New protein kinase A and G-dependent signaling pathways and networks in the regulation of platelet activation

血小板功能受激活和抑制信号通路的严格调控。环核苷酸依赖性蛋白激酶A （PKA）和G （PKG）在抑制血小板激活的重要步骤中发挥着关键作用，包括粘附、形状改变、整合素激活、颗粒释放、聚集和促凝活性。然而，我们对PKA/ pkg依赖性抑制信号调节不同血小板功能的复杂网络的理解是不完整的。根据我们最近发表的关于phospho-CalDAG-GEFI和整合素调控的数据[A9， A4]，我们假设新的cAMP/PKA和cGMP/PKG底物的鉴定和功能表征将确定血小板抑制的重要机制。此外，这些研究可能为选择性血小板激活或抑制提供有吸引力的新诊断和/或治疗靶点。基于初步的磷蛋白组学数据，我们打算通过建立的体外生化和功能方法来研究新发现的PKA/ pkg特异性和PKA/ pkg共同底物蛋白在抑制血小板功能中的作用，这些方法可以分析血小板中磷缺乏/模拟巨核细胞突变和敲低以及磷蛋白配体。新的PKA/ pkg共同底物亚胺- α (ENSA)的功能特征将通过生成巨核细胞和血小板特异性ENSA缺陷小鼠模型来进一步解决。基于lc - ms的定量磷蛋白组学和先进的血小板功能分析将用于全面阐明PKA或pkg介导的抑制和凝血酶或胶原介导的激活途径之间的信号串扰及其对血小板功能的影响。基于定量磷蛋白组学数据的生物信息学分析，我们旨在确定相关的抑制下游特征并建立动态血小板抑制模型。

Platelet function is tightly regulated by activatory and inhibitory signaling pathways. Cyclic nucleotide-dependent protein kinases A (PKA) and G (PKG) represent major key players in inhibition of important platelet activation steps, including adhesion, shape change, integrin activation, granule release, aggregation and pro-coagulant activity. However, our understanding of the complex network of PKA/PKG-dependent inhibitory signaling that modulates distinct platelet functions is incomplete. Based on our recently published data with phospho-CalDAG-GEFI and integrin regulation [A9, A4] we hypothesize that the identification and functional characterization of novel cAMP/PKA and cGMP/PKG substrates will identify important mechanisms of platelet inhibition. Furthermore, these studies may indicate attractive candidates for novel diagnostic and /or therapeutic targets for selective platelet activation or inhibition.Based on preliminary phosphoproteome data we intend to investigate the role of newly identified PKA/PKG-specific and PKA/PKG-common substrate proteins in inhibitory platelet function by using established biochemical and functional in vitro methods that enable analysis of phospho-deficient/-mimetic megakaryocytic mutants and knockdowns as well as phosphoprotein ligands in platelets. Functional characterization of the novel PKA/PKG-common substrate endosulfine-alpha (ENSA) will be additionally addressed by generation of a megakaryocyte- and platelet-specific ENSA-deficient mouse model. Quantitative LC-MS-based phosphoproteomic and advanced platelet function analysis will be used to comprehensively elucidate the signaling crosstalk between PKA- or PKG-mediated inhibiting, and thrombin- or collagen-mediated activating pathways and its consequences for platelet function. Based on bioinformatics analysis of quantitative phosphoproteomic data we aim to identify relevant inhibitory downstream signatures and to develop a dynamic platelet inhibitory model.