Novel Molecules in Calcium Signaling in Platelets

血小板钙信号传导的新分子

• 批准号: 8323641
• 负责人: Wolfgang Bergmeier
• 金额: $ 32.03万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-05 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8323641
• 关键词: 1,2-diacylglycerol; 1-Phosphatidylinositol 3-Kinase; ADP Receptors; Adhesions; Affect; Affinity; Agonist; Binding; Biology; Blood Platelets; Calcium; Calcium Signaling; Cell membrane; Cells; Coupled; Cytoplasmic Granules; Cytoskeleton; DAG/PE-Binding Domain; Data; Dependency; Diglycerides; Event; Extracellular Signal Regulated Kinases; Family; Feedback; Generations; Goals; Health; Hemorrhage; In Vitro; Individual; Inflammation; Integrins; Kinetics; Lead; Leukocytes; Link; Malignant Neoplasms; Mediating; Mediator of activation protein; Modeling; Monomeric GTP-Binding Proteins; Mus; Nature; Neurons; Pathway interactions; Pharmaceutical Preparations; Phosphorylation; Platelet Activation; Play; Process; Protein Isoforms; Protein Kinase C; Proteins; Regulation; Research; Role; Second Messenger Systems; Signal Pathway; Signal Transduction; Signaling Protein; Structure; Technology; Testing; Thrombin; Thrombosis; Thromboxane A2; Thrombus; Venous Thrombosis; Work; clopidogrel; in vivo; intravital microscopy; member; mutant; novel; platelet protein P47; ras Guanine Nucleotide Exchange Factors; receptor; release of sequestered calcium ion into cytoplasm; response; second messenger; sensor

DESCRIPTION (provided by applicant): Platelets are of great importance for many pathophysiological processes, including thrombosis, hemorrhage, inflammation, and cancer. The second messenger Ca2+ is critical for several facets of platelet activation. However, the nature of the molecule(s) linking calcium mobilization to the signaling pathways regulating platelet activation is largely unknown. The goal of this proposal is to establish CalDAG-GEFI (CD- GEFI) as a Ca2+ sensor that is central to integrin activation, thromboxane A2 (TxA2) generation, and granule release. CD-GEF proteins are guanine nucleotide exchange factors for Ras family small GTPases. They are regulated by both by Ca2+ and/or diacylglycerol (DAG). We have shown that CD-GEFI, the major isoform in platelets, is a central component of Ca2+-dependent activation of Rap1 and ¿1/¿3 integrins. Integrin activation in the absence of CD-GEFI required signaling by protein kinase C (PKC) and the Gai-coupled ADP receptor, P2Y12. The P2Y12 receptor is the target of one of the most successful anti-thrombotic drugs, clopidogrel. Unexpectedly, the PKC/P2Y12-dependent pathway was not able to support thrombus formation under arterial flow conditions in CD-GEFI-/- mice. With the current study, we aim to understand critical variables regulating both CD-GEFI- dependent and -independent platelet thrombosis. Three major unresolved questions will be asked. First, what is the role of CD-GEFI in Ca2+-dependent TxA2 generation and granule release, and how does it communicate with well-established signaling pathways such as PKC and PI3 kinase? It is our hypothesis that CD-GEFI directly affects TxA2 generation and ADP release through Rap1/2-mediated activation of ERK MAP kinases and the small GTPase Rac1, respectively. In platelets activated with weak agonists, CD-GEFI mediates the first wave of TxA2 release, which provides essential feedback for PKC- mediated granule release. PI3 kinase participates in CD-GEFI- and P2Y12-dependent Rap1/2 activation, depending on the agonist and mechanism of platelet activation. Second, how is CD-GEFI function regulated in platelets? We hypothesize that CD-GEFI is a high-affinity Ca2+ sensor in platelets, which does not rely on binding of DAG to its C1 domain (in contrast to other members of the CD-GEF family). We further propose that translocation of CD-GEFI to the plasma membrane during platelet activation depends on its direct association with the cytoskeleton, and that CD-GEFI serves as an adapter for Rap1/2. We will test these hypotheses by performing structure-function studies in platelets. Third, what are the conditions allowing for CD-GEFI- independent platelet adhesion and thrombus formation under flow? Using flow chamber and intravital microscopy approaches, we will test our hypothesis that CD-GEFI-independent adhesion is relevant in vivo when thrombus formation is driven by thrombin under low shear conditions, such as in venous thrombosis models. We have strong preliminary data supporting each of the above specific aims. In summary, our studies will identify CD-GEFI as a central sensor linking Ca2+ mobilization to integrin activation, TxA2 generation, and granule release. An in-depth analysis of how CD-GEFI regulates platelet function in vitro and in vivo will aid in its establishment as a target for antiplatelet therapy. PUBLIC HEALTH RELEVANCE: The proposed research investigates the mechanisms of calcium signaling in platelets, focusing on the role of CalDAG-GEFI as a calcium sensor that is central to integrin activation, thromboxane A2 generation, and granule release. Our work will be of great value for a better understanding of these processes in platelets and other cells, such as leukocytes or neurons, and it may lead to the identification of novel targets for antiplatelet therapy.

