Zhuang Mingfeng, Sun Bingwei, Liu Dadong, Shi Yuan
Department of Critical Care Medicine, Jiangyin People's Hospital, Jiangyin 214400, Jiangsu, China (Zhuang MF, Shi Y); Department of Burn and Plastic Surgery, Affiliated Hospital of Jiangsu University, Zhenjiang 212000, Jiangsu, China (Sun BW); Department of Critical Care Medicine, Affiliated Hospital of Jiangsu University, Zhenjiang 212000, Jiangsu, China (Liu DD). Corresponding author: Shi Yuan, Email:
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue. 2017 Feb;29(2):156-161. doi: 10.3760/cma.j.issn.2095-4352.2017.02.012.
To investigate the suppressive effect of carbon monoxide-releasing molecule II (CORM-2) on LPS induced platelet α-granule exocytosis in sepsis via soluble N-ethylmaleimide-sensitive factor attached protein receptor/mammalian uncoordinated 18b (SNARE/Munc18b) complex formation.
Blood was collected from healthy volunteers' cubital vein, then platelets were isolated by differential centrifugation. Platelets were randomly divided into 5 groups. The control group did not undergo any treatment, the LPS group received 10 mg/L LPS simulation, the CORM-2 group and iCORM-2 group underwent LPS simulation and immediate administration of CORM-2 (10 μmol/L and 50 μmol/L) or iCORM-2 (50 μmol/L), respectively. Samples were incubated in a CO-incubator at 37 °C, 95% humidity, and 5% CO. Platelet α-granule contents were detected by using standard enzyme linked immunosorbent assay (ELISA), including platelet factor 4 (PF4), platelet derived growth factor-BB (PDGF-BB), and matrix metalloproteinase-2 (MMP-2). The expression of P-selectin was detected by flow cytometer. Transmission electron microscope and immunofluorescence microscope was used to assess platelet α-granules distribution. Expressions of Munc18b and SNARE proteins including vesicle-associated membrane protein-8 (VAMP-8), synaptosomal-associated protein-23 (SNAP-23) and syntaxin-11 (STX-11) were detected by Western Bolt. The SNARE/Munc18b complex formation was detected by immunoprecipitation.
Compared with the control group, levels of PF4, PDGF-BB, MMP-2 and P-selectin in LPS induced platelets were found to markedly elevated, while CORM-2 (10 μmol/L and 50 μmol/L) could decrease platelet α-granule contents exocytosis: [PF4 (μg/L): 7.69±0.58, 6.03±0.71 vs. 10.13±0.82; PDGF-BB (μg/L): 112.71±1.79, 102.91±5.86 vs. 128.78±1.39; MMP-2 (ng/L): 32.94±2.73, 27.58±3.36 vs. 53.26±1.21; P-selectin: (17.14±0.57)%, (15.35±0.68)% vs. (23.78±0.62)%; all P < 0.01]. Transmission electron microscope and immunofluorescence microscope showed that the extent of platelet α-granules assembled to platelet plasma membrane was significantly decreased following CORM-2 treatment. Compared with the control group, the expressions of Munc18b and SNARE proteins and SNARE/Munc18b complex formation in LPS-stimulated platelets were significantly increased, while CORM-2 (10 μmol/L and 50 μmol/L) inhibited these elevations (Munc18b/GAPDH: 0.80±0.08, 0.69±0.01 vs. 0.99±0.09; VAMP-8/GAPDH: 0.72±0.09, 0.50±0.12 vs. 1.18±0.14; SNAP-23/GAPDH: 1.18±0.22, 0.63±0.10 vs. 1.90±0.08; STX-11/GAPDH: 0.76±0.02, 0.57±0.08 vs. 1.16±0.23; VAMP-8/Munc18b: 0.65±0.09, 0.53±0.07 vs. 1.21±0.20; SNAP-23/Munc18b: 0.85±0.07, 0.55±0.09 vs. 1.26±0.08; STX-11/Munc18b: 0.78±0.05, 0.61±0.10 vs. 1.39±0.16; all P < 0.01). Above all, the data showed a dose dependent change.
We could suggest that CORM-2 suppressed α-granule exocytosis in LPS-stimulated platelets and the potential mechanisms might involve SNARE/Munc18b complex formation.
通过可溶性N - 乙基马来酰亚胺敏感因子附着蛋白受体/哺乳动物不协调18b（SNARE/Munc18b）复合物形成，研究一氧化碳释放分子II（CORM - 2）对脓毒症中脂多糖（LPS）诱导的血小板α - 颗粒胞吐作用的抑制效果。
从健康志愿者肘静脉采集血液，通过差速离心法分离血小板。血小板随机分为5组。对照组不进行任何处理，LPS组接受10 mg/L LPS模拟，CORM - 2组和iCORM - 2组分别在LPS模拟后立即给予CORM - 2（10 μmol/L和50 μmol/L）或iCORM - 2（50 μmol/L）。样本在37℃、95%湿度、5% CO的CO培养箱中孵育。采用标准酶联免疫吸附测定（ELISA）检测血小板α - 颗粒内容物，包括血小板因子4（PF4）、血小板衍生生长因子 - BB（PDGF - BB）和基质金属蛋白酶 - 2（MMP - 2）。用流式细胞仪检测P - 选择素的表达。用透射电子显微镜和免疫荧光显微镜评估血小板α - 颗粒的分布。通过蛋白质免疫印迹法检测Munc18b和SNARE蛋白的表达，包括囊泡相关膜蛋白 - 8（VAMP - 8）、突触体相关蛋白 - 23（SNAP - 23）和 syntaxin - 11（STX - 11）。通过免疫沉淀检测SNARE/Munc18b复合物的形成。
与对照组相比，LPS诱导的血小板中PF4、PDGF - BB、MMP - 2和P - 选择素水平显著升高，而CORM - 2（10 μmol/L和50 μmol/L）可降低血小板α - 颗粒内容物的胞吐作用：[PF4（μg/L）：7.69±0.58，6.03±0.71对10.13±0.82；PDGF - BB（μg/L）：112.71±1.79，102.91±5.86对128.78±1.39；MMP - 2（ng/L）：32.94±2.73，27.58±3.36对53.26±1.21；P - 选择素：（17.14±0.57）%，（15.35±0.68）%对（23.78±0.62）%；所有P < 0.01]。透射电子显微镜和免疫荧光显微镜显示，CORM - 2处理后血小板α - 颗粒聚集到血小板质膜的程度显著降低。与对照组相比，LPS刺激的血小板中Munc18b和SNARE蛋白的表达以及SNARE/Munc18b复合物的形成显著增加，而CORM - 2（10 μmol/L和50 μmol/L）抑制了这些升高（Munc18b/GAPDH：0.80±0.08，0.69±0.01对0.99±0.09；VAMP - 8/GAPDH：0.72±0.09，0.50±0.12对1.18±0.14；SNAP - 23/GAPDH：1.18±0.22，0.63±0.10对1.90±0.08；STX - 11/GAPDH：0.76±0.02，0.57±0.08对1.16±0.23；VAMP - 8/Munc18b：0.65±0.09，0.53±0.07对1.21±0.20；SNAP - 23/Munc18b：0.85±0.07，0.55±0.09对1.26±0.08；STX - 11/Munc18b：0.78±0.05，0.61±0.10对1.39±0.16；所有P < 0.01）。综上所述，数据呈现剂量依赖性变化。
我们可以认为CORM - 2抑制LPS刺激的血小板中α - 颗粒的胞吐作用，其潜在机制可能涉及SNARE/Munc18b复合物的形成。
