Real-time analysis of cGMP signals in platelets in vitro and in vivo
Final Report Abstract
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 15 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 with regard to platelet interactions with immobilized substrates, other cell types, blood flow, and shear forces that affect platelet activity under in vivo conditions. It is possible that during platelet activation, spatiotemporally confined cGMP signals with different functional outcomes are generated. To test this hypothesis, we have established transgenic mice that express a fluorescence-based cGMP biosensor in platelets. These mice enabled us to “watch” for the first time cGMP signals in real time in living platelets subjected to physiological and pathophysiological flow conditions. Surprisingly, we discovered that NO-induced cGMP signals in platelets are strongly flow/shear-dependent. Mechanosensitive cGMP signaling was detected ex vivo in aggregated platelets in a flow chamber as well as in vivo during arterial thrombosis. Correlative profiling of cGMP dynamics and thrombus formation indicated that high cGMP concentrations in shear-exposed platelets at the thrombus periphery limit thrombosis. We propose that an increase in shear stress during thrombus growth activates the cGMP pathway in platelets, which acts as an autoregulatory brake to prevent vessel occlusion, while preserving wound closure under low shear. This work has improved our understanding of how cGMP controls platelet activity under in vivo conditions and how we could treat thrombotic conditions with cGMP-modulating drugs. Future studies are necessary to dissect the molecular mechanism behind mechanosensitive cGMP signaling in platelets and probably many other cell types, in particular how mechanical force is converted into a biochemical cGMP signal. The main publication resulting from this project has been covered in a press release of the University of Tübingen. It was received with great interest by the general public, which is reflected by reports in multiple news outlets and internet platforms.
Publications
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(2015). Intercellular signaling via cyclic GMP diffusion through gap junctions restarts meiosis in mouse ovarian follicles. Proc Natl Acad Sci USA 112, 5527-5532
Shuhaibar L.C., Egbert J.R., Norris R.P., Lampe P.D., Nikolaev V.O., Thunemann M., Wen L., Feil R., Jaffe L.A.
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(2015). Platelet-derived HMGB1 is a critical mediator for thrombosis. J Clin Invest 125, 4638-4654
Vogel S., Bodenstein R., Chen Q., Feil S., Feil R., Rheinlaender J., Schäffer T., Bohn E., Frick J.S., Borst O., Münzer P., Walker B., Markel J., Csanyi G., Pagano P.J, Loughran P., Jessup M.E., Watkins SC., Bullock G.C., Sperry J.L., Zuckerbraun B.S., Billiar T.R., Lotze M.T., Gawaz M., Neal M.D.
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(2016). DRG axon bifurcation tolerates increased cGMP levels: the role of phosphodiesterase 2A and scavenger receptor Npr3. Eur J Neurosci 44, 2991-3000
Schmidt H., Peters S., Frank K., Wen L., Feil R., Rathjen F.G.
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(2016). Sildenafil potentiates a cGMP-dependent pathway to promote melanoma growth. Cell Reports 14, 2599-2610
Dhayade S., Kaesler S., Sinnberg T., Dobrowinski H., Peters S., Naumann U., Liu H., Hunger R.E., Thunemann M., Biedermann T., Schittek B., Simon H.-U., Feil S., Feil R.
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(2018). A shear-dependent NO-cGMP-cGKI cascade in platelets acts as an auto-regulatory brake of thrombosis. Nat Commun 9, 4301
Wen L., Feil S., Wolters M., Thunemann M., Regler F., Schmidt K., Friebe A., Olbrich M., Langer H., Gawaz M., de Wit C., Feil R.
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(2018). cGMP imaging in brain slices reveals brain region-specific activity of NO-sensitive guanylyl cyclases (NO-GCs) and NO-GC stimulators. Int J Mol Sci 19, E2313
Peters S., Paolillo M., Mergia E., Koesling D., Kennel L., Schmidtko A., Russwurm M., Feil R.
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(2018). Chronic linaclotide treatment reduces colitis-induced neuroplasticity and reverses persistent bladder dysfunction. JCI Insight 3, e121841
Grundy L., Harrington A.M., Castro J., Garcia-Caraballo S., Deiteren A., Maddern J., Rychkov G.Y., Ge P., Peters S., Feil R., Miller P., Ghetti A., Hannig G., Kurtz C.B., Silos-Santiago I., Brierley S.M.
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(2018). Real-time imaging reveals augmentation of glutamate-induced Ca2+ transients by the NO-cGMP pathway in cerebellar granule neurons. Int J Mol Sci 19, E2185
Paolillo M., Peters S., Schramm A., Schlossmann J., Feil R.
