Eicosanoid generation and effects on the aggregation of thrombocytes from the rainbow trout, Oncorhynchus mykiss

Abstract

Fish blood lacks anucleate platelets but contains a nucleated cell type termed the thrombocyte that is thought to be functionally analogous. Thrombocytes were purified from the peripheral blood of the rainbow trout, Oncorhynchus mykiss, by a two step gradient centrifugation method. Following this procedure, the recovered thrombocytes were 78–86% pure as defined by immunoreactivity to a panel of monoclonal antibodies and were of variable morphology from round to spindle-shaped. Incubation of thrombocyte suspensions with either calcium ionophore, A23187, platelet-activating factor or a thromboxane (TX) mimetic, U-46619, generated a range of eicosanoids derived from arachidonic acid including 12-hydroxyeicosatetraenoic acid (12-HETE), TXB 2, prostaglandin (PG)E 2, leukotriene (LT) B 4 and lipoxin (LX) A 4. The equivalent products 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 any further new lipoxygenase products. Incubation of isolated thrombocytes in plasma-free conditions with U-46619 (0.03–10 μM) resulted in a rapid, dose-dependent aggregatory response. This effect was markedly augmented in the presence of mammalian fibrinogen (400 μgml −1). Thrombin (0.1–1.3 units ml −1), like U-46619, was also a potent proaggregatory compound for trout thrombocytes. LXA 4 and LTB 4 had limited aggregatory potential and then only at high concentrations (10 μM), while 12-HETE and PAF had no significant effect at all concentrations tested. These results demonstrate that some of the eicosanoids released during the activation of trout thrombocytes are involved in the aggregatory behaviour of this cell type.