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

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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.