Department of Pharmacology, Faculty of Medical Sciences, University of Campinas, Campinas (SP), Brazil.
Platelets. 2012;23(3):195-201. doi: 10.3109/09537104.2011.603065. Epub 2011 Aug 2.
High production of reactive-oxygen species (ROS) by blood cells is involved in damage of the vascular endothelium and multiple organ dysfunction in sepsis. However, little is known about the intraplatelet ROS production in sepsis and its consequences on platelet reactivity. In this study, we evaluated whether the treatment of rats with lipopolysaccharide (LPS) affects platelet aggregation through intraplatelet ROS generation. Rats were injected with LPS (1 mg/kg, i.p.), and at 2 to 72 h thereafter, adenosine diphosphate (ADP) (3-10 µM) induced platelet aggregation was evaluated. Production of ROS in platelets was measured by flow cytometry using 2',7'-dichlorofluorescein diacetate (DCFH-DA). Treatment of rats with LPS time-dependently inhibited ADP-induced platelet aggregation within 72 h. The inhibitory effect of LPS on platelet aggregation was further increased when the platelets were incubated with polyethylene glycol-superoxide dismutase (PEG-SOD; 30 U/mL), polyethylene glycol-catalase (PEG-CAT; 1000 U/mL) or the NADPH oxidase inhibitor diphenyleneiodonium (DPI; 10 µM). The ROS production in non-stimulated platelets did not differ between control and LPS-treated rats. However, in ADP-activated platelets, generation of ROS was increased by 3.0- and 7.0-fold, as evaluated at 8 and 48 h after LPS injection, respectively. This increased ROS production was significantly reduced when platelets were incubated in vitro with DPI, PEG-SOD or PEG-CAT. In contrast, treatment of rats with N-acetylcysteine (150 mg/kg, i.p.) significantly reduced the inhibitory effect of LPS on platelet aggregation, and prevented the increased ROS production by in vivo LPS. Our results indicate that the increased intraplatelet ROS production does not contribute to the inhibitory effect of LPS on platelet aggregation; however, the maintenance of redox balance in LPS-treated rats is fundamental to restore the normal platelet response in these animals.
血细胞中活性氧（ROS）的大量产生与败血症中血管内皮损伤和多器官功能障碍有关。然而，关于败血症中血小板内 ROS 的产生及其对血小板反应性的影响知之甚少。在这项研究中，我们评估了用脂多糖（LPS）治疗大鼠是否通过血小板内 ROS 的产生影响血小板聚集。大鼠腹腔注射 LPS（1mg/kg），之后在 2 至 72 小时，评估二磷酸腺苷（ADP）（3-10μM）诱导的血小板聚集。通过用 2'，7'-二氯荧光素二乙酸酯（DCFH-DA）使用流式细胞术测量血小板中的 ROS 产生。LPS 处理大鼠在 72 小时内时间依赖性地抑制 ADP 诱导的血小板聚集。当将血小板与聚乙二醇超氧化物歧化酶（PEG-SOD；30U/mL）、聚乙二醇过氧化氢酶（PEG-CAT；1000U/mL）或 NADPH 氧化酶抑制剂二苯基碘（DPI；10μM）孵育时，LPS 对血小板聚集的抑制作用进一步增加。与对照组相比，LPS 处理大鼠的非刺激血小板中 ROS 产生没有差异。然而，在 ADP 激活的血小板中，ROS 的产生在 LPS 注射后 8 小时和 48 小时分别增加了 3.0 倍和 7.0 倍。当将血小板在体外与 DPI、PEG-SOD 或 PEG-CAT 孵育时，这种增加的 ROS 产生明显减少。相比之下，用 N-乙酰半胱氨酸（150mg/kg，腹腔注射）治疗大鼠可显著降低 LPS 对血小板聚集的抑制作用，并防止 LPS 在体内增加 ROS 的产生。我们的结果表明，血小板内 ROS 的增加产生不会导致 LPS 对血小板聚集的抑制作用；然而，维持 LPS 处理大鼠的氧化还原平衡对于恢复这些动物的正常血小板反应至关重要。
