Nicergoline inhibits human platelet Ca2+ signalling through triggering a microtubule-dependent reorganization of the platelet ultrastructure

Abstract

Background and Purpose Recently, we demonstrated that a pericellular Ca2+ recycling system potentiates agonist-evoked Ca2+ signalling and granule secretion in human platelets and hypothesized a role for the membrane complex (MC) in orchestrating the accumulation of Ca2+ 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 Ca2+ fluxes and dense granule secretion. Experimental Approach Thrombin-evoked Ca2+ 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 Ca2+ store distribution in TubulinTracker- and Fluo-5N-loaded platelets respectively. Dense granule secretion was monitored using luciferin-luciferase. Key Results Nicergoline treatment inhibited thrombin-evoked Ca2+ signalling and induced alterations in the microtubule structure and the distribution of intracellular Ca2+ stores in platelets. Nicergoline altered the generation and spreading of thrombin-induced pericellular Ca2+ signals and almost completely prevented dense granule secretion. Stabilization of microtubules using taxol reversed most effects of nicergoline on platelet Ca2+ signalling and partially reversed its effects on dense granule secretion. Conclusions and Implications Nicergoline-induced alterations to platelet ultrastructure disrupt platelet Ca2+ 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.