Calpain-1 Signaling Pathways in Platelets

血小板中的 Calpain-1 信号通路

• 批准号: 7582882
• 负责人: Athar H. Chishti
• 金额: $ 39.25万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 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/7582882
• 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; Cytoskeleton; Data; Defect; Development; Enzymes; Equilibrium; Evaluation; Event; Fibrinogen; Gel; Gene Deletion; Genetic; Glass; 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; public health relevance; 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）：Calpains是半胱氨酸蛋白酶，涉及各种细胞过程，例如细胞骨架组织，细胞增殖，凋亡，细胞运动性和止血性。我们建议测试Calpain-1通过以下特定目的调节PTP1B活性来调节血小板信号的假设：（1）通过Calpain-1调节PTP1B。我们将使用域特异性抗体和免疫复合磷酸酶测定后，在激活后，我们将确定野生型PTP1B裂解的状态。我们将测量PTP1B的降解和降解片段的特定活性，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，并为增加的Calpain-I Null小鼠血小板中酪氨酸磷酸酶活性增加提供了解释。 （2）calpain-1依赖性β-3整合素与血小板细胞骨架的相互作用的机理。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。 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.这些实验在BIACORE技术的协助下，将有助于阐明PTP1B的下游效应子，这对于血小板中的外部信号至关重要。 （3）Calpain-1信号传导在血小板扩散中的作用。使用具有纯C57BL/6J遗传背景的Calpain-1 NULL小鼠，我们将通过鉴定小鼠血小板中CALPAIN-1的潜在calpain-1的潜在ptp1b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 NULL小鼠是否在血小板分泌和体内血栓形成状态中显示异常。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常见和不同方面，揭示PTP1B调节的新方面，并更好地理解血小板聚集和凝块缩回途径的机理。最终，这些研究可能导致发展针对血栓形成的新治疗策略。公共卫生相关性：Calpains是半胱氨酸蛋白酶，涉及各种细胞过程，例如细胞增殖，凋亡，细胞运动和止血。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缩回途径的机理。最终，这些研究可能导致发展针对血栓形成的新治疗策略。