摘要目的探讨外源性一氧化碳（CO）对脓毒症时血小板异常释放的抑制作用及其可能分子机制。方法采集健康成年志愿者空腹肘静脉血，采用差速离心法获得富含血小板血浆（PRP），按随机数字表法分为正常对照组、脂多糖（LPS）组（10mg/LLPS刺激）、无活性外源性一氧化碳释放分子2（iCORM-2）组（10mg/LLPS+50μmol/LiCORM-2干预）、外源性一氧化碳释放分子2（CORM-2）10μmol/L和50μmol/L组（10mg/LLPS+CORM-210μmol/L或50μmol/L干预）。30min后用酶联免疫吸附试验（ELISA）检测上清液中血小板源性生长因子BB（PDGF-BB）和基质金属蛋白酶2（MMP-2）水平；化学荧光素法检测血小板三磷酸腺苷（ATP）水平；流式细胞仪检测血小板膜糖蛋白P-选择素水平；蛋白质免疫印迹试验（WesternBlot）检测血小板表面信号分子Toll样受体4（TLR4）表达及关键信号分子蛋白激酶Cθ亚型（PKCθ）和突触融合蛋白结合蛋白1（STXBP-1）磷酸化；免疫共沉淀法检测STXBP-1介导的可溶性N-乙基马来酰亚胺敏感因子黏附受体（SNAREs）复合体突触融合蛋白2-突触相关蛋白23-囊泡相关膜蛋白8（STX2-SNAP23-VAMP8）的形成。结果①与正常对照组相比，LPS刺激后血小板释放PDGF-BB、MMP-2和ATP均显著增加，血小板表面P-选择素表达明显上调〔PDGF-BB（μg/L）：127.53±1.78比94.35±5.84，MMP-2（ng/L）：51.87±9.20比35.83±3.17，ATP（μmol/L）：1.288±0.056比0.975±0.010，P-选择素：（3.93±0.19）%比（0.44±0.10）%，均P＜0.05〕；10μmol/L和50μmol/LCORM-2干预均能有效抑制LPS诱导血小板释放PDGF-BB、MMP-2、ATP和P-选择素的高表达，并呈剂量依赖性〔PDGF-BB（μg/L）：114.68±1.35、97.08±6.14比127.53±1.78，MMP-2（ng/L）：32.67±8.00、24.63±1.63比51.87±9.20，ATP（μmol/L）：0.999±0.015、0.965±0.008比1.288±0.056，P-选择素：（1.95±0.27）%、（0.94±0.11）%比（3.93±0.19）%，均P＜0.05〕。②与正常对照组相比，LPS刺激后血小板TLR4表达以及PKCθ和STXBP-1的磷酸化水平均显著增加〔TLR4（灰度值）：1.21±0.38比0.67±0.06，p-PKCθ（灰度值）：1.36±0.20比0.44±0.03，p-STXBP-1（灰度值）：1.13±0.06比0.59±0.04，均P＜0.05〕；10μmol/L和50μmol/LCORM-2干预均能有效抑制血小板各指标，并呈剂量依赖性〔TLR4（灰度值）：0.76±0.05、0.65±0.04比1.21±0.38，p-PKCθ（灰度值）：0.71±0.07、0.47±0.10比1.36±0.20，p-STXBP-1（灰度值）：0.56±0.02、0.48±0.01比1.13±0.06，均P＜0.05〕。③与正常对照组相比，LPS刺激后血小板中与STXBP-1耦联的STX2、SNAP23和VAMP8均显著增加〔STX2（灰度值）：1.35±0.06比0.57±0.04，SNAP23（灰度值）：0.97±0.04比0.30±0.12，VAMP8（灰度值）：1.37±0.12比0.77±0.10，均P＜0.05〕；10μmol/L和50μmol/LCORM-2干预均能够有效抑制血小板SNAREs复合体的形成，并呈剂量依赖性〔STX2（灰度值）：0.77±0.02、0.39±0.03比1.35±0.06），SNAP23（灰度值）：0.41±0.03、0.22±0.08比0.97±0.04，VAMP8（灰度值）：0.85±0.07、0.66±0.07比1.37±0.12，均P＜0.05〕。iCORM-2组与LPS组上述各指标比较差异均无统计学意义。结论脓毒症时血小板释放功能异常活跃，CORM-2干预能有效抑制血小板的过度释放，其机制可能与TLR4/PKCθ/STXBP-1信号途径介导的SNAREs复合体形成有关。

abstractsObjective To investigate the suppressive effect of exogenous carbon monoxide (CO) on abnormal platelet exocytosis and its possible molecular mechanism. Methods Venous blood was collected from healthy volunteers. Platelet-rich plasma (PRP) was isolated from the blood by differential centrifugation. The PRP was randomly divided into five groups by random number table, namely normal control group, lipopolysaccharide (LPS) group (challenged with 10 mg/L LPS), inactively exogenous carbon monoxide releasing molecule 2 (iCORM-2) group (given 10 mg/L LPS + 50 μmol/L iCORM-2 for intervention), exogenous carbon monoxide releasing molecule 2 (CORM-2) 10 μmol/L and 50 μmol/L groups (given 10 mg/L LPS + CORM-2 10 μmol/L or 50 μmol/L for intervention). After 30 minutes, enzyme linked immunosorbent assay (ELISA) was used to determine the platelet-derived growth factor BB (PDGF-BB) and matrix metalloproteinase 2 (MMP-2). Chemical fluorescein method was used to determine the platelet adenosine triphosphate (ATP). Flow cytometer was used to