Platelet Integrin Structure and Function

血小板整合素结构和功能

• 批准号: 10434810
• 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/10434810
• 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; 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; Transmembrane Domain; Trauma; Vinculin; Work; base; cohesion; extracellular; human disease; in vivo evaluation; induced pluripotent stem cell; integrin alphavbeta8; interfacial; intermolecular interaction; mutant; novel; optical traps; protein data bank; protein protein interaction; single molecule; 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被认为是原型整合素的典型特征是 几乎瞬时的转换，从无效的便当会议上的有关循环血小板的会议到 血管创伤后的扩展配体结合考虑因素。这种全球重组是 由血小板激动剂刺激的生化反应发起的，破坏了分子内扣 由αIIB和β3胞质，跨膜和细胞外茎的部分组成 域。最近的工作表明，αIIBβ3并不代表所有整合素，并且 整联蛋白在其法规的严格性方面有所不同。与αIIBβ3不同，某些整合素可能是 组成性活动。项目2解决了与蛋白质蛋白质相互作用有关的主题 在其基本状态下维持整合素，特定目标1的分子内相互作用和互相互作用 特定目标2中的分子相互作用。在特定目标1中，分子内约束位于 αIIB和β3细胞外茎之间的界面将使用新型结构进行鉴定 预测相互作用的界面“热点”的生物信息学方法。相对重要的 然后，预测的热点将通过在CHO细胞中表达热点突变体和在 与项目4合作生产的IPSC衍生的人类巨核细胞。 然后，方法将用于将αIIBβ3与整联蛋白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 NBEAL2在旨在了解灰色缺陷的发病机理的研究中 血小板综合征。位于跨膜结构域中的第二组整联蛋白约束 界面将根据初步数据进行研究，表明β3使用不同的基序 与αIIB和αV相互作用。然后，新型计算方法将用于得出两种 与项目3合作的这些相互作用的维数动力学参数。最后，我们 将使用高分辨率的冷冻电子显微镜来关联我们的计算和实验 全长整合素的全局构象的结果。具体目标2将标识和 定量评估负责αIIBβ3介导的纤维蛋白克隆的蛋白质蛋白质相互作用 收缩。这些研究基于以下观察结果，即激动剂刺激会导致血小板 钙蛋白酶激活和血小板胞质蛋白的降解，尤其是蛋白质塔林 将αIIBβ3与肌动蛋白细胞骨架联系起来的Vinculin。拟议的研究将检验假设 钙蛋白酶的塔林裂解可以使Vinculin结合，从而产生足够的牵引力 收缩αIIBβ3结合的纤维蛋白血块。该假设也将使用鼠标在体内进行测试 与项目3合作的血栓形成模型和钙蛋白酶缺陷小鼠。