Novel Molecules in Calcium Signaling in Platelets

描述（由申请人提供）：血小板在许多病理生理过程中都很重要，包括血栓形成、出血、炎症和癌症。第二信使Ca2+对血小板活化的几个方面至关重要。然而，连接钙动员和调节血小板激活的信号通路的分子的性质在很大程度上是未知的。本提案的目标是建立CalDAG-GEFI （CD- GEFI）作为Ca2+传感器，是整合素激活、血栓素A2 （TxA2）生成和颗粒释放的核心。CD-GEF蛋白是Ras家族小gtpase的鸟嘌呤核苷酸交换因子。它们由Ca2+和/或二酰基甘油（DAG）调节。我们已经证明CD-GEFI，血小板中的主要亚型，是Ca2+依赖性激活Rap1和¿1/¿3整合素的核心成分。在CD-GEFI缺失的情况下，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标。出乎意料的是，在CD-GEFI-/-小鼠动脉血流条件下，PKC/ p2y12依赖通路不能支持血栓形成。在目前的研究中，我们旨在了解CD-GEFI依赖性和非依赖性血小板血栓形成的关键变量。三个主要的未解决的问题将被提出。首先，CD-GEFI在Ca2+依赖性TxA2生成和颗粒释放中的作用是什么，以及它如何与PKC和PI3激酶等成熟的信号通路进行通信？我们假设CD-GEFI分别通过rap1 /2介导的ERK MAP激酶和小GTPase Rac1的激活直接影响TxA2的生成和ADP的释放。在被弱激动剂激活的血小板中，CD-GEFI介导TxA2释放的第一波，这为PKC介导的颗粒释放提供了必要的反馈。PI3激酶参与CD-GEFI-和p2y12依赖性Rap1/2活化，取决于血小板活化的激动剂和机制。第二，CD-GEFI在血小板中的功能是如何调节的？我们假设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）。我们进一步提出，在血小板活化过程中，CD-GEFI向质膜的易位取决于其与细胞骨架的直接关联，CD-GEFI作为Rap1/2的适配器。我们将通过对血小板进行结构-功能研究来检验这些假设。第三，血流过程中不依赖CD-GEFI的血小板粘附和血栓形成的条件是什么？使用流动室和活体显微镜方法，我们将验证我们的假设，即cd - gefi独立粘附在体内与低剪切条件下凝血酶驱动的血栓形成有关，例如在静脉血栓形成模型中。我们有强有力的初步数据支持上述每一个具体目标。总之，我们的研究将确定CD-GEFI作为连接Ca2+动员、整合素激活、TxA2生成和颗粒释放的中心传感器。深入分析CD-GEFI如何在体外和体内调节血小板功能将有助于将其作为抗血小板治疗的靶点。公共卫生相关性：拟议的研究调查了血小板中钙信号传导的机制，重点关注CalDAG-GEFI作为钙传感器在整合素激活、血栓素A2生成和颗粒释放中的作用。我们的工作将对更好地理解血小板和其他细胞（如白细胞或神经元）中的这些过程具有重要价值，并可能导致发现抗血小板治疗的新靶点。

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.