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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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Blockade of P2Y12 and P2Y1 as well as PI3K p110β inhibition profoundly inhibits hypothermic ECC-induced P-selectin expression on platelets and platelet microparticles.Prior to (“before circ.”) and after hypothermic ECC (28°C, 30 minutes) flow cytometric analysis of P-selectin expression on platelets and PMPs was performed in groups treated with either PBS as control, 2-MeSAMP and MRS2179 (100 µM each; “P2Y block”), propylene glycol (“PG”) as control, TGX-221 to inhibit PI3K p110β (2.2 µM; “TGX”) or a combination of 2-MeSAMP and MRS2179 (100 µM each) as well as TGX-221 (2.2 µM; “P2Y block + TGX”). Representative dot plot indicating the identification of PMPs, single platelets and aggregates according to their size and granularity (A). Representative histogram overlay including a marker to identify percentages of P-selectin expressing platelets before circulation without additional stimulation (grey filled), before circulation with addition of ADP (20 µM; grey solid line) as well as 30 minutes after hypothermic ECC (black solid line) (B). Percentages of platelets (C) and PMPs (D) expressing P-selectin are depicted. Data in (C) and (D) are given as means (n = 6) and SD; groups 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-g003: Blockade of P2Y12 and P2Y1 as well as PI3K p110β inhibition profoundly inhibits hypothermic ECC-induced P-selectin expression on platelets and platelet microparticles.Prior to (“before circ.”) and after hypothermic ECC (28°C, 30 minutes) flow cytometric analysis of P-selectin expression on platelets and PMPs was performed in groups treated with either PBS as control, 2-MeSAMP and MRS2179 (100 µM each; “P2Y block”), propylene glycol (“PG”) as control, TGX-221 to inhibit PI3K p110β (2.2 µM; “TGX”) or a combination of 2-MeSAMP and MRS2179 (100 µM each) as well as TGX-221 (2.2 µM; “P2Y block + TGX”). Representative dot plot indicating the identification of PMPs, single platelets and aggregates according to their size and granularity (A). Representative histogram overlay including a marker to identify percentages of P-selectin expressing platelets before circulation without additional stimulation (grey filled), before circulation with addition of ADP (20 µM; grey solid line) as well as 30 minutes after hypothermic ECC (black solid line) (B). Percentages of platelets (C) and PMPs (D) expressing P-selectin are depicted. Data in (C) and (D) are given as means (n = 6) and SD; groups were compared using RM-ANOVA with Bonferroni’s multiple comparison test; *p<0.05; **p<0.01, ***p<0.001.

Mentions: As an indicator for α-granule release and platelet activation, P-selectin expression on the surface of platelets and platelet-derived microparticles was measured before and after hypothermic ECC. Single platelets and PMPs were identified according to their size and granularity (Figure 3A). As a positive control P-selectin expression on ADP-stimulated platelets was measured to confirm platelet reactivity before and after circulation in the control group (Figure 3B).


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)

Blockade of P2Y12 and P2Y1 as well as PI3K p110β inhibition profoundly inhibits hypothermic ECC-induced P-selectin expression on platelets and platelet microparticles.Prior to (“before circ.”) and after hypothermic ECC (28°C, 30 minutes) flow cytometric analysis of P-selectin expression on platelets and PMPs was performed in groups treated with either PBS as control, 2-MeSAMP and MRS2179 (100 µM each; “P2Y block”), propylene glycol (“PG”) as control, TGX-221 to inhibit PI3K p110β (2.2 µM; “TGX”) or a combination of 2-MeSAMP and MRS2179 (100 µM each) as well as TGX-221 (2.2 µM; “P2Y block + TGX”). Representative dot plot indicating the identification of PMPs, single platelets and aggregates according to their size and granularity (A). Representative histogram overlay including a marker to identify percentages of P-selectin expressing platelets before circulation without additional stimulation (grey filled), before circulation with addition of ADP (20 µM; grey solid line) as well as 30 minutes after hypothermic ECC (black solid line) (B). Percentages of platelets (C) and PMPs (D) expressing P-selectin are depicted. Data in (C) and (D) are given as means (n = 6) and SD; groups were compared using RM-ANOVA with Bonferroni’s multiple comparison test; *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-g003: Blockade of P2Y12 and P2Y1 as well as PI3K p110β inhibition profoundly inhibits hypothermic ECC-induced P-selectin expression on platelets and platelet microparticles.Prior to (“before circ.”) and after hypothermic ECC (28°C, 30 minutes) flow cytometric analysis of P-selectin expression on platelets and PMPs was performed in groups treated with either PBS as control, 2-MeSAMP and MRS2179 (100 µM each; “P2Y block”), propylene glycol (“PG”) as control, TGX-221 to inhibit PI3K p110β (2.2 µM; “TGX”) or a combination of 2-MeSAMP and MRS2179 (100 µM each) as well as TGX-221 (2.2 µM; “P2Y block + TGX”). Representative dot plot indicating the identification of PMPs, single platelets and aggregates according to their size and granularity (A). Representative histogram overlay including a marker to identify percentages of P-selectin expressing platelets before circulation without additional stimulation (grey filled), before circulation with addition of ADP (20 µM; grey solid line) as well as 30 minutes after hypothermic ECC (black solid line) (B). Percentages of platelets (C) and PMPs (D) expressing P-selectin are depicted. Data in (C) and (D) are given as means (n = 6) and SD; groups were compared using RM-ANOVA with Bonferroni’s multiple comparison test; *p<0.05; **p<0.01, ***p<0.001.
Mentions: As an indicator for α-granule release and platelet activation, P-selectin expression on the surface of platelets and platelet-derived microparticles was measured before and after hypothermic ECC. Single platelets and PMPs were identified according to their size and granularity (Figure 3A). As a positive control P-selectin expression on ADP-stimulated platelets was measured to confirm platelet reactivity before and after circulation in the control group (Figure 3B).

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