PI3 kinase dependent regulation of ion channels and carriers in platelets

血小板是血管损伤部位主要止血的关键，同样也是急性血栓形成和血栓性血管闭塞发展的决定性因素。血小板活化涉及几种信号传导途径，包括磷酸肌醇-3-激酶（PI 3 K）的活化，其对于活化的血小板的功能至关重要。激活最终导致胞浆Ca 2+浓度增加，这对于触发分泌、整合素激活和血栓形成是决定性的。在第一个资助期，申请人鉴定了几种PI 3 K依赖性信号通路，这是血小板激活和血栓形成所必需的。他们可以表明，PI 3 K敏感的血清和糖皮质激素诱导的激酶SGK 1调节核因子κ B（NF-κ B），这反过来又控制几种巨核细胞蛋白的表达。具体而言，NF-κ B被鉴定为通道蛋白Orai 1及其调节因子STIM 1的强大转录调节因子。Orai 1和STIM 1完成钙库操纵的钙内流（SOCE）。此外，申请人可以证明类似的PI 3 K依赖性蛋白激酶B/Akt在炎症触发的血小板活化中起决定性作用，第二个资助期应解决与PI 3 K信号通路相互作用的其他激酶的影响和调节。申请人计划产生各自的血小板特异性敲除小鼠模型，以揭示对血栓闭塞性疾病模型如中风和心肌梗死的影响。特别研究的信号分子包括酪蛋白激酶2 β（CK 2-β）、磷酸肌醇依赖性激酶1（PDK 1）、I κ激酶β（IKK-β）和p38促分裂原活化蛋白激酶α（p38 MAPK-α）。此外，将进行进一步的实验以确定在第一资助期鉴定的那些信号分子，如SGK 1、chorein、Akt 1、Akt 2和酸性鞘磷脂酶（aSM）在血栓形成中的意义。预期的实验特别说明了SGK 1在粒生物发生中的作用，SGK 1对巨核细胞和血小板中的Pendrin、Na+/H+交换器、Na+/K+ ATP酶和Na+/Ca 2+交换器的敏感性调节，以及已知与SGK 1表达强烈上调相关的疾病的血小板功能和血栓闭塞并发症（例如慢性肾衰竭中的醛固酮过多症和糖尿病中的胰岛素过多症）。应通过研究aSM敲除小鼠（smpd 1-/-）和尼曼-皮克病患者的血小板，确定酸性鞘磷脂酶（aSM）对血小板细胞膜组成和血小板功能的重要性。

Platelets are pivotal for primary hemostasis at sites of vascular injury and by the same token decisive for the development of acute thrombosis and thrombotic vascular occlusion. Platelet activation involves several signaling pathways including activation of phosphoinositide-3-kinase (PI3K), which is critically important for the function of activated platelets. Activation eventually leads to increase of cytosolic Ca2+-concentration, which is decisive for triggering of secretion, integrin activation and thrombus formation.In the first funding period the applicants identified several PI3K-dependent signaling pathways, which are required for platelet activation and thrombus formation. They could show that the PI3K-sensitive serum- and glucocorticoid-inducible kinase SGK1 regulates the nuclear factor kappa B (NF-kB), which in turn governs the expression of several megakaryocytic proteins. Specifically, NF-kB was identified as powerful transcriptional regulator of the channel protein Orai1 and its regulator STIM1. Orai1 and STIM1 accomplish store operated Ca2+-entry (SOCE). Moreover, the applicants could show that the similarly PI3K-dependent protein kinase B/Akt plays a decisive role for inflammation-triggered platelet activation.The second funding period shall address the impact and regulation of further kinases, which interact with the PI3K signaling pathway. The applicants plan to generate respective platelet specific knockout- mouse models in order to reveal the impact on thrombo-occlusive disease models such as stroke and myocardial infarction. The signaling molecules particularly studied include casein kinase 2 beta (CK2-beta), phosphoinositide-dependent kinase 1 (PDK1), I kappa kinase beta (IKK-beta) and p38 mitogen activated protein kinase alpha (p38MAPK-alpha). Moreover, further experiments will be done to the define the significance of those signaling molecules identified in the first funding period, such as SGK1, chorein, Akt1, Akt2 and acid sphingomyelinase (aSM), for the development of thrombi. The intended experiments particularly address the role of SGK1 for the granule biogenesis, SGK1-sensitive regulation of Pendrin, Na+/H+ exchanger, Na+/K+ ATPase and Na+/Ca2+ exchanger in megakaryocytes and platelets, as well as platelet function and thrombo-occlusive complications at diseases known to be associated with strong upregulation of SGK1 expression (e.g. hyperaldosteronism in chronic renal failure and hyperinsulinism in diabetes mellitus). The significance of acid sphingomyelinase (aSM) for the composition of the platelet cell membrane and the function of platelets shall be defined by studying platelets from aSM knockout mice (smpd1-/-) and from patients suffering from Niemann-Pickdisease.