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Inhibition of platelet activation and thrombus formation by adenosine and inosine: studies on their relative contribution and molecular modeling.

Fuentes E, Pereira J, Mezzano D, Alarcón M, Caballero J, Palomo I - PLoS ONE (2014)

Bottom Line: The mechanisms of antiplatelet action of adenosine and inosine in vitro and in vivo, and their differential biological effects by molecular modeling were investigated.Adenosine and inosine reduced collagen-induced platelet adhesion and aggregate formation under flow.Therefore, adenosine and inosine may represent novel agents lowering the risk of arterial thrombosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Biochemistry and Immunohematology, Faculty of Health Sciences, Interdisciplinary Excellence Research Program on Healthy Aging (PIEI-ES), Universidad de Talca, Talca, Chile; Centro de Estudios en Alimentos Procesados (CEAP), CONICYT-Regional, Gore Maule, Talca, Chile.

ABSTRACT

Background: The inhibitory effect of adenosine on platelet aggregation is abrogated after the addition of adenosine-deaminase. Inosine is a naturally occurring nucleoside degraded from adenosine.

Objectives: The mechanisms of antiplatelet action of adenosine and inosine in vitro and in vivo, and their differential biological effects by molecular modeling were investigated.

Results: Adenosine (0.5, 1 and 2 mmol/L) inhibited phosphatidylserine exposure from 52±4% in the control group to 44±4 (p<0.05), 29±2 (p<0.01) and 20±3% (p<0.001). P-selectin expression in the presence of adenosine 0.5, 1 and 2 mmol/L was inhibited from 32±4 to 27±2 (p<0.05), 14±3 (p<0.01) and 9±3% (p<0.001), respectively. At the concentrations tested, only inosine to 4 mmol/L had effect on platelet P-selectin expression (p<0.05). Adenosine and inosine inhibited platelet aggregation and ATP release stimulated by ADP and collagen. Adenosine and inosine reduced collagen-induced platelet adhesion and aggregate formation under flow. At the same concentrations adenosine inhibited platelet aggregation, decreased the levels of sCD40L and increased intraplatelet cAMP. In addition, SQ22536 (an adenylate cyclase inhibitor) and ZM241385 (a potent adenosine receptor A2A antagonist) attenuated the effect of adenosine on platelet aggregation induced by ADP and intraplatelet level of cAMP. Adenosine and inosine significantly inhibited thrombosis formation in vivo (62±2% occlusion at 60 min [n = 6, p<0.01] and 72±1.9% occlusion at 60 min, [n = 6, p<0.05], respectively) compared with the control (98±2% occlusion at 60 min, n = 6). A2A is the adenosine receptor present in platelets; it is known that inosine is not an A2A ligand. Docking of adenosine and inosine inside A2A showed that the main difference is the formation by adenosine of an additional hydrogen bond between the NH2 of the adenine group and the residues Asn253 in H6 and Glu169 in EL2 of the A2A receptor.

Conclusion: Therefore, adenosine and inosine may represent novel agents lowering the risk of arterial thrombosis.

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Related in: MedlinePlus

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).
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pone-0112741-g005: 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).

Mentions: We investigated whether the effects of adenosine and inosine on platelet function were mediated by changes in platelet cAMP levels. As shown in Figure 5, levels of cAMP in resting platelets were significantly lower compared with PGE1 (0.02 mmol/L)-treated platelets (p<0.001). At the same concentrations adenosine significantly inhibits platelet aggregation and concentration-dependently (0.5 to 2 mmol/L) increased the intraplatelet levels of cAMP (p<0.001). On the other hand, inosine (1 to 4 mmol/L) did not show any effect on intraplatelet levels of cAMP.


Inhibition of platelet activation and thrombus formation by adenosine and inosine: studies on their relative contribution and molecular modeling.

Fuentes E, Pereira J, Mezzano D, Alarcón M, Caballero J, Palomo I - PLoS ONE (2014)

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).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4231063&req=5

pone-0112741-g005: 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).
Mentions: We investigated whether the effects of adenosine and inosine on platelet function were mediated by changes in platelet cAMP levels. As shown in Figure 5, levels of cAMP in resting platelets were significantly lower compared with PGE1 (0.02 mmol/L)-treated platelets (p<0.001). At the same concentrations adenosine significantly inhibits platelet aggregation and concentration-dependently (0.5 to 2 mmol/L) increased the intraplatelet levels of cAMP (p<0.001). On the other hand, inosine (1 to 4 mmol/L) did not show any effect on intraplatelet levels of cAMP.

Bottom Line: The mechanisms of antiplatelet action of adenosine and inosine in vitro and in vivo, and their differential biological effects by molecular modeling were investigated.Adenosine and inosine reduced collagen-induced platelet adhesion and aggregate formation under flow.Therefore, adenosine and inosine may represent novel agents lowering the risk of arterial thrombosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Biochemistry and Immunohematology, Faculty of Health Sciences, Interdisciplinary Excellence Research Program on Healthy Aging (PIEI-ES), Universidad de Talca, Talca, Chile; Centro de Estudios en Alimentos Procesados (CEAP), CONICYT-Regional, Gore Maule, Talca, Chile.

ABSTRACT

Background: The inhibitory effect of adenosine on platelet aggregation is abrogated after the addition of adenosine-deaminase. Inosine is a naturally occurring nucleoside degraded from adenosine.

Objectives: The mechanisms of antiplatelet action of adenosine and inosine in vitro and in vivo, and their differential biological effects by molecular modeling were investigated.

Results: Adenosine (0.5, 1 and 2 mmol/L) inhibited phosphatidylserine exposure from 52±4% in the control group to 44±4 (p<0.05), 29±2 (p<0.01) and 20±3% (p<0.001). P-selectin expression in the presence of adenosine 0.5, 1 and 2 mmol/L was inhibited from 32±4 to 27±2 (p<0.05), 14±3 (p<0.01) and 9±3% (p<0.001), respectively. At the concentrations tested, only inosine to 4 mmol/L had effect on platelet P-selectin expression (p<0.05). Adenosine and inosine inhibited platelet aggregation and ATP release stimulated by ADP and collagen. Adenosine and inosine reduced collagen-induced platelet adhesion and aggregate formation under flow. At the same concentrations adenosine inhibited platelet aggregation, decreased the levels of sCD40L and increased intraplatelet cAMP. In addition, SQ22536 (an adenylate cyclase inhibitor) and ZM241385 (a potent adenosine receptor A2A antagonist) attenuated the effect of adenosine on platelet aggregation induced by ADP and intraplatelet level of cAMP. Adenosine and inosine significantly inhibited thrombosis formation in vivo (62±2% occlusion at 60 min [n = 6, p<0.01] and 72±1.9% occlusion at 60 min, [n = 6, p<0.05], respectively) compared with the control (98±2% occlusion at 60 min, n = 6). A2A is the adenosine receptor present in platelets; it is known that inosine is not an A2A ligand. Docking of adenosine and inosine inside A2A showed that the main difference is the formation by adenosine of an additional hydrogen bond between the NH2 of the adenine group and the residues Asn253 in H6 and Glu169 in EL2 of the A2A receptor.

Conclusion: Therefore, adenosine and inosine may represent novel agents lowering the risk of arterial thrombosis.

Show MeSH
Related in: MedlinePlus