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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。
To investigate the inhibitory effect of snake venom tripeptide (p ENW) on platelet adhesion to fibrinogen and its mechanism. The inhibitory effect of p ENW (116.5 ~ 466.2μmol·L-1) on platelet adhesion to fibrinogen and the inhibition of fibrin clot retraction were detected by plate assay. MTT assay was used to determine the effect of p ENW on platelet vitality; the change of platelet cytoplasmic [Ca2 +] i was measured by dual-wavelength fluorescence spectrophotometer; the content of reactive oxygen species (ROS) in platelets was measured by flow cytometry; CGMP, cAMP and TXA2 were detected by ELISA. The effect of p-ENW on the phosphorylation of Akt, ERK and p38 in platelets was determined by Western blot. The results showed that p ENW significantly inhibited platelet adhesion to fibrinogen and thrombin-induced fibrin clot retraction; p ENW increased platelet c GMP / c AMP content, inhibition of TXA2 production and inhibition of platelet cytoplasm Within the [Ca2] i increased. p ENW inhibits thrombin-induced Akt signaling in platelets, phosphorylation of ERK and p38 signaling, and inhibition of platelet by p ENW has nothing to do with ROS. The results showed that p ENW can effectively inhibit platelet adhesion to fibrinogen and fibrin clot retraction, so p ENW can both stop the initial link of thrombosis, but also slow the thrombus formation process has been cured.