Novel Molecules in Calcium Signaling in Platelets

血小板钙信号传导的新分子

• 批准号: 8069934
• 负责人: Wolfgang Bergmeier
• 金额: $ 6.6万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 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 2011-05-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8069934
• 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（由适用提供）：对于许多病理生理过程，包括血栓形成，出血，炎症和癌症非常重要。第二个Messenger Ca2+对于血小板激活的几个方面至关重要。然而，将钙动员与调节血小板激活的信号通路连接的分子的性质在很大程度上未知。该提案的目的是将Caldag-Gefi（CD-GEFI）建立为CA2+传感器，该传感器是整联蛋白激活，Thromboxane A2（TXA2）生成和颗粒释放的核心。 CD-GEF蛋白是RAS家族小GTPase的鸟嘌呤核交换因子。它们受CA2+和/或二酰基甘油（DAG）的调节。我们已经表明，血小板中的主要同工型CD-GEFI是Ca2+依赖性rap1和1/€3整联蛋白的核心组成部分。在没有CD-GEFI的情况下，整联蛋白激活需要蛋白激酶C（PKC）和GAI耦合ADP受体P2Y12的信号传导。 P2Y12受体是最成功的抗血栓形成药物之一，氯吡格雷的靶标。出乎意料的是，PKC/P2Y12依赖性途径无法支持CD-GEFI - / - 小鼠中动脉流条件下的血栓形成。通过当前的研究，我们旨在理解控制CD-GEFI-CEFI-CHEFI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。首先，CD-GEFI在Ca2+依赖性TXA2生成和颗粒释放中的作用是什么？它如何与诸如PKC和PI3激酶等良好的信号通路进行通信？我们的假设是，CD-GEFI分别通过RAP1/2介导的ERK MAP激酶和小的GTPase Rac1直接影响TXA2生成和ADP释放。在用弱激动剂激活的血小板中，CD-GEFI介导了第一波TXA2释放，这为PKC-介导的颗粒释放提供了必不可少的反馈。 PI3激酶参与CD-GEFI-和P2Y12依赖性RAP1/2激活，具体取决于血小板激活的激动剂和机制。其次，如何在血小板中调节CD-GEFI功能？我们假设CD-GEFI是血小板中的高亲和力Ca2+传感器，它不依赖DAG与其C1结构域的结合（与CD-GEF家族的其他成员相反）。我们进一步提出，在血小板激活期间，CD-GEFI转移到质膜取决于其与细胞骨架的直接关联，并且该CD-GEFI是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。第三，在流动下允许CD-GEFI独立的血小板粘合剂和血栓形成的条件是什么？使用流动室和插入显微镜方法，我们将测试我们的假设，即当在低剪切条件下，例如在静脉血栓模型中，当血栓形成由凝血酶驱动时，凝血酶形成驱动时，与CD-GEFI依赖性粘合剂相关。我们有强大的初步数据支持上述特定目标。总而言之，我们的研究将CD-GEFI确定为中央传感器，将Ca2+动员与整合素激活，TXA2生成和颗粒释放联系起来。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其建立作为抗血小板治疗的靶标。公共卫生相关性：拟议的研究调查了血小板中钙信号的机制，重点是Caldag-Gefi作为钙激活，血栓烷A2生成和颗粒释放的钙传感器的作用。我们的工作将具有很大的价值，可以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，并且可能导致鉴定出抗血小板治疗的新靶标。