Mechanisms of Ca2+ regulation in platelets

胞质Ca 2+浓度的变化调节几乎所有细胞的基本过程。在血小板中，激动剂诱导的Ca 2+内流的机制仅部分了解。我们已经表明，钙池操纵的钙内流（SOCE）是血小板中Ca 2+内流的主要途径，这对ITAM/PLC γ 2诱导的细胞活化以及闭塞性动脉血栓形成和血栓炎症过程至关重要，而它对正常止血并不重要。我们已经确定了Ca 2+传感器STIM 1和SOC通道蛋白Orai 1作为血小板SOCE的关键介质，并通过Ca 2+通道蛋白Orai 1和TRPC 6表征了SOCE和ROCE之间的分子相互作用。新产生的具有STIM 2同种型（Stim 2deltaEF）功能获得性突变的小鼠将很快上市。在生物化学方法中，我们已经确定了衔接蛋白桥接整合子2（BIN 2）作为血小板中STIM 1的相互作用伴侣。用新产生的BIN 2缺陷小鼠进行的初步实验显示，激动剂诱导的Ca 2+储存耗竭存在明显缺陷，但动物血小板中的SOCE也存在明显缺陷。有趣的是，BIN 2在SOCE过程中以钙蛋白酶依赖性方式被有效切割，但该过程的功能相关性尚不清楚。在这个项目中，我们的目的是研究BIN 2和BIN 1的功能，这也是在血小板中表达，在体外和体内。我们将利用条件性BIN 2和BIN 1单和双KO小鼠进行巨核细胞生成/血小板生物发生和血小板功能的研究，以鉴定BIN蛋白的细胞功能及其（病理）生理相关性。在进一步的实验中，这些小鼠将与SOCE机制中携带确定缺陷的小鼠杂交，以进一步分析BIN同种型在血小板Ca 2+信号传导中的功能作用。使用生物化学和细胞生物学方法，我们希望识别和功能特性的BIN 2（和BIN 1）相互作用的合作伙伴，并分析钙蛋白酶依赖的BIN 2切割的潜在机制和功能意义。我们期望这些研究将为血小板中Ca 2+信号传导的分子机制提供新的见解，这也可能成为开发新型抗血栓治疗药物的基础。

Changes in the cytosolic Ca2+ concentration regulate fundamental processes in virtually all cells. In platelets, the mechanisms underlying agonist-induced Ca2+ influx are only partially understood. We have shown that store operated calcium entry (SOCE) is the principal route of Ca2+ influx in platelets that is of major importance for ITAM/PLCgamma2-induced cellular activation and thus occlusive arterial thrombus formation and thrombo-inflammatory processes whereas it is not essential for normal hemostasis. We have identified the Ca2+ sensor STIM1 and the SOC channel protein Orai1 as key mediators of platelet SOCE and characterized the molecular interaction between SOCE and ROCE through the Ca2+-channel proteins Orai1 and TRPC6. Newly generated mice with a gain-of-function mutation in the STIM2 isoform (Stim2deltaEF) will be available soon. In a biochemical approach we have identified the adapter protein Bridging Integrator 2 (BIN2) as an interaction partner of STIM1 in platelets. Initial experiments with newly generated BIN2-deficient mice revealed a pronounced defect of agonist-induced Ca2+ store depletion, but also of SOCE in the platelets of the animals. Interestingly, BIN2 is efficiently cleaved in a calpain-dependent manner in the course of SOCE, but the functional relevance of this process is unknown. In this project we aim to study the function of BIN2 and BIN1, which is also expressed in platelets, in vitro and in vivo. We will utilize conditional BIN2 and BIN1 single and double KO mice for studies on megakaryopoiesis/platelet biogenesis and platelet function to identify the cellular functions of the BIN proteins and their (patho)physiological relevance. In further experiments, these mice will be crossed with mice carrying defined defects in the SOCE machinery to further analyze the functional role of the BIN isoforms in platelet Ca2+ signaling. Using biochemical and cell biological approaches, we want to identify and functionally characterize BIN2 (and BIN1) interaction partners and analyze the underlying mechanisms and functional significance of calpain-dependent BIN2 cleavage. We expect that these studies will provide fundamental new insights into the molecular mechanisms underlying Ca2+ signaling in platelets that may also serve as a basis for the development of novel antithrombotic therapeutics.