N-ethylmaleimide-stimulated arachidonic acid release in human platelets

Abstract

Treatment of human platelets with the alkylating agent N-ethylmaleimide (NEM) induces arachidonic acid release. The effect was time- and dose-dependent. NEM-stimulated arachidonic acid mobilisation could be prevented by pretreating platelets with the cytosolic phospholipase A 2 (cPLA 2)-specific inhibitor arachidonyltrifluoromethyl ketone. Moreover, the tyrosine kinase inhibitor genistein was able to significantly inhibit arachidonic acid mobilisation. NEM-stimulated release of arachidonic acid appears to be a Ca 2+-dependent mechanism, as shown by the observation that arachidonic acid mobilisation was significantly reduced by platelet treatment with EGTA and abolished by preloading platelets with the intracellular chelator 1,2- bis ( o-aminophenoxy) ethane- N, N, N′, N′-tetraacetic acid tetra (acetoxymethyl) ester (BAPTA/AM). In Fura-2-loaded platelets, NEM was able to significantly increase the intracellular Ca 2+ level. The Ca 2+ elevation was significantly reduced in the presence of EGTA and suppressed by cell treatment with BAPTA/AM. Arachidonic acid released by NEM produced a significant increase in reactive oxygen species (ROS) intracellular levels, which was partially inhibited by diphenyleneiodonium and almost completely suppressed by 5,8,11,14-eicosatetraynoic acid. In conclusion, the results in this study demonstrate that NEM stimulates arachidonic acid release by cPLA 2 activation through intracellular Ca 2+ elevation. In addition, tyrosine specific protein kinases seem to be involved in arachidonic acid release. ROS was also shown to be formed during arachidonic acid metabolisation.