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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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GPIIb/IIIa and GPIbα expression as well as vWF binding are not influenced by ex vivo hypothermic ECC and antiplatelet agents, while platelet PI3K p110β inhibition alone or in combination with P2Y12/P2Y1 receptor blockade reduce GPIIb/IIIa activation induced by hypothermic ECC.Human blood was left untreated (“before circ.”) or treated ex vivo with PBS (control), a combination of 2-MeSAMP and MRS2179 to block P2Y receptors (100 µM each; “P2Y block”), propylene glycol (“PG”, control), TGX-221 to inhibit PI3K p110β (2.2 µM; “TGX”) or a combination of 2-MeSAMP, MRS2179 (100 µM each) and TGX-221 (2.2 µM; “P2Y block + TGX”). All treated samples were circulated in an ECC model for 30 minutes at 28°C. Expression of GPIIb (A; n = 4), activated GPIIb/IIIa (B; n = 6), GPIbα (C; n = 6) as well as vWF binding (D; n = 4) were evaluated in flow cytometry using specific antibodies. ADP stimulation (20 µM) before and after hypothermic ECC was performed as positive control for GPIbα expression (C) and vWF binding (D) in the PBS group. Geometric mean fluorescence values of fluorescently labeled antibodies are given in diagrams as means and SD; not normally distributed data were analyzed using a non-parametrical test (Friedman test with Dunǹs multiple comparison test; A, D); normally distributed data (B, C) were compared using RM-ANOVA with Bonferroni’s multiple comparison test; *p<0.05; **p<0.01; ***p<0.001.
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pone-0038455-g002: GPIIb/IIIa and GPIbα expression as well as vWF binding are not influenced by ex vivo hypothermic ECC and antiplatelet agents, while platelet PI3K p110β inhibition alone or in combination with P2Y12/P2Y1 receptor blockade reduce GPIIb/IIIa activation induced by hypothermic ECC.Human blood was left untreated (“before circ.”) or treated ex vivo with PBS (control), a combination of 2-MeSAMP and MRS2179 to block P2Y receptors (100 µM each; “P2Y block”), propylene glycol (“PG”, control), TGX-221 to inhibit PI3K p110β (2.2 µM; “TGX”) or a combination of 2-MeSAMP, MRS2179 (100 µM each) and TGX-221 (2.2 µM; “P2Y block + TGX”). All treated samples were circulated in an ECC model for 30 minutes at 28°C. Expression of GPIIb (A; n = 4), activated GPIIb/IIIa (B; n = 6), GPIbα (C; n = 6) as well as vWF binding (D; n = 4) were evaluated in flow cytometry using specific antibodies. ADP stimulation (20 µM) before and after hypothermic ECC was performed as positive control for GPIbα expression (C) and vWF binding (D) in the PBS group. Geometric mean fluorescence values of fluorescently labeled antibodies are given in diagrams as means and SD; not normally distributed data were analyzed using a non-parametrical test (Friedman test with Dunǹs multiple comparison test; A, D); normally distributed data (B, C) were compared using RM-ANOVA with Bonferroni’s multiple comparison test; *p<0.05; **p<0.01; ***p<0.001.

Mentions: Platelet activation is accompanied by a conformational change of the platelet GPIIb/IIIa receptor into a high-affinity conformation, which enables binding of ligands including fibrinogen and vWF [19]. Using specific antibodies we analyzed (1) the general expression of GPIIb/IIIa on the platelet surface and (2) the GPIIb/IIIa activation state. Basic GPIIb/IIIa expression was neither influenced by hypothermic ECC nor by antiplatelet agents (Figure 2A), while hypothermic ECC induced a significant increase in GPIIb/IIIa activation in controls (p<0.01; Figure 2B). This effect was inhibited by PI3K p110β inhibition using TGX-221 alone (p<0.05) and more profoundly by PI3K p110β inhibition combined with P2Y blockade (2-MeSAMP and MRS2179; p<0.001).


