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Combined blockade of ADP receptors and PI3-kinase p110β fully prevents platelet and leukocyte activation during hypothermic extracorporeal circulation.

Krajewski S, Kurz J, Geisler T, Peter K, Wendel HP, Straub A - PLoS ONE (2012)

Bottom Line: Under clinical conditions these processes are associated with life-threatening complications including thromboembolism and inflammation.Further inhibition of ADP-mediated effects was achieved with MRS2179.This novel finding warrants further studies and the development of suitable pharmacological agents to decrease ECC- and hypothermia-associated complications in clinical applications.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesiology and Intensive Care Medicine, University of Tübingen, Tübingen, Germany. krajewski@thg-lab.de

ABSTRACT
Extracorporeal circulation (ECC) and hypothermia are used to maintain stable circulatory parameters and improve the ischemia tolerance of patients in cardiac surgery. However, ECC and hypothermia induce activation mechanisms in platelets and leukocytes, which are mediated by the platelet agonist ADP and the phosphoinositide-3-kinase (PI3K) p110β. Under clinical conditions these processes are associated with life-threatening complications including thromboembolism and inflammation. This study analyzes effects of ADP receptor P(2)Y(12) and P(2)Y(1) blockade and PI3K p110β inhibition on platelets and granulocytes during hypothermic ECC. Human blood was treated with the P(2)Y(12) antagonist 2-MeSAMP, the P(2)Y(1) antagonist MRS2179, the PI3K p110β inhibitor TGX-221, combinations thereof, or PBS and propylene glycol (controls). Under static in vitro conditions a concentration-dependent effect regarding the inhibition of ADP-induced platelet activation was found using 2-MeSAMP or TGX-221. Further inhibition of ADP-mediated effects was achieved with MRS2179. Next, blood was circulated in an ex vivo ECC model at 28°C for 30 minutes and various platelet and granulocyte markers were investigated using flow cytometry, ELISA and platelet count analysis. GPIIb/IIIa activation induced by hypothermic ECC was inhibited using TGX-221 alone or in combination with P(2)Y blockers (p<0.05), while no effect of hypothermic ECC or antiplatelet agents on GPIIb/IIIa and GPIbα expression and von Willebrand factor binding was observed. Sole P(2)Y and PI3K blockade or a combination thereof inhibited P-selectin expression on platelets and platelet-derived microparticles during hypothermic ECC (p<0.05). P(2)Y blockade alone or combined with TGX-221 prevented ECC-induced platelet-granulocyte aggregate formation (p<0.05). Platelet adhesion to the ECC surface, platelet loss and Mac-1 expression on granulocytes were inhibited by combined P(2)Y and PI3K blockade (p<0.05). Combined blockade of P(2)Y(12), P(2)Y(1) and PI3K p110β completely inhibits hypothermic ECC-induced activation processes. This novel finding warrants further studies and the development of suitable pharmacological agents to decrease ECC- and hypothermia-associated complications in clinical applications.

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Concentration-dependent inhibition of ADP-induced P-selectin expression using 2-MeSAMP and TGX-221.Heparinized human whole blood (n = 4) was treated for 5 minutes at 37°C with (A) PBS as control, 2-MeSAMP (10 and 100 µM; “2-MeS”) or a combination (100 µM each) of 2-MeSAMP and MRS2179 and (B) propylene glycol (“PG”) as control or TGX-221 (0.5 or 2.2 µM; “TGX”). Afterwards platelets were activated using ADP (final concentration: 20 µM) and the percentage of P-selectin expressing platelets under a pre-set histogram marker was analyzed in flow cytometry using an anti-P-selectin mAb. Data are given as means and SD.
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pone-0038455-g001: Concentration-dependent inhibition of ADP-induced P-selectin expression using 2-MeSAMP and TGX-221.Heparinized human whole blood (n = 4) was treated for 5 minutes at 37°C with (A) PBS as control, 2-MeSAMP (10 and 100 µM; “2-MeS”) or a combination (100 µM each) of 2-MeSAMP and MRS2179 and (B) propylene glycol (“PG”) as control or TGX-221 (0.5 or 2.2 µM; “TGX”). Afterwards platelets were activated using ADP (final concentration: 20 µM) and the percentage of P-selectin expressing platelets under a pre-set histogram marker was analyzed in flow cytometry using an anti-P-selectin mAb. Data are given as means and SD.

Mentions: Treatment of whole blood with different concentrations of 2-MeSAMP (10 and 100 µM) showed that ADP-induced (final ADP concentration: 20 µM) P-selectin expression is more potently inhibited with higher antagonist concentrations (Figure 1A). The addition of MRS2179 (100 µM) in the 2-MeSAMP-treated group further decreased the expression of platelet P-selectin expression upon ADP activation (Figure 1A).


