Real-time analysis of cGMP signals in platelets in vitro and in vivo

环核苷酸cGMP是真核生物中的中心细胞内信号分子。在哺乳动物中，它介导一氧化氮（NO）的许多作用，包括调节血管张力和血小板活性。药理学和遗传学研究表明，NO-cGMP途径可能是抗血栓药物的一个有吸引力的靶点。然而，由于过去10年的结果相互矛盾，血小板cGMP增加是否对止血具有有益和/或有害影响是一个有争议的问题。许多以前的研究是在体外条件下进行的，可能无法完全模拟体内情况。在血小板活化过程中，可能产生具有不同功能结果的时空限制的cGMP信号。为了验证这一假设，我们将首次“观察”活血小板中的cGMP信号，并详细描述这些信号。为此，我们建立了转基因小鼠，表达基于荧光的cGMP生物传感器在血小板。cGMP信号将在流动室模型中体外血小板血栓形成之前、期间和之后以及在各种血栓形成小鼠模型中体内血小板血栓形成之前、期间和之后的尽可能生理条件下在真实的时间内可视化。这些实验的重点是分析在血栓形成过程中，cGMP的时空分布与血小板活性的功能变化之间的关系。这项研究将提高我们对哺乳动物体内血小板NO-cGMP信号转导功能和治疗相关性的理解。

The cyclic nucleotide cGMP is a central intracellular signaling molecule in eukaryotes. In mammals, it mediates many effects of nitric oxide (NO) including the regulation of vascular tone and platelet activity. Pharmacologic and genetic studies have indicated that the NO-cGMP pathway could be an attractive target for anti-thrombotic drugs. However, due to contradictory results over the last 10 years it is a controversial issue, whether an increase in platelet cGMP has beneficial and/or detrimental effects on hemostasis. Many previous studies were performed under in vitro conditions, which do probably not completely mimic the in vivo situation. It is possible that during platelet activation spatiotemporally confined cGMP signals with different functional outcomes are generated. To test this hypothesis, we will "watch" for the first time cGMP signals in living platelets and characterize these signals in detail. To this end, we have established transgenic mice that express a fluorescence-based cGMP biosensor in platelets. The cGMP signals will be visualized in real time under as physiological as possible conditions before, during and after the formation of platelet thrombi in vitroin a flow chamber model as well as in vivoin various mouse models of thrombosis. The focus of these experiments is to analyze how the spatiotemporal cGMP profile is associated with functional changes in platelet activity during thrombus formation. This study should improve our understanding of the in vivo functions and therapeutic relevance of platelet NO-cGMP signaling in mammals.