Novel Molecules in Calcium Signaling in Platelets

描述(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Ca~(2+)对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联系起来的分子(S)的性质在很大程度上尚不清楚。这项建议的目标是建立CalDAG-GEFI(CD-GEFI)作为钙离子感受器，对整合素激活、血栓素A2(TXA2)产生和颗粒释放起中心作用。CD-gef蛋白是RAS家族小GTP酶的鸟嘌呤核苷酸交换因子。它们同时受钙离子和/或甘油二酯(DAG)的调节。我们已经证明，血小板中的主要异构体CD-GEFI是依赖钙激活的RAP1和整合素1/3的中心成分。在缺乏CD-GEFI的情况下，整合素的激活需要通过蛋白激酶C(PKC)和GAI偶联的ADP受体P2Y12来传递信号。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，依赖PKC/P2Y12的途径不能支持CD-GEFI-/-小鼠在动脉血流条件下的血栓形成。通过目前的研究，我们的目标是了解调控CD-GEFI依赖和非依赖的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在钙依赖的TXA2生成和颗粒释放中起什么作用，以及它如何与PKC和PI3激酶等成熟的信号通路联系？我们的假设是，CD-GEFI分别通过Rap1/2介导的ERK信号通路和小的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介导了TXA2的第一波释放，为PKC介导的颗粒释放提供了必要的反馈。PI3激酶参与依赖CD-GEFI和P2Y12的RAP1/2激活，这取决于激动剂和激活血小板的机制。第二，CD-GEFI在血小板中的功能是如何调节的？我们假设CD-GEFI是一种高亲和力的血小板内钙离子感受器，它不依赖于DAG与其C1域的结合(与CD-gef家族的其他成员不同)。我们进一步提出，在血小板激活过程中，CD-GEFI向质膜的移位依赖于它与细胞骨架的直接联系，CD-GEFI作为RAP1/2的适配器。我们将通过对血小板进行结构-功能研究来验证这些假设。第三，在流动状态下，允许CD-GEFI非依赖性血小板黏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，我们将检验我们的假设，即当血栓形成是由凝血酶在低切变条件下驱动时，例如在静脉血栓形成模型中，CD-GEFI非依赖性黏附在体内是相关的。我们有强有力的初步数据支持上述每一个具体目标。综上所述，我们的研究将确定CD-GEFI是一种中央感受器，将钙离子动员与整合素激活、TXA2产生和颗粒释放联系起来。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确立为抗血小板治疗的靶点。与公共健康相关：这项拟议的研究调查了血小板中钙信号的机制，重点是CalDAG-GEFI作为钙传感器的作用，它是整合素激活、血栓素A2产生和颗粒释放的中心。我们的工作将对更好地了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，并可能导致识别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.