Platelet activation at the onset of human endotoxemia is undetectable <i>in vivo</i>

Infection induces platelet activation and consumption, which leads to thrombocytopenia, enhances microvascular thrombosis, impairs microcirculation and eventually triggers disseminated intravascular coagulation (DIC). It is well characterized that endotoxemia results in a pro-inflammatory and pro-coagulatory state, which favors platelet activation. However the early, direct effects of endotoxemia on platelets have not been investigated so far. Therefore we aimed to determine the early effects of the endotoxin lipopolysaccharide (LPS) on platelet function in vivo. In a human endotoxemia model, 15 healthy volunteers were stimulated with LPS (2 ng/kg). Blood was drawn before, 10, 30 and 60 min after LPS challenge and platelet activation analyzed by flow cytometry (GPIIb/IIIa activation, surface CD62P and CD40L, intraplatelet reactive oxygen formation and platelet–leukocyte aggregates) and ELISA (sCD40L, sCD62P and CXCL4). In parallel, blood samples and platelets were spiked with LPS (50 pg/ml) in vitro and monitored over 60 min for the same platelet activation markers. In vitro platelet stimulation with LPS activated platelets independent of the presence of leukocytes and enhanced their adhesion to endothelial cells. In contrast, in vivo no increase in GPIIb/IIIa activation or surface expression of CD62P was observed. However, endotoxemia resulted in a significant drop in platelet count and elevated the plasma CXCL4 levels already 10 min after the LPS challenge. These data indicate that LPS rapidly activates platelets, leading to α-granule release and endothelial adhesion. This might explain the drop in platelet count observed at the onset of endotoxemia.感染可诱导血小板活化与消耗，进而引发血小板减少症，加重微血管血栓形成，损害微循环功能，最终诱发弥散性血管内凝血（disseminated intravascular coagulation，DIC）。现有研究已明确，内毒素血症可促发促炎与促凝状态，从而促进血小板活化。然而截至目前，内毒素血症对血小板的早期直接作用尚未得到研究。因此本研究旨在明确内毒素脂多糖（lipopolysaccharide，LPS）在体内对血小板功能的早期影响。本研究采用人体内毒素血症模型，对15名健康志愿者给予脂多糖（2 ng/kg）刺激。分别于脂多糖攻毒前、攻毒后10、30及60分钟采集血液样本，通过流式细胞术（flow cytometry）分析血小板活化相关指标，包括GPIIb/IIIa活化、细胞膜表面CD62P与CD40L表达、血小板内活性氧生成水平以及血小板-白细胞聚集物形成情况，并采用酶联免疫吸附试验（enzyme-linked immunosorbent assay，ELISA）检测sCD40L、sCD62P及CXCL4的表达水平。 同时，体外实验中向血液样本与血小板悬液中加入50 pg/ml的脂多糖，并于60分钟内持续监测上述同一组血小板活化标志物。体外实验中，脂多糖刺激可直接活化血小板，且该过程不依赖白细胞的存在，同时可增强血小板与内皮细胞的黏附能力。与之相反，体内实验未观察到GPIIb/IIIa活化或CD62P表面表达水平升高。但内毒素血症可导致血小板计数显著下降，并在脂多糖攻毒后10分钟即出现血浆CXCL4水平升高。 上述结果表明，脂多糖可快速活化血小板，引发α颗粒释放并增强血小板与内皮细胞的黏附能力，这或可解释内毒素血症发病初期观察到的血小板计数下降现象。