Department of Pharmacology, State University of Campinas (UNICAMP), Campinas (SP), Brazil.
Platelets. 2010;21(4):260-9. doi: 10.3109/09537101003637240.
Alterations in platelet aggregation in septic conditions are well established. However, little is known about the effects of lipopolysaccharide (LPS) on platelet adhesion. We have therefore investigated the effects of LPS in human platelet adhesion, using an in vitro model of platelet adhesion to fibrinogen-coated wells. Microtiter plates were coated with human fibrinogen, after which washed platelets (6 x 10(8) platelets/ml) were allowed to adhere. Adherent platelets were quantified through measurement of acid phosphatase activity. Calcium mobilization in Fura2-AM-loaded platelets was monitored with a spectrofluorimeter. Platelet flow cytometry in thrombin-stimulated platelets was performed using monoclonal mouse anti-platelet GPIIb/IIIa antibody (PAC-1). Prior incubation of washed platelets with LPS (0.01-300 microg/ml) for 5 to 60 min concentration- and time-dependently inhibited non-activated platelet adhesion. In thrombin-activated (50 mU/ml) platelets, LPS inhibited the adhesion to a significantly lesser extent than non-activated platelets. Cyclohexamide, superoxide dismutase polyethylene glycol (PEG-SOD) or catalase polyethylene glycol did not affect the LPS responses. No alterations in cyclic GMP levels were seen after platelet incubation with LPS, except with the highest concentration employed (300 microg/ml) where an increase of 36% (P < 0.05) was observed. Thrombin increased by 7.5-fold the internal Ca(2+) platelet levels, an effect markedly inhibited by LPS. Thrombin induced concentration-dependent platelet GPIIb/IIIa activation, but LPS failed to affect the activation state of this membrane glycoprotein. In conclusion, LPS inhibits human platelet adhesion to fibrinogen by mechanisms involving blockade of external Ca(2+), independently of cGMP generation and activation of GPIIb/IIIa complex.
在感染条件下血小板聚集的改变是公认的。然而，关于脂多糖 (LPS) 对血小板黏附的影响知之甚少。因此，我们使用人纤维蛋白原包被的平板模型研究了 LPS 对人血小板黏附的影响。用人类纤维蛋白原包被微量滴定板，然后加入已清洗的血小板（6×10(8)个血小板/ml），使其黏附。通过测量酸性磷酸酶活性来定量测定黏附的血小板。用荧光分光光度计监测 Fura2-AM 负载血小板中的钙动员。在凝血酶刺激的血小板中，通过单克隆鼠抗血小板 GPIIb/IIIa 抗体（PAC-1）进行血小板流式细胞术。用 LPS（0.01-300μg/ml）孵育清洗后的血小板 5-60 分钟，浓度和时间依赖性地抑制非激活血小板的黏附。在凝血酶激活（50mU/ml）的血小板中，LPS 对黏附的抑制作用明显小于非激活血小板。环已亚胺、超氧化物歧化酶聚乙二醇（PEG-SOD）或过氧化氢酶聚乙二醇均不影响 LPS 反应。除了使用的最高浓度（300μg/ml）外，LPS 孵育后血小板中环鸟苷酸（cGMP）水平没有变化，其中观察到增加 36%（P<0.05）。凝血酶使血小板内 Ca(2+)水平增加 7.5 倍，LPS 明显抑制该作用。凝血酶诱导浓度依赖性血小板 GPIIb/IIIa 激活，但 LPS 未能影响该膜糖蛋白的激活状态。总之，LPS 通过阻断外 Ca(2+)，独立于 cGMP 生成和 GPIIb/IIIa 复合物激活，抑制人血小板黏附至纤维蛋白原。
