蛇毒三肽pENW的抗血小板黏附作用及其机制研究  

Inhibitory effects and mechanisms of snake venom tripeptide pENW on platelet adhesion

作　　者：白莉[1] 方伟蓉[2] 孔毅[2] 李运曼[2] 

机构地区：[1]河南中医学院,河南郑州450046 [2]中国药科大学药学院,江苏南京210009

出　　处：《药学学报》2015年第9期1107-1115,共9页Acta Pharmaceutica Sinica

基　　金：国家自然科学基金资助项目(81173088);河南省科技攻关重点项目(102102310320);河南中医学院博士科研基金资助项目(BSJJ2012-01)

摘　　要：考察蛇毒三肽（p ENW）对血小板与纤维蛋白原黏附的抑制作用及其机制。采用平板法检测p ENW（116.5∽466.2μmol·L-1）对血小板与纤维蛋白原黏附的抑制作用以及纤维蛋白凝块回缩的抑制作用。MTT法测定p ENW对血小板活力的影响;双波长荧光分光光度计法测定血小板胞浆[Ca2＋]i变化;流式细胞术测定血小板内活性氧族（ROS）含量;ELISA法测定血小板内环磷酸鸟苷（c GMP）、环磷酸腺苷（c AMP）、血栓烷A2（TXA2）的水平;Western blot法测定p ENW对血小板内Akt、ERK与p38磷酸化水平的影响。实验结果显示,p ENW显著抑制血小板与纤维蛋白原的黏附及凝血酶诱导纤维蛋白凝块的回缩;p ENW可升高血小板内c GMP/c AMP含量,抑制TXA2的生成及抑制血小板胞浆内[Ca2＋]i升高。p ENW抑制凝血酶诱导血小板内的Akt信号通路、ERK与p38信号通路的磷酸化,p ENW对血小板的抑制作用与ROS无关。结果表明,p ENW能够有效抑制血小板与纤维蛋白原的黏附及纤维蛋白凝块的回缩,因此p ENW既可以阻止血栓形成的始动环节,又能减缓已形成血栓的固化过程。This study was designed to investigate inhibitory effects and possible mechanisms of snake venom tripeptide（p ENW） on platelet adhesion in order to promote the development of a novel anti-platelet therapy. To study the inhibitory effects of p ENW on platelet adhesion, washed platelets pre-incubated with p ENW（116.5-466.2 μmol·L-1） were used to test the ability of platelet adhesion to fibrinogen. Effect of p ENW on fibrin clot retraction was also tested. Effect of p ENW on platelets viability was tested by MTT assay. Effect of p ENW on reactive-oxygen species（ROS） levels of platelet was studied by flow cytometry assay. Calcium mobilization in Fura-2/AM-loaded platelets was monitored with a spectrofluorimeter. Cyclic guanosine monophosphate（c GMP） and cyclic adenosine monophosphate（c AMP）, thromboxane A2（determined as its metabolite thromboxane B2） were measured using enzyme immunoassay kits. Akt, ERK and p38 phosphorylation were tested by Western blot. The results showed that p ENW inhibited platelet adhesion and fibrin clot retraction in a concentration-dependent manner without cytotoxicity. Intracellular c GMP and c AMP in both resting and thrombin-activated platelets were increased by p ENW. In addition, p ENW attenuated intracellular Ca2＋mobilization and TXA2 production in platelets stimulated by thrombin. As shown by Western blot assay, Akt, ERK and p38 phosphorylation in thrombin-induced platelet were attenuated by p ENW. However, inhibitory effects of p ENW had nothing to do with ROS. Thus, p ENW exhibited a significant inhibition on platelet adhesion to fibrinogen, which means p ENW could block the first step of thrombosis as while as retard the more stable clot formation. The mechanisms of p ENW on inhibition platelet adhesion might be related to instant regulations, such as protein kinases.

关 键 词：蛇毒 血小板黏附 血栓形成 

分 类 号：R965[医药卫生—药理学]