Effect of membrane-permeable sulfhydryl reagents and depletion of glutathione on calcium mobilisation in human platelets.

R.M.A. van Gorp Beisser, M.C.E. Mieras-van Dam, G. Hornstra, J.W.M. Heemskerk

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

Department of Human Biology, Maastricht University, The Netherlands. R.vanGorp@HB.unimaas.NL

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.
Original languageEnglish
Pages (from-to)1533-1542
Number of pages10
JournalBiochemical Pharmacology
Volume53
Issue number10
DOIs
Publication statusPublished - 1 Jan 1997

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