Mechanosensitive cGMP signaling in platelets and thrombus formation

Thrombotic events remain one of the main causes of death worldwide. Although several antithrombotic therapies are available, most of them bear the risk of bleeding. Nitric oxide (NO) is an important endogenous inhibitor of platelet aggregation. Recent work indicated that NO drives the generation of cGMP in platelets via the NO-sensitive guanylyl cyclase (NO-GC) in a flow/shear-sensitive manner, also referred to as mechanosensitive cGMP signaling (mechano-cGMP). In the present work, the cGMP pathway was induced in murine and human platelets via the application of the NO donor DEA/NO and/or the clinically used NO-GC stimulator riociguat. Compared to control conditions, this treatment resulted in a significant reduction of thrombus formation and a faster dissolution of preformed thrombi ex vivo. Furthermore, using a laser-induced thrombosis model in arteries of the mouse cremaster muscle, we found a faster thrombus dissolution and stabilization after treatment with riociguat in vivo. To investigate the molecular mechanism behind flow-regulated cGMP signals in platelets, we used co-immunoprecipitation followed by mass spectrometry, as well as high-resolution fluorescence microscopy. We identified a cGMP signaling complex at the human platelet membrane consisting of NO-GC, cGMP-dependent protein kinase I, and several integrins. Blocking integrin αIIbβ3 led to an attenuation of mechano-cGMP as measured by real-time imaging of cGMP signals in platelet thrombi ex vivo. Our findings suggest that integrins play a key role in mechanosensitive cGMP signaling and that pharmacological targeting of mechano-cGMP in platelets with riociguat may be a valuable strategy to limit thrombosis while avoiding life-threatening bleeding side effects.