A shear-dependent NO-cGMP-cGKI cascade in platelets acts as an auto-regulatory brake of thrombosis

Fig. 1. A flow/shear-dependent NO-cGMP-cGKI cascade inhibits platelet thrombus formation ex vivo. Thrombi were formed ex vivo by perfusion of mouse blood through a flow chamber over a collagen-coated surface at a shear rate of 500 s−1. a Thrombus formation by DiOC6-labeled platelets from control mice (SM22+/Iβ; cGKI+/L−) or SM-Iβ rescue mice lacking cGKI in platelets (SM22+/Iβ; cGKIL−/L−) in the absence (left) or presence (right) of 1 μM DEA/NO. Data are shown as mean ± SEM (n = 3). Scale bars, 50 μm. b FRET/cGMP imaging in cGi500-expressing platelet thrombi superfused with 100 nM DEA/NO at a shear rate of 500 s−1. Thrombi were subjected to different shear conditions by switching the buffer flow off and on. Data are shown as mean ± SEM (n = 11 thrombi from one representative experiment out of 10 independent experiments). The cGMP concentration scale (upper right) is derived from in-cell calibration of VSMCs25. c Western blot detection of VASP phosphorylation (p-VASP) in platelets exposed to buffer without (−) or with (+) 100 nM DEA/NO for 3 min under static or flow (500 s−1) conditions. The ratio of p-VASP/VASP was determined in three experiments; GAPDH was used as loading control. d…

Fig. 2. NO-GC activity is regulated by fluid shear stress and lowers the intracellular Ca2+ concentration. FRET/cGMP imaging of cGi500-expressing platelet thrombi formed on collagen was performed under flow on (500 s−1) and off (0 s−1) conditions. a–c Thrombi were exposed to a DEA/NO (100 nM), b SPER/NO (500 nM), or c DETA/NO (10 μM). At the end of each experiment, 20 μM ODQ was applied in the presence of the respective NO donor and continuous flow. d–f Thrombi were superfused with the NO-GC stimulators d BAY 41-2272 (5 µM) or e riociguat (5 µM), or f with the NO-GC activator cinaciguat (100 nM). g DEA/NO (100 nM)-induced cGMP signals in the presence of the MRP4 inhibitor, MK-571 (1 µM). Data are shown as mean ± SEM (n = 18 thrombi in a, b, g; n = 10 thrombi in c, d; n = 15 thrombi in e, f). h Western blot analysis of NO-GC β1, cGKI, and PECAM-1 in cytosolic and membrane fractions of mouse platelets. Data are shown as mean ± SEM (n = 4). i Simultaneous imaging of cGMP (green trace) and Ca2+ (red trace) in the presence and absence of flow and 100 nM DEA/NO. These measurements were performed in the presence of 0.2 mM EGTA. Data are shown as mean ± SEM (n = 25 thrombi). j Traces between…

Fig. 3. Thrombus growth is associated with dynamic changes of intraplatelet cGMP in vivo. Platelet cGMP (black traces) and thrombus growth (red traces) were visualized by intravital spinning disk FRET microscopy of cremaster arterioles. Vessel walls are outlined by broken white lines, and white arrows indicate the direction of blood flow. a–f cGMP imaging in thrombi formed in platelet-specific sensor mice (cGi500-L2fl/fl; Pf4-Cretg/+) after mechanical vessel injury. a Sensor fluorescence confirms its specific expression in platelets. b, c cGMP concentration shown by CFP/YFP emission ratio (R = F480/F535) in a stable thrombus before (t1) and after (t2) application of 10 μM DEA/NO. The cGMP level in the entire thrombus is shown in c. The signals at time point t1 and t2 correspond to the images shown in b. d–f Endogenous cGMP and thrombus area during mechanical injury-induced thrombosis. Note that no exogenous NO was applied. The cGMP level in the periphery of the thrombus is shown in f. The signals at time point t1 and t2 correspond to the images shown in e. g–k Endogenous cGMP and thrombus area during laser-induced thrombus formation. Representative results obtained with sensor control…

Fig. 4. Increased thrombosis and thrombus dissolution time in platelet-specific NO-GC knockout mice. a–g Laser-induced thrombosis in cremaster arterioles of control mice (Ctrl) (cGi500-L2fl/fl; Pf4-Cretg/+; NO-GC β1+/fl) and platelet-specific NO-GC β1 knockout mice (NO-GC KO) (cGi500-L2fl/fl; Pf4-Cretg/+; NO-GC β1fl/fl). a Representative fluorescence (YFP) images of platelet thrombi at indicated time points after laser injury. Vessel walls are outlined by broken white lines, and white arrows indicate the direction of blood flow. Scale bars, 10 μm. b Profile of thrombus growth shown in a depicted as thrombus area over time in Ctrl (black) and NO-GC KO (red) mice. Various aspects of thrombus formation were evaluated: c Time to maximum thrombus size; d Maximal thrombus size; e Thrombus half dissolution time, i.e. the time required after peak maximum to decline to 50%; f Area under curve, which reflects integrated thrombus size over time; g Size of stabilized thrombi at the end of the experiment (300 s). Data are shown as mean ± SEM; n = 31 and 37 thrombi in b–f generated in 10 Ctrl and 7 NO-GC KO mice, respectively; n = 27 and 33 thrombi in g generated in 10 Ctrl and 7 NO-GC KO mice,…