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)

GPIIb/IIIa and GPIbα expression as well as vWF binding are not influenced by ex vivo hypothermic ECC and antiplatelet agents, while platelet PI3K p110β inhibition alone or in combination with P2Y12/P2Y1 receptor blockade reduce GPIIb/IIIa activation induced by hypothermic ECC.Human blood was left untreated (“before circ.”) or treated ex vivo with PBS (control), a combination of 2-MeSAMP and MRS2179 to block P2Y receptors (100 µM each; “P2Y block”), propylene glycol (“PG”, control), TGX-221 to inhibit PI3K p110β (2.2 µM; “TGX”) or a combination of 2-MeSAMP, MRS2179 (100 µM each) and TGX-221 (2.2 µM; “P2Y block + TGX”). All treated samples were circulated in an ECC model for 30 minutes at 28°C. Expression of GPIIb (A; n = 4), activated GPIIb/IIIa (B; n = 6), GPIbα (C; n = 6) as well as vWF binding (D; n = 4) were evaluated in flow cytometry using specific antibodies. ADP stimulation (20 µM) before and after hypothermic ECC was performed as positive control for GPIbα expression (C) and vWF binding (D) in the PBS group. Geometric mean fluorescence values of fluorescently labeled antibodies are given in diagrams as means and SD; not normally distributed data were analyzed using a non-parametrical test (Friedman test with Dunǹs multiple comparison test; A, D); normally distributed data (B, C) were compared using RM-ANOVA with Bonferroni’s multiple comparison test; *p<0.05; **p<0.01; ***p<0.001.
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getmorefigures.php?uid=PMC3368839&req=5

pone-0038455-g002: GPIIb/IIIa and GPIbα expression as well as vWF binding are not influenced by ex vivo hypothermic ECC and antiplatelet agents, while platelet PI3K p110β inhibition alone or in combination with P2Y12/P2Y1 receptor blockade reduce GPIIb/IIIa activation induced by hypothermic ECC.Human blood was left untreated (“before circ.”) or treated ex vivo with PBS (control), a combination of 2-MeSAMP and MRS2179 to block P2Y receptors (100 µM each; “P2Y block”), propylene glycol (“PG”, control), TGX-221 to inhibit PI3K p110β (2.2 µM; “TGX”) or a combination of 2-MeSAMP, MRS2179 (100 µM each) and TGX-221 (2.2 µM; “P2Y block + TGX”). All treated samples were circulated in an ECC model for 30 minutes at 28°C. Expression of GPIIb (A; n = 4), activated GPIIb/IIIa (B; n = 6), GPIbα (C; n = 6) as well as vWF binding (D; n = 4) were evaluated in flow cytometry using specific antibodies. ADP stimulation (20 µM) before and after hypothermic ECC was performed as positive control for GPIbα expression (C) and vWF binding (D) in the PBS group. Geometric mean fluorescence values of fluorescently labeled antibodies are given in diagrams as means and SD; not normally distributed data were analyzed using a non-parametrical test (Friedman test with Dunǹs multiple comparison test; A, D); normally distributed data (B, C) were compared using RM-ANOVA with Bonferroni’s multiple comparison test; *p<0.05; **p<0.01; ***p<0.001.
Mentions: Platelet activation is accompanied by a conformational change of the platelet GPIIb/IIIa receptor into a high-affinity conformation, which enables binding of ligands including fibrinogen and vWF [19]. Using specific antibodies we analyzed (1) the general expression of GPIIb/IIIa on the platelet surface and (2) the GPIIb/IIIa activation state. Basic GPIIb/IIIa expression was neither influenced by hypothermic ECC nor by antiplatelet agents (Figure 2A), while hypothermic ECC induced a significant increase in GPIIb/IIIa activation in controls (p<0.01; Figure 2B). This effect was inhibited by PI3K p110β inhibition using TGX-221 alone (p<0.05) and more profoundly by PI3K p110β inhibition combined with P2Y blockade (2-MeSAMP and MRS2179; p<0.001).

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