Calpain-1 Signaling Pathways in Platelets

血小板中的 Calpain-1 信号通路

• 批准号: 8007400
• 负责人: Athar H. Chishti
• 金额: $ 41.25万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-01-21 至 2012-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8007400
• 关键词: Adaptor Signaling Protein; Agonist; Antibodies; Antigen-Antibody Complex; Apoptosis; Binding; Biological Assay; Blood Platelets; Calcium; Calpain; Calpain I; Cell Proliferation; Cell physiology; Cleaved cell; Clot retraction; Cysteine Protease; Cytoplasmic Tail; Cytoskeletal Modeling; Cytoskeletal Proteins; Cytoskeleton; Data; Defect; Development; Enzymes; Equilibrium; Evaluation; Event; Fibrinogen; Gel; Gene Deletion; Genetic; Glass; Health; Hemostatic function; In Vitro; Integrin beta3; Integrins; Kinetics; Knockout Mice; Lead; Length; Mass Spectrum Analysis; Measures; Mediating; Membrane; Modeling; Molecular; Monomeric GTP-Binding Proteins; Mus; Mutation; Myosin ATPase; PTPN1 gene; Pathway interactions; Phosphoric Monoester Hydrolases; Phosphorylation; Physiology; Platelet Activation; Platelet aggregation; Predisposition; Protein Dephosphorylation; Protein Tyrosine Phosphatase; Proteins; Proteolysis; Recombinant Fusion Proteins; Recombinants; Regulation; Role; SRC gene; Signal Pathway; Signal Transduction; Structure; Surface; Technology; Testing; Thrombin; Thrombosis; Two-Dimensional Gel Electrophoresis; Tyrosine; Tyrosine Phosphorylation; adapter protein; base; cell motility; enzyme activity; gel electrophoresis; in vivo; insight; mouse model; mutant; novel; novel therapeutics; polypeptide; protein activation; protein protein interaction; research study; response; stoichiometry

DESCRIPTION (provided by applicant): Calpains are cysteine proteases implicated in a wide variety of cellular processes such as cytoskeletal organization, cell proliferation, apoptosis, cell motility, and hemostasis. We propose to test the hypothesis that calpain-1 regulates platelet signaling by modulating the activity of PTP1B under the following specific aims: (1) Regulation of PTP1B by calpain-1. We will determine the status of PTP1B cleavage in the wild type and calpain-1 null mouse platelets upon activation using domain-specific antibodies and immune complex phosphatase assays. We will measure the degradation of PTP1B and specific activity of degraded fragments and investigate the effect of PTP1B on c-Src activation in platelets. These studies will help to clarify the role of calpain-1 in the regulation of PTP1B and provide an explanation for the increased tyrosine phosphatase activity in calpain-I null mouse platelets. (2) Mechanism of calpain-1 dependent beta-3 integrin interactions with the platelet cytoskeleton. We plan to determine the identity of PTP1B substrates in platelets by gel electrophoresis and mass spectrometry. Using beta-3 integrin cytoplasmic domain constructs carrying conservative and radical replacement of tyrosines at 747 and 759, we will determine their phosphorylation stoichiometry, susceptibility to dephosphorylation by PTP1B, and quantify their interactions with Shc, myosin, and newly identified PTP1B substrates. These experiments, assisted by the BIAcore technology, will help to clarify the downstream effectors of PTP1B that are critical for outside-in signaling in platelets. (3) Role of calpain-1 signaling in platelet spreading. Using calpain-1 null mice with pure C57BL/6J genetic background, we will investigate the mechanism of enhanced platelet spreading by identifying the potential PTP1B-independent substrates of calpain-1 in mouse platelets. We will also test whether the calpain-1 null mice with the pure C57BL/6J genetic background display abnormalities in the platelet secretion and in vivo thrombosis status. Together, these studies will help to clarify the role of calpain-1 in integrating common and divergent aspects of platelet signaling, reveal novel aspects of PTP1B regulation, and provide a better understanding of the mechanism of platelet aggregation and clot retraction pathways. Ultimately, these studies may lead to the development of new therapeutic strategies against thrombosis. PUBLIC HEALTH RELEVANCE: Calpains are cysteine proteases implicated in a wide variety of cellular processes such as cell proliferation, apoptosis, cell motility, and hemostasis. The proposed studies will clarify the role of calpain-1 in platelet signaling and provide a better understanding of the mechanism of platelet aggregation and clot retraction pathways. Ultimately, these studies may lead to the development of new therapeutic strategies against thrombosis.

描述（由申请人提供）：钙蛋白酶是涉及多种细胞过程的半胱氨酸蛋白酶，例如细胞骨架组织、细胞增殖、细胞凋亡、细胞运动和止血。我们建议测试 calpain-1 通过调节 PTP1B 的活性来调节血小板信号传导的假设，具体目标如下：（1）calpain-1 对 PTP1B 的调节。我们将使用域特异性抗体和免疫复合物磷酸酶测定来确定野生型和 calpain-1 null 小鼠血小板激活后 PTP1B 裂解的状态。我们将测量 PTP1B 的降解情况和降解片段的比活性，并研究 PTP1B 对血小板中 c-Src 激活的影响。这些研究将有助于阐明 calpain-1 在 PTP1B 调节中的作用，并为 calpain-I 缺失小鼠血小板中酪氨酸磷酸酶活性增加提供解释。 (2) calpain-1依赖性β-3整合素与血小板细胞骨架相互作用的机制。我们计划通过凝胶电泳和质谱法确定血小板中 PTP1B 底物的身份。使用在 747 和 759 处携带酪氨酸保守和激进替换的 β-3 整联蛋白胞质结构域构建体，我们将确定它们的磷酸化化学计量、对 PTP1B 去磷酸化的敏感性，并量化它们与 Shc、肌球蛋白和新鉴定的 PTP1B 底物的相互作用。这些实验在 BIAcore 技术的协助下，将有助于阐明 PTP1B 的下游效应器，这些效应器对于血小板中由外而内的信号传导至关重要。 (3) calpain-1信号在血小板扩散中的作用。使用具有纯 C57BL/6J 遗传背景的 calpain-1 缺失小鼠，我们将通过鉴定小鼠血小板中 calpain-1 的潜在 PTP1B 独立底物来研究增强血小板扩散的机制。我们还将测试具有纯C57BL/6J遗传背景的calpain-1缺失小鼠是否表现出血小板分泌和体内血栓形成状态的异常。总之，这些研究将有助于阐明 calpain-1 在整合血小板信号传导的共同和不同方面中的作用，揭示 PTP1B 调节的新方面，并更好地理解血小板聚集和血栓回缩途径的机制。最终，这些研究可能会导致开发新的抗血栓治疗策略。公共健康相关性：钙蛋白酶是半胱氨酸蛋白酶，与细胞增殖、细胞凋亡、细胞运动和止血等多种细胞过程有关。拟议的研究将阐明 calpain-1 在血小板信号传导中的作用，并更好地理解血小板聚集和凝块收缩途径的机制。最终，这些研究可能会导致开发新的抗血栓治疗策略。