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：摘要 血小板αIIbβ3被认为是典型的整合素，其典型特征是其在血小板膜上的表达。 几乎是瞬间从循环血小板上的非活性弯曲构象转化为 血管创伤后的配体结合构象扩展。此次全球重组是 由血小板激动剂刺激的生物化学反应引发， 由αIIb和β3胞质、跨膜和细胞外柄部分组成 域.最近的研究表明，αIIbβ3并不能代表所有的整合素， 整联蛋白的调节严格性不同。与αIIbβ3不同，一些整合素可能是 组成活跃。项目2涉及与蛋白质-蛋白质相互作用相关的主题， 维持整合素在其基础状态，在特异性目的1中的分子内相互作用和 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的结构来识别， 生物信息学方法来预测相互作用的界面“热点”。的相对重要性 然后通过在CHO细胞中表达热点突变体并在 与项目4合作产生的iPSC衍生的人巨核细胞。该实验 然后将使用一种方法将αIIbβ3与整合素αvβ3、α2β1和αvβ8进行比较， 与项目1合作，以表征PH和海滩域之间的相互作用 Nbeal 2在旨在了解灰质α颗粒缺陷发病机制的研究中的作用 血小板综合征第二组整合素约束位于跨膜结构域 界面将根据初步数据进行研究，这些数据表明β3使用不同的基序， 与αIIb和αv相互作用。新的计算方法，然后将被用来获得两个- 这些相互作用的三维动力学参数与项目3合作。最后我们 将使用高分辨率低温电子显微镜将我们的计算和实验 结果与全长整联蛋白的整体构象。具体目标2将确定和 定量评价αIIbβ3介导的纤维蛋白凝块的蛋白质-蛋白质相互作用 收缩。这些研究是基于激动剂刺激引起血小板聚集的观察结果。 钙蛋白酶活化和血小板胞质蛋白，特别是蛋白质talin的降解 以及连接αIIbβ3和肌动蛋白细胞骨架的黏着斑蛋白。拟议中的研究将检验这一假设 钙蛋白酶对塔林蛋白的切割使黏着斑蛋白结合，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在体内进行测试。 血栓模型和钙蛋白酶缺陷小鼠与项目3合作。