title: Effect of membrane-permeable sulfhydryl reagents and depletion of glutathione on calcium mobilisation in human platelets.
abstract: Exposure to peroxides is known to increase the sensitivity of platelets towards activation by agonists. Similar platelet-activating effects are induced by sulfhydryl reagents that evoke Ca2+-induced Ca2+ release (CICR) by stimulating the Ca2+-releasing property of the inositol-1,4,5-trisphosphate receptor. We questioned whether these compounds may act by mobilising intracellular calcium in platelets by altering the intracellular glutathione redox state. Using FURA2-loaded, aspirin-treated platelets, Ca2+ signals were studied following exposure to the membrane-permeable sulfhydryl reagents, thimerosal and disulfiram, the glutathione peroxidase substrate, tert-butyl hydroperoxide, and the inhibitor of glutathione reductase, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). In single platelets monitored by fluorescence imaging techniques, thimerosal and disulfiram elicited repetitive spiking in [Ca2+]i after variable lag times, indicating that these compounds stimulated CICR. BCNU caused [Ca2+]i spiking of only low amplitude, whereas tert-butyl hydroperoxide was inactive. In platelets in suspension devoid of extracellular CaCl2, the sulfhydryl reagents, at concentrations which decreased glutathione by 25%, strongly increased the Ca2+ responses of agonists that stimulated phospholipase C (thrombin) or acted independently of phospholipase C stimulation (thapsigargin). However, Ca2+ release was only slightly promoted by concentrations of BCNU that resulted in substantial depletion of the glutathione level. Tert-butyl hydroperoxide was without effect on glutathione, but partially inhibited Ca2+ mobilisation with these agonists. It is concluded that, in platelets, the potent CICR-promoting effects of sulfhydryl reagents are not solely due to their reaction with intracellular glutathione, but that extensive reduction in glutathione content is associated with Ca2+ mobilisation and CICR.
authors.affiliation: Department of Human Biology, Maastricht University, The Netherlands. R.vanGorp@HB.unimaas.NL
authors.forename: R M
authors.initials: RM
authors.lastname: van Gorp
authors.forename: M C
authors.initials: MC
authors.lastname: van Dam-Mieras
authors.forename: G
authors.initials: G
authors.lastname: Hornstra
authors.forename: J W
authors.initials: JW
authors.lastname: Heemskerk
mesh_terms: D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002118:Calcium / Q000097:blood*; D002330:Carmustine / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D004221:Disulfiram / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016257:Fura-2 / Q000378:metabolism; D005978:Glutathione / Q000097:blood*; D005979:Glutathione Peroxidase / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D010545:Peroxides / Q000494:pharmacology; D017382:Reactive Oxygen Species; D013439:Sulfhydryl Reagents / Q000494:pharmacology*; D019284:Thapsigargin / Q000494:pharmacology; D013849:Thimerosal / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology; D020122:tert-Butylhydroperoxide
chemical_list: D004791:Enzyme Inhibitors; D010545:Peroxides; D017382:Reactive Oxygen Species; D013439:Sulfhydryl Reagents; D013849:Thimerosal; D019284:Thapsigargin; D020122:tert-Butylhydroperoxide; D005979:Glutathione Peroxidase; D013917:Thrombin; D005978:Glutathione; D002118:Calcium; D004221:Disulfiram; D016257:Fura-2; D002330:Carmustine