Figure 1.
Effects of adenosine and inosine on platelet activation.
Blood sample was incubated with saline, adenosine (2 mmol/L) or inosine (4 mmol/L) for 10 min, prior to measuring human platelet activation induced by agonists (collagen/ADP). Phosphatidylserine externalization (n = 9 experiments, panel A) and P-selectin expression (n = 6 experiments, panel B) were determined by flow cytometry. The basal bar represents the fluorescence in unstimulated sample. The graph depicts the mean ± SEM, *p<0.05 and ***p<0.001. The results presented are from 6 separate volunteers (each donors performed as single triplicates).
Figure 2.
Effects of adenosine and inosine on ADP (8 µmol/L), collagen (1.5 µg/mL), induced platelet ATP secretion (A) and aggregation (B).
PRP was pre-incubated with saline (control), adenosine (0.5 to 2 mmol/L) or inosine (1 to 4 mmol/L). After 3 min of incubation, 20 µL of agonist (ADP 8 µmol/L or collagen 1.5 µg/mL) was added to initiate platelet aggregation, which was measured for 6 min. The inhibition of the maximal platelet ATP secretion and aggregation were expressed as a percentage with respect to control (mean ± SEM; n = 6). The results presented are from 6 separate volunteers (each donors performed as single triplicates).
Figure 3.
Effects of adenosine and inosine on collagen-induced platelet adhesion and aggregation under arterial flow conditions.
Panel A shows the time-lapse of 10 min at 10 dyne/cm2. Panel B shows the surface covered by platelets expressed as the percentage of the total surface observed, values are mean ± SEM; n = 6. **p<0.01 and ***p<0.001. The results presented are from 6 separate volunteers (each donors performed as single triplicates).
Figure 4.
Effect of adenosine receptor A2A antagonist (ZM241385) and adenylyl cyclase inhibitor (SQ22536) on ADP-induced platelet aggregation.
PRP suspension was incubated with ADP, adenosine or inosine plus ADP or pretreated with SQ22536 or ZM241385 for 3 min, followed by addition of adenosine or inosine and ADP. The graph depicts the mean ± SEM of n = 6 experiments. ***p<0.001. The results presented are from 6 separate volunteers (each donors performed as single triplicates).
Figure 5.
Effects of adenosine and inosine on intraplatelet levels of cAMP.
Platelets were incubated with PGE1 (0.02 mmol/L, positive control), adenosine (0.5 to 2 mmol/L) or inosine (1 to 4 mmol/L) for measurement of cAMP formations as described in Materials and methods. ***p<0.001 as compared with resting platelets (n = 6). The results presented are from 6 separate volunteers (each donors performed as single triplicates).
Figure 6.
Molecular modeling of adenosine and inosine on adenosine receptor A2A. (top) Molecular conformations of adenosine (green) and inosine (cyan) obtained using docking inside adenosine receptor A2A binding pocket are represented.
The X-ray reference structure of adenosine is represented in purple (PDB code: 2YDO). A comparison between the conformations reflects that inosine and adenosine adopt the same binding orientation inside adenosine receptor A2A (bottom). Alignment of the A2A motifs that contain residues Glu169 and Asn253 with A2B, A1, and A3.
Figure 7.
Adenosine and inosine inhibited arterial thrombosis formation.
Panel A shows thrombus formation after laser irradiation in the saline control group (n = 6); ASA (acetylsalicylic acid) (200 mg/kg, n = 6); adenosine (200 mg/kg, n = 6) and inosine (200 mg/kg, n = 6). The figures below each photograph represent the percentages of occlusions caused by thrombus formation. Panel B shows the time course changes of thrombus size (µm2). Panel C shows maximum percentage of occlusion at 60 min after laser irradiation. For panels A and C the percentages of occlusions were calculated using the percentage of a certain area where the occlusion is greater. The graph depicts the mean± SEM of n = 6 experiments. *p<0.05, **p<0.01 and ***p<0.001.
Figure 8.
Effect of adenosine and inosine on release of sCD40L from platelets.
Washed platelets were incubated with saline, adenosine, inosine or ASA (acetylsalicylic acid) and then stimulated with thrombin. The graph depicts the mean± SEM of n = 6 experiments. *p<0.05, **p<0.01 and ***p<0.001 as compared with the active (saline and then stimulated with thrombin). The results presented are from 6 separate volunteers (each donors performed as single triplicates).