描述（由申请人提供）：血小板对于许多病理生理过程非常重要，包括血栓形成、出血、炎症和癌症。然而，第二信使Ca2+对于血小板活化的多个方面至关重要。将钙动员与调节血小板激活的信号通路联系起来很大程度上是未知的。该提案的目标是建立 CalDAG-GEFI (CD-GEFI) 作为 Ca2+ 传感器。整合素激活、血栓素 A2 (TxA2) 生成和 CD-GEF 蛋白是 Ras 家族小 GTP 酶的鸟嘌呤核苷酸交换因子，它们受到 Ca2+ 和/或二酰基甘油 (DAG) 的调节。 CD-GEFI 是血小板中的主要亚型，是 Ca2+ 依赖性 Rap1 和 ¿ 激活的核心成分1/¿ 3 整合素。在缺乏 CD-GEFI 的情况下，整合素的激活需要蛋白激酶 C (PKC) 和 Gai 偶联 ADP 受体 P2Y12 的信号传导。P2Y12 受体是最成功的抗血栓药物之一氯吡格雷的靶点。出乎意料的是，在 CD-GEFI-/- 小鼠的动脉血流条件下，PKC/P2Y12 依赖性途径无法支持血栓形成。通过当前的研究，我们的目标是了解调节 CD-GEFI 依赖性和非依赖性血小板血栓形成的关键变量，将提出三个主要未解决的问题：CD-GEFI 在 Ca2+ 依赖性 TxA2 生成和颗粒中的作用是什么。我们假设 CD-GEFI 通过 Rap1/2 介导的 TxA2 介导的激活直接影响 TxA2 的生成和 ADP 的释放？ ERK MAP 激酶和小 GTP 酶 Rac1 分别在用弱激动剂激活的血小板中，CD-GEFI 介导第一波 TxA2 释放，这为 PKC 介导的颗粒释放提供重要反馈，参与 CD-GEFI- 和 P2Y12。 - 依赖性 Rap1/2 激活，取决于血小板激活的激动剂和机制 其次，我们着迷的是血小板中的 CD-GEFI 功能是如何调节的？ CD-GEFI 是血小板中的高亲和力 Ca2+ 传感器，它不依赖于 DAG 与其 C1 结构域的结合（与 CD-GEF 家族的其他成员相反）。血小板活化过程中的质膜依赖于其与细胞骨架的直接关联，并且 CD-GEFI 作为 Rap1/2 的适配器，我们将通过在血小板中进行结构功能研究来检验这些假设。流动下不依赖CD-GEFI的血小板粘附和血栓形成？使用流动室和活体显微镜方法，我们将检验我们的假设，即当血栓形成在低剪切条件下由凝血酶驱动时，不依赖CD-GEFI的粘附在体内是相关的，例如与静脉血栓形成模型一样，我们有强有力的初步数据支持上述每个具体目标。总而言之，我们的研究将确定 CD-GEFI 作为将 Ca2+ 动员与钙离子流动联系起来的中心传感器。整合素激活、TxA2 生成和颗粒释放对 CD-GEFI 如何在体外和体内调节血小板功能的深入分析将有助于将其确立为抗血小板治疗的靶标：拟议的研究调查了其机制。血小板中钙信号传导的研究，重点关注 CalDAG-GEFI 作为钙传感器的作用，该传感器对于整合素激活、血栓素 A2 生成和释放至关重要。对于更好地了解血小板和其他细胞（例如白细胞或神经元）中的这些过程具有重要价值，并且可能导致抗血小板治疗新靶点的识别。