Department of Anesthesiology and Intensive Care Medicine, University of Tübingen, Tübingen, Germany.
PLoS One. 2012;7(6):e38455. doi: 10.1371/journal.pone.0038455. Epub 2012 Jun 6.
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.
体外循环 (ECC) 和低温用于维持心脏手术患者循环参数的稳定并提高其对缺血的耐受性。然而，ECC 和低温会诱导血小板和白细胞的激活机制，这些机制是由血小板激动剂 ADP 和磷酸肌醇-3-激酶 (PI3K) p110β介导的。在临床条件下，这些过程与包括血栓栓塞和炎症在内的危及生命的并发症有关。本研究分析了在低温 ECC 期间，ADP 受体 P(2)Y(12)和 P(2)Y(1)阻断以及 PI3K p110β 抑制对血小板和粒细胞的影响。用 P(2)Y(12)拮抗剂 2-MeSAMP、P(2)Y(1)拮抗剂 MRS2179、PI3K p110β 抑制剂 TGX-221、它们的组合或 PBS 和丙二醇（对照）处理人血。在静态体外条件下，使用 2-MeSAMP 或 TGX-221 发现了对 ADP 诱导的血小板激活的浓度依赖性抑制作用。用 MRS2179 进一步抑制 ADP 介导的作用。接下来，血液在 28°C 的体外 ECC 模型中循环 30 分钟，并使用流式细胞术、ELISA 和血小板计数分析研究各种血小板和粒细胞标志物。单独使用 TGX-221 或与 P(2)Y 阻滞剂联合使用可抑制低温 ECC 诱导的 GPIIb/IIIa 激活（p<0.05），而低温 ECC 或抗血小板药物对 GPIIb/IIIa 和 GPIbα表达和 von Willebrand 因子结合没有影响。单独的 P(2)Y 和 PI3K 阻断或两者的组合抑制了低温 ECC 期间血小板上的 P-选择素表达和血小板衍生的微粒（p<0.05）。单独的 P(2)Y 阻断或与 TGX-221 联合使用可防止 ECC 诱导的血小板-粒细胞聚集体形成（p<0.05）。血小板与 ECC 表面的粘附、血小板损失和粒细胞上的 Mac-1 表达均被联合的 P(2)Y 和 PI3K 阻断抑制（p<0.05）。联合阻断 P(2)Y(12)、P(2)Y(1)和 PI3K p110β 可完全抑制低温 ECC 诱导的激活过程。这一新发现需要进一步研究，并开发合适的药理学药物来减少临床应用中与 ECC 和低温相关的并发症。