determine the expression of P-selectin. The expressions of Toll-like receptor 4 (TLR4), phosphorylation of protein kinase Cθ (PKCθ) and syntaxin binding protein 1 (STXBP-1) were determined by Western Bolt. The soluble N-ethylmaleimide-sensitive factor-attachment protein receptors (SNAREs) complex formation [syntaxin 2-synaptosomal-associated protein 23-vesicle associated membrane protein 8 (STX2-SNAP23-VAMP8)] mediated by STXBP-1 was determined by immunoprecipitation. Results ① Compared with normal control group, the platelet release of PDGF-BB, MMP-2 and ATP was significantly increased after LPS challenge, and the P-selectin expression of platelet was also obviously up-regulated [PDGF-BB (μg/L): 127.53±1.78 vs. 94.35±5.84, MMP-2 (ng/L): 51.87±9.20 vs. 35.83±3.17, ATP (μmol/L): 1.288±0.056 vs. 0.975±0.010, P-selectin: (3.93±0.19)% vs. (0.44±0.10)%, all P < 0.05]. The increases in platelet release of PDGF-BB, MMP-2 and ATP were suppressed by 10 μmol/L or 50 μmol/L CORM-2 administration, as well as high-expression of P-selectin in a dose-dependent manner [PDGF-BB (μg/L): 114.68±1.35, 97.08±6.14 vs. 127.53±1.78, MMP-2 (ng/L): 32.67±8.00, 24.63±1.63 vs. 51.87±9.20, ATP (μmol/L): 0.999±0.015, 0.965±0.008 vs. 1.288±0.056, P-selectin: (1.95±0.27)%, (0.94±0.11)% vs. (3.93±0.19)%, all P < 0.05]. ② Compared with normal control group, LPS challenge resulted in a significant increase in the expression of TLR4 and the phosphorylation of PKCθ and STXBP-1 [TLR4 (gray value): 1.21±0.38 vs. 0.67±0.06, p-PKCθ (gray value): 1.36±0.20 vs. 0.44±0.03, p-STXBP-1 (gray value): 1.13±0.06 vs. 0.59±0.04, all P < 0.05]. The increases in above parameters were suppressed by 10 μmol/L or 50 μmol/L CORM-2 administration in a dose-dependent manner [TLR4 (gray value): 0.76±0.05, 0.65±0.04 vs. 1.21±0.38; p-PKCθ (gray value): 0.71±0.07, 0.47±0.10 vs. 1.36±0.20; p-STXBP-1 (gray value): 0.56±0.02, 0.48±0.01 vs. 1.13±0.06, all P < 0.05]. ③ Compared with normal control group, the SNAREs proteins in platelet that combined with STXBP-1, including STX2, SNAP23 and VAMP8, were obviously increased after LPS challenge [STX2 (gray value): 1.35±0.06 vs. 0.57±0.04, SNAP23 (gray value): 0.97±0.04 vs. 0.30±0.12, VAMP8 (gray value): 1.37±0.12 vs. 0.77±0.10, all P < 0.05]. The increases in SNAREs complex formation were suppressed by 10 μmol/L or 50 μmol/L CORM-2 administration in a dose-dependent manner [STX2 (gray value): 0.77±0.02, 0.39±0.03 vs. 1.35±0.06, SNAP23 (gray value): 0.41±0.03, 0.22±0.08 vs. 0.97±0.04, VAMP8 (gray value): 0.85±0.07, 0.66±0.07 vs. 1.37±0.12, all P < 0.05]. There was no significant difference in the above mentioned parameters between iCORM-2 group and LPS group. Conclusions LPS-induced abnormal secretion of platelet was suppressed by CORM-2 administration. The mechanism may involve the TLR4/PKCθ/STXBP-1 signaling pathway activation and the SNAREs complex formation.