessential feature is its nearly instantaneous conversion from an inactive bent conformation on circulating platelets to an extended ligand binding conformation following vascular trauma. This global reorganization is initiated by platelet agonist-stimulated biochemical reactions that disrupt an intramolecular clasp composed of portions of the αIIb and β3 cytosolic, transmembrane, and extracellular stalk domains. Recent work suggests that αIIbβ3 is not representative of all integrins and that various integrins differ in the stringency of their regulation. Unlike αIIbβ3, some integrins may be constitutively active. Project 2 addresses topics related to the protein-protein interactions that maintain integrins in their basal states, intra-molecular interactions in Specific Aim 1 and inter- molecular interactions in Specific Aim 2. In Specific Aim 1, intramolecular constraints located in the interface between the αIIb and β3 extracellular stalks will be identified using a novel structural bioinformatics method to predict interacting interfacial “hot spots”. The relative importance of the predicted hot spots will then be determined by expressing hot spot mutants in CHO cells and in iPSC-derived human megakaryocytes produced in collaboration with Project 4. This experimental approach will then be used to compare αIIbβ3 to the integrins αvβ3, α2β1, and αvβ8 and in collaboration with Project 1, to characterize the interaction between the PH and BEACH domains of Nbeal2 in studies designed to understand the pathogenesis of α granule defect in the gray platelet syndrome. A second set of integrin constraints located in the transmembrane domain interface will be studied based on preliminary data indicating that β3 uses different motifs to interact with αIIb and αv. Novel computational methods will then be used to derive two- dimensional kinetic parameters for these interactions in collaboration with Project 3. Lastly, we will use high-resolution cryo-electron microscopy to correlate our computational and experimental results with the global conformation of full-length integrins. Specific Aim 2 will the identify and quantitively evaluate the protein-protein interactions responsible for αIIbβ3-mediated fibrin clot contraction. The studies are based on the observation that agonist stimulation causes platelet calpain activation and the degradation of platelet cytosolic proteins, in particular the proteins talin and vinculin that link αIIbβ3 to the actin cytoskeleton. Proposed studies will test the hypothesis that talin cleavage by calpain enables vinculin binding, thereby generating sufficient traction force to contract αIIbβ3-bound fibrin clots. This hypothesis will also be tested in vivo using mouse thrombosis models and calpain-deficient mice in collaboration with Project 3.

项目2：总结 血小板 αIIbβ3 被认为是原型整合素，其典型特征是 循环血小板上的非活性弯曲构象几乎瞬时转变为 血管创伤后配体结合构象延长。此次全球重组是 由血小板激动剂刺激的生化反应引发，破坏分子内的扣环 由 αIIb 和 β3 胞质、跨膜和胞外柄的部分组成 域。最近的研究表明 αIIbβ3 并不能代表所有整合素，并且各种整合素 整合素的不同之处在于其调控的严格程度。与 αIIbβ3 不同，一些整合素可能是 构成性活跃。项目 2 涉及与蛋白质-蛋白质相互作用相关的主题 将整合素维持在其基础状态，特定目标 1 中的分子内相互作用和分子间相互作用 具体目标 2 中的分子相互作用。在具体目标 1 中，分子内约束位于 αIIb 和 β3 细胞外柄之间的界面将使用一种新颖的结构来识别 生物信息学方法预测相互作用的界面“热点”。的相对重要性 然后通过在 CHO 细胞和细胞中表达热点突变体来确定预测的热点。 与 Project 4 合作生产的 iPSC 衍生的人类巨核细胞。该实验 然后使用该方法将 αIIbβ3 与整合素 αvβ3、α2β1 和 αvβ8 进行比较，并在 与项目 1 合作，描述 PH 和 BEACH 域之间的交互 Nbeal2 在旨在了解灰质中 α 颗粒缺陷发病机制的研究中的作用 血小板综合症。位于跨膜域的第二组整合素约束 将根据初步数据研究界面，表明β3使用不同的基序 与αIIb和αv相互作用。然后将使用新颖的计算方法来导出两个 与项目 3 合作研究这些相互作用的维度动力学参数。最后，我们 将使用高分辨率冷冻电子显微镜将我们的计算和实验关联起来 结果与全长整合素的整体构象。具体目标 2 将确定并 定量评估导致 αIIbβ3 介导的纤维蛋白凝块的蛋白质-蛋白质相互作用 收缩。这些研究基于激动剂刺激引起血小板的观察 钙蛋白酶的激活和血小板胞质蛋白的降解，特别是talin蛋白 以及将 αIIbβ3 连接到肌动蛋白细胞骨架的纽蛋白。拟议的研究将检验假设 钙蛋白酶切割踝蛋白使纽蛋白结合，从而产生足够的牵引力 收缩 αIIbβ3 结合的纤维蛋白凝块。该假设也将使用小鼠进行体内测试 与项目 3 合作开发血栓模型和钙蛋白酶缺陷小鼠。