N-ethylmaleimide-stimulated arachidonic acid release in human platelets

Treatment of human platelets with the alkylating agent N-ethylmaleimide (NE M) induces arachidonic acid release. The effect was time- and dose-dependen t. NEM-stimulated arachidonic acid mobilisation could be prevented by pretr eating platelets with the cytosolic phospholipase A(2) (cPLA(2))-specific i nhibitor arachidonyltrifluoromethyl ketone. Moreover, the tyrosine kinase i nhibitor genistein was able to significantly inhibit arachidonic acid mobil isation. NEM-stimulated release of arachidonic acid appears to be a Ca2+-de pendent mechanism, as shown by the observation that arachidonic acid mobili sation was significantly reduced by platelet treatment with EGTA and abolis hed by preloading platelets with the intracellular chelator 1,2-bis (o-amin ophenoxy) ethane-N,N,N',N'-tetraacetic acid tetra (acetoxymethyl) ester (BA PTA/AM). In Fura-2 loaded platelets, NEM was able to significantly increase the intracellular Ca2+ level. The Ca2+ elevation was significantly reduced in the presence of EGTA and suppressed by cell treatment with BAPTA/AM. Ar achidonic 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-eicosate traynoic acid. In conclusion, the results in this study demonstrate that NE M stimulates arachidonic acid release by cPLA(2) activation through intrace llular Ca2+ 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. (C) 1999 Elsevier Science Inc.