EICOSANOID GENERATION AND EFFECTS ON THE AGGREGATION OF THROMBOCYTES FROM THE RAINBOW-TROUT, ONCORHYNCHUS-MYKISS

Fish blood lacks anucleate platelets but contains a nucleated cell typ e termed the thrombocyte that is thought to be functionally analogous. Thrombocytes were purified from the peripheral blood of the rainbow t rout, Oncorhynchus mykiss, by a two step gradient centrifugation metho d. Following this procedure, the recovered thrombocytes were 78-86% pu re as defined by immunoreactivity to a panel of monoclonal antibodies and were of variable morphology from round to spindle-shaped. Incubati on of thrombocyte suspensions with either calcium ionophore, A23187, p latelet-activating factor or a thromboxane (TX) mimetic, U-46619, gene rated a range of eicosanoids derived from arachidonic acid including 1 2-hydroxyeicosatetraenoic acid (12-HETE), TXB(2), prostaglandin (PG) E (2), leukotriene (LT) B-4 and lipoxin (LX) A(4). The equivalent produc ts derived from eicosapentaenoic acid were also formed. Co-incubation of thrombocytes with either erythrocytes or granulocytes/monocytes in the presence of calcium ionophore did not result in the formation of a ny further new lipoxygenase products. Incubation of isolated thrombocy tes in plasma-free conditions with U-46619 (0.03-10 mu M) resulted in a rapid, dose-dependent aggregatory response. This effect was markedly augmented in the presence of mammalian fibrinogen (400 mu g ml(-1)). Thrombin (0.1-1.3 units ml(-1)), like U-46619, was also a potent proag gregatory compound for trout thrombocytes. LXA(4) and LTB(4) had limit ed aggregatory potential and then only at high concentrations (10 mu M ), while 12-HETE and PAF had no significant effect at an concentration s tested. These results demonstrate that some of the eicosanoids relea sed during the activation of trout thrombocytes are involved in the ag gregatory behaviour of this cell type.