Combined blockade of ADP receptors and PI3-kinase p110β fully prevents platelet and leukocyte activation during hypothermic extracorporeal circulation.

Krajewski S, Kurz J, Geisler T, Peter K, Wendel HP, Straub A - PLoS ONE (2012)

Concentration-dependent inhibition of ADP-induced P-selectin expression using 2-MeSAMP and TGX-221.Heparinized human whole blood (n = 4) was treated for 5 minutes at 37°C with (A) PBS as control, 2-MeSAMP (10 and 100 µM; “2-MeS”) or a combination (100 µM each) of 2-MeSAMP and MRS2179 and (B) propylene glycol (“PG”) as control or TGX-221 (0.5 or 2.2 µM; “TGX”). Afterwards platelets were activated using ADP (final concentration: 20 µM) and the percentage of P-selectin expressing platelets under a pre-set histogram marker was analyzed in flow cytometry using an anti-P-selectin mAb. Data are given as means and SD.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3368839&req=5

pone-0038455-g001: Concentration-dependent inhibition of ADP-induced P-selectin expression using 2-MeSAMP and TGX-221.Heparinized human whole blood (n = 4) was treated for 5 minutes at 37°C with (A) PBS as control, 2-MeSAMP (10 and 100 µM; “2-MeS”) or a combination (100 µM each) of 2-MeSAMP and MRS2179 and (B) propylene glycol (“PG”) as control or TGX-221 (0.5 or 2.2 µM; “TGX”). Afterwards platelets were activated using ADP (final concentration: 20 µM) and the percentage of P-selectin expressing platelets under a pre-set histogram marker was analyzed in flow cytometry using an anti-P-selectin mAb. Data are given as means and SD.
Mentions: Treatment of whole blood with different concentrations of 2-MeSAMP (10 and 100 µM) showed that ADP-induced (final ADP concentration: 20 µM) P-selectin expression is more potently inhibited with higher antagonist concentrations (Figure 1A). The addition of MRS2179 (100 µM) in the 2-MeSAMP-treated group further decreased the expression of platelet P-selectin expression upon ADP activation (Figure 1A).

Bottom Line: Under clinical conditions these processes are associated with life-threatening complications including thromboembolism and inflammation.Further inhibition of ADP-mediated effects was achieved with MRS2179.This novel finding warrants further studies and the development of suitable pharmacological agents to decrease ECC- and hypothermia-associated complications in clinical applications.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesiology and Intensive Care Medicine, University of Tübingen, Tübingen, Germany. krajewski@thg-lab.de

ABSTRACT
Extracorporeal circulation (ECC) and hypothermia are used to maintain stable circulatory parameters and improve the ischemia tolerance of patients in cardiac surgery. However, ECC and hypothermia induce activation mechanisms in platelets and leukocytes, which are mediated by the platelet agonist ADP and the phosphoinositide-3-kinase (PI3K) p110β. Under clinical conditions these processes are associated with life-threatening complications including thromboembolism and inflammation. This study analyzes effects of ADP receptor P(2)Y(12) and P(2)Y(1) blockade and PI3K p110β inhibition on platelets and granulocytes during hypothermic ECC. Human blood was treated with the P(2)Y(12) antagonist 2-MeSAMP, the P(2)Y(1) antagonist MRS2179, the PI3K p110β inhibitor TGX-221, combinations thereof, or PBS and propylene glycol (controls). Under static in vitro conditions a concentration-dependent effect regarding the inhibition of ADP-induced platelet activation was found using 2-MeSAMP or TGX-221. Further inhibition of ADP-mediated effects was achieved with MRS2179. Next, blood was circulated in an ex vivo ECC model at 28°C for 30 minutes and various platelet and granulocyte markers were investigated using flow cytometry, ELISA and platelet count analysis. GPIIb/IIIa activation induced by hypothermic ECC was inhibited using TGX-221 alone or in combination with P(2)Y blockers (p<0.05), while no effect of hypothermic ECC or antiplatelet agents on GPIIb/IIIa and GPIbα expression and von Willebrand factor binding was observed. Sole P(2)Y and PI3K blockade or a combination thereof inhibited P-selectin expression on platelets and platelet-derived microparticles during hypothermic ECC (p<0.05). P(2)Y blockade alone or combined with TGX-221 prevented ECC-induced platelet-granulocyte aggregate formation (p<0.05). Platelet adhesion to the ECC surface, platelet loss and Mac-1 expression on granulocytes were inhibited by combined P(2)Y and PI3K blockade (p<0.05). Combined blockade of P(2)Y(12), P(2)Y(1) and PI3K p110β completely inhibits hypothermic ECC-induced activation processes. This novel finding warrants further studies and the development of suitable pharmacological agents to decrease ECC- and hypothermia-associated complications in clinical applications.

Show MeSH
Related in: MedlinePlus