Figure 1.
Prior incubation with ODQ potentiates the inhibitory effects of BAY 60-2770 (0.001–10 µM) in human washed platelet aggregation.
Platelet suspension (1.2×108 platelets/ml) was pre-incubated for 3 min with the soluble guanylyl cyclase inhibitor ODQ (10 µM) or its correspondent vehicle DMSO (0.5% v/v). Platelets were incubated with BAY 60-2770 (0.001 to 10 µM), DMSO (0.5% v/v) or sodium nitroprusside (SNP, 3 µM) for another 3 min. Platelets were then stimulated with either collagen (2 µg/ml; Panel A) or thrombin (0.1 U/ml; Panel B) to perform the aggregation assays. Note that 1% DMSO (vehicle used for BAY 60-2770 and ODQ) had no effect on collagen- and thrombin-induced platelet aggregation. Data are shown as mean values ± SEM (n = 4–5 individuals). *p<0.05 compared with control values. #p<0.05 compared with respective values in the absence of ODQ.
Figure 2.
Original tracings showing human washed platelet aggregation stimulated with collagen or thrombin.
Platelet suspension (1.2×108 platelets/ml) was pre-incubated with the soluble guanylyl cyclase inhibitor ODQ (10 µM, 3 min). Platelets were then incubated with BAY 60-2770 (0.01 μM for collagen and 10 μM for thrombin), after which they were stimulated with either collagen (2 µg/ml) or thrombin (0.1 U/ml).
Figure 3.
Inhibitory effect of BAY 60-2770 and sodium nitroprusside (SNP) on human platelet adhesion to fibrinogen-coated plates.
Platelets (1.2×108 platelets/ml) were pre-incubated or not with ODQ (10 µM, 3 min) and subsequently with BAY 60-2770 (0.1–10 µM) or SNP (3 µM). Platelets were allowed to adhere to the wells for 30 min at 37°C. Note that 1% DMSO (vehicle used for BAY 60-2770 and ODQ) had no effect on platelet adhesion. Data are shown as percent of adhered platelets relative to the maximum adhesion in untreated platelets. Results are shown as mean ± SEM values (n = 4–6 individuals, each performed in triplicate). *p<0.05 compared with control values (in absence of BAY 60-2770 or SNP). #p<0.05 compared with respective values in absence of ODQ.
Figure 4.
Effect of BAY 60-2770 on cyclic GMP levels in human washed platelets activated with collagen or thrombin.
Platelets (1.2×108 platelet/ml) were incubated with BAY 60-2770 (0.01–3 µM for collagen; 1–10 µM for thrombin) or its correspondent vehicle (1% DMSO) in the absence and in the presence of ODQ (10 µM). Platelets were then activated with collagen (2 μg/ml; Panel A) or thrombin (0.1 U/ml; Panel B). Results represent the mean values ± SEM (n = 3 individuals, each performed in triplicate). *p<0.05 compared with control values (in absence of BAY 60-2770); #p<0.05 compared with respective values in absence of ODQ.
Table 1.
Levels of cyclic AMP in human activated platelets (1.2×108 platelet/ml) treated with BAY 60-2770 or iloprost in the absence and in the presence of the adenylyl cyclase inhibitor SQ-22536 (SQ, 100 μM).
Figure 5.
Inhibitory effect of BAY 60-2770 and sodium nitroprusside (SNP) on intracellular Ca2+ levels in human washed platelets activated with collagen or thrombin.
Platelets (1.2×108 platelets/ml) loaded with Fluorfort (10 µM) were pre-incubated or not with ODQ (10 µM, 3 min) and subsequently with BAY 60-2770 (0.01–10 µM, 3 min) or SNP (3 µM, 3 min) before addition of either collagen (2 μg/ml; Panels A and B) or thrombin (0.1 U/ml; Panels C and D). Assays were carried out in the presence of Ca2+ (1 mM) to yield total influx of Ca2+ (Panels A and C) or in Ca2+-free medium with EGTA to yield Ca2+ mobilization from internal storage sites (Panels B and D). Results represent the mean values ± SEM (n = 4–7 individuals). *p<0.05 compared with control values (in absence of BAY 60-2770 or SNP); #p<0.05 compared with respective values in absence of ODQ. †p<0.05 compared with collagen values in panels A and B.
Figure 6.
Inhibitory effect of 8-bromo-cyclic GMP on intracellular Ca2+ levels in human washed platelets activated with collagen or thrombin.
Platelets (1.2×108 platelets/ml) loaded with Fluorfort (10 µM) were pre-incubated or not with ODQ (10 µM, 3 min) and subsequently with 8-Br-cGMP (100 µM, 20 min) before addition of either collagen (2 μg/ml; Panels A and B) or thrombin (0.1 U/ml; Panels C and D). Assays were carried out in the presence of Ca2+ (1 mM) to yield total influx of Ca2+ (Panels A and C) or in Ca2+-free medium with EGTA to yield Ca+2 mobilization from internal storage sites (Panels B and D). Results represent the mean values ± SEM (n = 3–5 individuals). *p<0.01, **p<0.001 compared with respective baseline; #p<0.01 compared with respective vehicle. †p<0.01 compared with respective baseline.
Figure 7.
Inhibitory effect of BAY 60-2770 upon PAC-1 mean fluorescence (activated αIIbβ3).
Platelet suspension (20 µl; 1.2×108 platelets/ml) was pre-incubated with ODQ (10 µM) and BAY 60-2770 or respective vehicle (1% DMSO). Platelets were incubated with PAC-1 solution (FITCPAC-1; 10 µl of 25 µg/ml) or control antibody solution (FITC Mouse IgM, same dilution of PAC-1 solution). Platelets were activated with either collagen (2 μg/ml) or thrombin (0.1 U/ml). Mean fluorescence was acquired in flow cytometer (FACSCalibur) equipped with a 488 nm wavelength argon laser using the FL1 channel. Mean fluorescence was considered as a parameter to describe binding intensity to FITC labeled PAC-1. Panels A, B and C show, respectively, the fluorescence intensity in non-activated, collagen- and thrombin-activated platelets. Panels D and E show the mean fluorescence ± SEM (n = 3–4 individuals). *p<0.05 compared with untreated platelets. #p<0.05 compared with respective values in the absence of ODQ. †p<0.05 compared with respective collagen values in panel D.
Figure 8.
Protective effect of Bay 60-2770 on the ODQ-induced sGC degradation.
Isolated platelets were incubated with ODQ (10 µM) in the absence and in the presence of BAY 60-2770 (10 µM) for 2.5 h, after which protein expression of α1 (Panel A) and β1 (Panel B) subunits of sGC subunits were determined by Western blotting. Results represent the mean values ± SEM (n = 4 individuals). *p<0.05 compared with other groups.