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 P2Y12 and P2Y1 blockade and PI3K p110β inhibition on platelets and granulocytes during hypothermic ECC. Human blood was treated with the P2Y12 antagonist 2-MeSAMP, the P2Y1 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 P2Y 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 P2Y and PI3K blockade or a combination thereof inhibited P-selectin expression on platelets and platelet-derived microparticles during hypothermic ECC (p<0.05). P2Y 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 P2Y and PI3K blockade (p<0.05). Combined blockade of P2Y12, P2Y1 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.
Highlights
Under physiological conditions, platelets play a fundamental role in hemostasis, prevention of blood loss, and healing of vascular injury
PI3K p110b Inhibition Alone or in Combination with ADP Receptor Blockade has no Effect on Expression Levels of GPIIb/IIIa and GPIba as Well as on von Willebrand factor (vWF) Binding of Platelets, but Reduces GPIIb/IIIa Activation during Hypothermic extracorporeal circulation (ECC)
We investigate the effects of ADP receptor blockade combined with PI3K p110b inhibition on platelets and granulocytes in an ex vivo model simulating hypothermic ECC
Summary
Platelets play a fundamental role in hemostasis, prevention of blood loss, and healing of vascular injury. Contact of blood with the artificial surfaces of the ECC circuit as well as hypothermia are all known to be associated with platelet activation, which results in disturbed platelet function and associated complications [1,3,4]. Activated platelets can trigger an inflammatory response through interactions with leukocytes [5]. These platelet-leukocyte interactions are mainly mediated by binding of the platelet surface receptor P-selectin to its counter receptor P-selectin glycoprotein ligand-1 (PSGL-1) on leukocytes. Upregulation and activation of the Mac receptor (CD11b/CD18) on leukocytes is induced as a result of the P-selectin-PSGL-1 interaction [5,6]. It has been shown that CD40 ligand, which is shed from platelets upon activation, promotes Mac-1 upregulation [7]
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