Novel Molecules in Calcium Signaling in Platelets

描述（由申请人提供）：血小板对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。第二信使Ca2+对血小板活化的几个方面至关重要。然而，将钙动员与调节血小板活化的信号传导途径联系起来的分子的性质在很大程度上是未知的。该提案的目标是建立CalDAG-GEFI（CD-GEFI）作为对整合素活化、血栓烷A2（TxA2）生成和颗粒释放至关重要的Ca2+传感器。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Ca 2+和/或二酰基甘油（DAG）的调节。我们已经表明，CD-GEFI，血小板中的主要亚型，是Rap1和<$1/<$3整合素的Ca 2+依赖性激活的核心组成部分。在不存在CD-GEFI的情况下，整合素活化需要通过蛋白激酶C（PKC）和Gai偶联的ADP受体P2Y12进行信号传导。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，PKC/P2Y12依赖性途径不能支持CD-GEFI-/-小鼠在动脉血流条件下的血栓形成。通过目前的研究，我们旨在了解调节CD-GEFI依赖性和非依赖性血小板血栓形成的关键变量。将提出三个尚未解决的主要问题。首先，CD-GEFI在Ca2+依赖性TxA2产生和颗粒释放中的作用是什么，它如何与已建立的信号通路如PKC和PI3激酶进行通信？我们假设CD-GEFI分别通过Rap1/2介导的ERK MAP激酶和小GTdR Rac1的激活直接影响TxA2的产生和ADP的释放。在用弱激动剂活化的血小板中，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P2Y1/2-依赖性Rap1/2活化，这取决于血小板活化的激动剂和机制。第二，CD-GEFI功能在血小板中是如何调节的？我们假设CD-GEFI是血小板中的高亲和力Ca2+传感器，其不依赖于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，易位的CD-GEFI血小板活化过程中的质膜依赖于它的直接协会与细胞骨架，CD-GEFI作为一个适配器Rap1/2。我们将通过在血小板中进行结构-功能研究来验证这些假设。第三，在流动条件下，允许CD-GEFI非依赖性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，我们将测试我们的假设，即CD-GEFI独立的粘附是相关的，在体内血栓形成时，凝血酶在低剪切条件下，如在静脉血栓形成模型。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作为一个中央传感器连接Ca2+动员整合素激活，TxA2的生成，和颗粒释放。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，重点是CalDAG-GEFI作为钙传感器的作用，该传感器对整合素活化，血栓烷A2生成和颗粒释放至关重要。我们的工作对于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，并且可能导致抗血小板治疗的新靶点的鉴定。

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.