Walford T, Musa F I, Harper A G S
Institute for Science and Technology in Medicine, Keele University, Guy Hilton Research Centre, Stoke-on-Trent, Staffordshire, UK.
Br J Pharmacol. 2016 Jan;173(1):234-47. doi: 10.1111/bph.13361. Epub 2015 Dec 5.
Recently, we demonstrated that a pericellular Ca(2+) recycling system potentiates agonist-evoked Ca(2+) signalling and granule secretion in human platelets and hypothesized a role for the membrane complex (MC) in orchestrating the accumulation of Ca(2+) in the pericellular region. Previous work has demonstrated that treatment with high concentrations of nicergoline may disrupt the MC through an ability to trigger a re-organization of the dense tubular system. Experiments were therefore performed to assess whether nicergoline-induced changes in platelet ultrastructure affects thrombin-evoked Ca(2+) fluxes and dense granule secretion.
Thrombin-evoked Ca(2+) fluxes were monitored in Fura-2- or Fluo-5N-loaded human platelets, or using platelet suspensions containing Fluo-4 or Rhod-5N K(+) salts. Fluorescence microscopy was utilized to monitor microtubule structure and intracellular Ca(2+) store distribution in TubulinTracker- and Fluo-5N-loaded platelets respectively. Dense granule secretion was monitored using luciferin-luciferase.
Nicergoline treatment inhibited thrombin-evoked Ca(2+) signalling and induced alterations in the microtubule structure and the distribution of intracellular Ca(2+) stores in platelets. Nicergoline altered the generation and spreading of thrombin-induced pericellular Ca(2+) signals and almost completely prevented dense granule secretion. Stabilization of microtubules using taxol reversed most effects of nicergoline on platelet Ca(2+) signalling and partially reversed its effects on dense granule secretion.
Nicergoline-induced alterations to platelet ultrastructure disrupt platelet Ca(2+) signalling in a manner that would be predicted if the MC had been disrupted. These data suggest that nicergoline may be a useful prototype for the discovery of novel MC-disrupting anti-thrombotics.
最近，我们证明了细胞周缘Ca(2+)循环系统可增强激动剂诱发的人血小板Ca(2+)信号传导和颗粒分泌，并推测膜复合物（MC）在协调细胞周缘区域Ca(2+)积累中发挥作用。先前的研究表明，高浓度麦角隐亭治疗可能通过触发致密管状系统的重新组织来破坏MC。因此，进行了实验以评估麦角隐亭诱导的血小板超微结构变化是否会影响凝血酶诱发的Ca(2+)通量和致密颗粒分泌。
在加载Fura-2或Fluo-5N的人血小板中，或使用含有Fluo-4或Rhod-5N钾盐的血小板悬浮液监测凝血酶诱发的Ca(2+)通量。分别利用荧光显微镜监测加载微管追踪染料和Fluo-5N的血小板中的微管结构和细胞内Ca(2+)储存分布。使用荧光素-荧光素酶监测致密颗粒分泌。
麦角隐亭治疗抑制了凝血酶诱发的Ca(2+)信号传导，并诱导了血小板微管结构和细胞内Ca(2+)储存分布的改变。麦角隐亭改变了凝血酶诱导细胞周缘Ca(2+)信号的产生和扩散，并几乎完全阻止了致密颗粒分泌。使用紫杉醇稳定微管可逆转麦角隐亭对血小板Ca(2+)信号传导的大部分影响，并部分逆转其对致密颗粒分泌的影响。
麦角隐亭诱导的血小板超微结构改变以一种如果MC被破坏就可以预测的方式破坏血小板Ca(2+)信号传导。这些数据表明，麦角隐亭可能是发现新型MC破坏型抗血栓药物的有用原型。
