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被认为是典型的整合素，其典型特征是 从循环中的血小板上不活跃的弯曲构象几乎瞬间转变为 血管损伤后延长的配体结合构象。这一全球重组是 由血小板激动剂刺激的生化反应启动，破坏分子内卡环 由部分αIIb和β3胞质、跨膜和胞外柄组成 域名。最近的研究表明，αIIbβ3并不代表所有的整合素，而且各种不同的整合素 整合素在其调控的严格程度上有所不同。与αIIbβ3不同，一些整合素可能是 天生活跃的。项目2涉及与蛋白质-蛋白质相互作用有关的主题 保持整合素的基础状态，分子内的相互作用，在特定的目标1和之间 特定目标中的分子相互作用2.在特定目标1中，分子内约束位于 αIIb和β3胞外茎之间的界面将使用一种新的结构进行鉴定 预测界面“热点”的生物信息学方法。中国经济发展的相对重要性 预测的热点将通过在CHO细胞和在 与Project 4合作生产的IPSC来源的人巨核细胞。这项实验 然后将使用一种方法来比较αIIbβ3与整合素αvβ3、α2β1和αvβ8以及in 与项目1合作，描述PH域和海滩域之间的相互作用 Nbeal2在旨在了解α颗粒缺陷发病机制的研究中的应用 血小板综合征。位于跨膜区的第二组整合素限制 将根据初步数据研究界面，这些数据表明β3使用不同的基序 与αIIb和αv进行交互，然后使用新的计算方法得出两个- 这些相互作用的维度动力学参数与项目3合作。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联系起来 结果与全长整合素的整体构象一致。具体目标2将确定和 定量评估αIIbβ3介导的纤维蛋白凝块的蛋白质-蛋白质相互作用 收缩。这些研究的基础是观察到激动剂刺激会导致血小板 钙激活与血小板胞浆蛋白，特别是Talin蛋白的降解 以及将αIIbβ3连接到肌动蛋白细胞骨架的纽蛋白。拟议的研究将检验这一假设 钙蛋白被钙蛋白裂解使纽蛋白结合，从而产生足够的牵引力。 以收缩αIIbβ3结合的纤维蛋白凝块。这一假说也将在体内用小鼠进行验证 血栓模型和钙蛋白缺乏症小鼠与项目3合作。