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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: Further inhibition of ADP-mediated effects was achieved with MRS2179.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.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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Effects of hypothermic ECC and platelet inhibitor treatment on platelet adhesion to the ECC surface, platelet-granulocyte aggregate formation and platelet counts.Human blood samples were either left untreated or treated with PBS (control group), a combination of 2-MeSAMP and MRS2179 (100 µM each; “P2Y block”), propylene glycol (“PG”, control group), TGX-221 (2.2 µM; “TGX”) or a combination of 2-MeSAMP, MRS2179 and TGX-221 (“P2Y block + TGX”). Blood, which was treated accordingly, was circulated in an ex vivo ECC model at 28°C for 30 minutes. A specific ELISA method was employed to detect platelet adhesion to the ECC surface (A; n = 6). Platelet-granulocyte aggregate formation was measured in flow cytometry before circulation (“before circ.”) and after circulation in all treatment groups (B; n = 4) according to the fluorescence of an anti-CD15-PE antibody on aggregates (aggregate region in figure 3A) under a pre-set histogram marker. Platelet counts were measured in all samples (C; n = 6). Data are given as means and SD; normally distributed data were compared using RM-ANOVA with Bonferroni’s multiple comparison test (A, B); not normally distributed data were analyzed using a non-parametrical test (Friedman test with Dunǹs multiple comparison test; C); *p<0.05; **p<0.01; ***p<0.001.
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pone-0038455-g004: Effects of hypothermic ECC and platelet inhibitor treatment on platelet adhesion to the ECC surface, platelet-granulocyte aggregate formation and platelet counts.Human blood samples were either left untreated or treated with PBS (control group), a combination of 2-MeSAMP and MRS2179 (100 µM each; “P2Y block”), propylene glycol (“PG”, control group), TGX-221 (2.2 µM; “TGX”) or a combination of 2-MeSAMP, MRS2179 and TGX-221 (“P2Y block + TGX”). Blood, which was treated accordingly, was circulated in an ex vivo ECC model at 28°C for 30 minutes. A specific ELISA method was employed to detect platelet adhesion to the ECC surface (A; n = 6). Platelet-granulocyte aggregate formation was measured in flow cytometry before circulation (“before circ.”) and after circulation in all treatment groups (B; n = 4) according to the fluorescence of an anti-CD15-PE antibody on aggregates (aggregate region in figure 3A) under a pre-set histogram marker. Platelet counts were measured in all samples (C; n = 6). Data are given as means and SD; normally distributed data were compared using RM-ANOVA with Bonferroni’s multiple comparison test (A, B); not normally distributed data were analyzed using a non-parametrical test (Friedman test with Dunǹs multiple comparison test; C); *p<0.05; **p<0.01; ***p<0.001.

Mentions: Hypothermic ECC induced a significant increase in platelet adhesion to the artificial ECC surface in controls (p<0.001; Figure 4A). This was only prevented by a combination of P2Y blockade and PI3K p110β inhibition (p<0.05), but not by P2Y blockade or PI3K p110β inhibition alone.


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)

Effects of hypothermic ECC and platelet inhibitor treatment on platelet adhesion to the ECC surface, platelet-granulocyte aggregate formation and platelet counts.Human blood samples were either left untreated or treated with PBS (control group), a combination of 2-MeSAMP and MRS2179 (100 µM each; “P2Y block”), propylene glycol (“PG”, control group), TGX-221 (2.2 µM; “TGX”) or a combination of 2-MeSAMP, MRS2179 and TGX-221 (“P2Y block + TGX”). Blood, which was treated accordingly, was circulated in an ex vivo ECC model at 28°C for 30 minutes. A specific ELISA method was employed to detect platelet adhesion to the ECC surface (A; n = 6). Platelet-granulocyte aggregate formation was measured in flow cytometry before circulation (“before circ.”) and after circulation in all treatment groups (B; n = 4) according to the fluorescence of an anti-CD15-PE antibody on aggregates (aggregate region in figure 3A) under a pre-set histogram marker. Platelet counts were measured in all samples (C; n = 6). Data are given as means and SD; normally distributed data were compared using RM-ANOVA with Bonferroni’s multiple comparison test (A, B); not normally distributed data were analyzed using a non-parametrical test (Friedman test with Dunǹs multiple comparison test; C); *p<0.05; **p<0.01; ***p<0.001.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3368839&req=5

pone-0038455-g004: Effects of hypothermic ECC and platelet inhibitor treatment on platelet adhesion to the ECC surface, platelet-granulocyte aggregate formation and platelet counts.Human blood samples were either left untreated or treated with PBS (control group), a combination of 2-MeSAMP and MRS2179 (100 µM each; “P2Y block”), propylene glycol (“PG”, control group), TGX-221 (2.2 µM; “TGX”) or a combination of 2-MeSAMP, MRS2179 and TGX-221 (“P2Y block + TGX”). Blood, which was treated accordingly, was circulated in an ex vivo ECC model at 28°C for 30 minutes. A specific ELISA method was employed to detect platelet adhesion to the ECC surface (A; n = 6). Platelet-granulocyte aggregate formation was measured in flow cytometry before circulation (“before circ.”) and after circulation in all treatment groups (B; n = 4) according to the fluorescence of an anti-CD15-PE antibody on aggregates (aggregate region in figure 3A) under a pre-set histogram marker. Platelet counts were measured in all samples (C; n = 6). Data are given as means and SD; normally distributed data were compared using RM-ANOVA with Bonferroni’s multiple comparison test (A, B); not normally distributed data were analyzed using a non-parametrical test (Friedman test with Dunǹs multiple comparison test; C); *p<0.05; **p<0.01; ***p<0.001.
Mentions: Hypothermic ECC induced a significant increase in platelet adhesion to the artificial ECC surface in controls (p<0.001; Figure 4A). This was only prevented by a combination of P2Y blockade and PI3K p110β inhibition (p<0.05), but not by P2Y blockade or PI3K p110β inhibition alone.

Bottom Line: Further inhibition of ADP-mediated effects was achieved with MRS2179.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.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