Mechanisms of Ca2+ regulation in platelets

细胞内钙离子浓度的变化调节着几乎所有细胞的基本过程。在血小板中，激动剂诱导钙内流的机制还只有部分了解。我们已经证明，储存操作的钙内流(SOCE)是血小板内钙内流的主要途径，对于ITAM/PLCGamma2诱导的细胞激活，从而阻断动脉血栓形成和血栓炎症过程具有重要作用，而对正常止血并不是必需的。我们已经确定钙离子感受器STIM1和SOC通道蛋白Orai1是血小板SOCE的关键介质，并通过钙通道蛋白Orai1和TRPC6表征了SOCE和ROCE之间的分子相互作用。新生的具有STIM2亚型(Stim2deltaEF)功能获得突变的小鼠将很快上市。在生化方法中，我们已经确定适配器蛋白桥接整合子2(BIN2)是STIM1在血小板中的相互作用伙伴。对新生的BIN2缺陷小鼠的初步实验显示，激动剂诱导的钙储存耗尽，但动物血小板中的SOCE也存在明显的缺陷。有趣的是，在SOCE过程中，BIN2以钙蛋白依赖的方式被有效切割，但这一过程的功能相关性尚不清楚。在本项目中，我们旨在研究BIN2和BIN1在体外和体内的功能，BIN2和BIN1也在血小板中表达。我们将利用条件性BIN2和BIN1单、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，以确定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机制中存在明确缺陷的小鼠杂交，以进一步分析BIN亚型在血小板钙信号转导中的功能作用。利用生物化学和细胞生物学方法，我们想要确定和功能表征BIN2(和BIN1)相互作用伙伴，并分析依赖于钙蛋白的BIN2裂解的潜在机制和功能意义。我们期望这些研究将为研究血小板内钙信号的分子机制提供新的基础，这也可能成为开发新的抗血栓药物的基础。

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.