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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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P2Y12/P2Y1 blockade in combination with TGX-221 prevents upregulation of the Mac-1 receptor on granulocytes.Granulocyte activation was measured before (“before circ.”) and 30 minutes after hypothermic ECC in groups treated with PBS (control group), 2-MeSAMP and MRS2179 (“P2Y block”; 100 µM each), propylene glycol (“PG”, control group), TGX-221 (“TGX”; 2.2 µM) and a combination of P2Y block (100 µM each) and PI3K p110β inhibition with TGX-221 (2.2 µM; “P2Y block + TGX”;). Mac-1 expression on granulocytes was evaluated using geometric mean values of antibody fluorescence in flow cytometry. Data are given as means (n = 6) and SD; groups were compared using RM-ANOVA with Bonferroni’s multiple comparison test; *p<0.05; ***p<0.001.
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pone-0038455-g005: P2Y12/P2Y1 blockade in combination with TGX-221 prevents upregulation of the Mac-1 receptor on granulocytes.Granulocyte activation was measured before (“before circ.”) and 30 minutes after hypothermic ECC in groups treated with PBS (control group), 2-MeSAMP and MRS2179 (“P2Y block”; 100 µM each), propylene glycol (“PG”, control group), TGX-221 (“TGX”; 2.2 µM) and a combination of P2Y block (100 µM each) and PI3K p110β inhibition with TGX-221 (2.2 µM; “P2Y block + TGX”;). Mac-1 expression on granulocytes was evaluated using geometric mean values of antibody fluorescence in flow cytometry. Data are given as means (n = 6) and SD; groups were compared using RM-ANOVA with Bonferroni’s multiple comparison test; *p<0.05; ***p<0.001.

Mentions: Interaction of activated platelets with leukocytes triggers the upregulation of the Mac-1 receptor on leukocytes. Mac-1 mediates leukocyte adhesion and migration as well as interaction and binding of leukocytes and platelets [30]. To evaluate granulocyte activation, we measured surface CD11b expression on granulocytes. Hypothermic ECC induced a profound increase in CD11b expression on granulocytes in controls (p<0.05; Figure 5). This was significantly decreased by P2Y blockade in combination with PI3K p110β inhibition (p<0.001). P2Y and PI3K p110β inhibition alone had no effect on hypothermic ECC-induced CD11b expression on granulocytes.


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)

P2Y12/P2Y1 blockade in combination with TGX-221 prevents upregulation of the Mac-1 receptor on granulocytes.Granulocyte activation was measured before (“before circ.”) and 30 minutes after hypothermic ECC in groups treated with PBS (control group), 2-MeSAMP and MRS2179 (“P2Y block”; 100 µM each), propylene glycol (“PG”, control group), TGX-221 (“TGX”; 2.2 µM) and a combination of P2Y block (100 µM each) and PI3K p110β inhibition with TGX-221 (2.2 µM; “P2Y block + TGX”;). Mac-1 expression on granulocytes was evaluated using geometric mean values of antibody fluorescence in flow cytometry. Data are given as means (n = 6) and SD; groups were compared using RM-ANOVA with Bonferroni’s multiple comparison test; *p<0.05; ***p<0.001.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3368839&req=5

pone-0038455-g005: P2Y12/P2Y1 blockade in combination with TGX-221 prevents upregulation of the Mac-1 receptor on granulocytes.Granulocyte activation was measured before (“before circ.”) and 30 minutes after hypothermic ECC in groups treated with PBS (control group), 2-MeSAMP and MRS2179 (“P2Y block”; 100 µM each), propylene glycol (“PG”, control group), TGX-221 (“TGX”; 2.2 µM) and a combination of P2Y block (100 µM each) and PI3K p110β inhibition with TGX-221 (2.2 µM; “P2Y block + TGX”;). Mac-1 expression on granulocytes was evaluated using geometric mean values of antibody fluorescence in flow cytometry. Data are given as means (n = 6) and SD; groups were compared using RM-ANOVA with Bonferroni’s multiple comparison test; *p<0.05; ***p<0.001.
Mentions: Interaction of activated platelets with leukocytes triggers the upregulation of the Mac-1 receptor on leukocytes. Mac-1 mediates leukocyte adhesion and migration as well as interaction and binding of leukocytes and platelets [30]. To evaluate granulocyte activation, we measured surface CD11b expression on granulocytes. Hypothermic ECC induced a profound increase in CD11b expression on granulocytes in controls (p<0.05; Figure 5). This was significantly decreased by P2Y blockade in combination with PI3K p110β inhibition (p<0.001). P2Y and PI3K p110β inhibition alone had no effect on hypothermic ECC-induced CD11b expression on granulocytes.

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