EXOGENOUS DIACYLGLYCEROLS SYNERGIZE WITH PAF WITH HUMAN PLATELETS, BUT INHIBIT PAF-INDUCED RESPONSES OF RABBIT PLATELETS

To investigate whether diacylglycerol (DAG) has a role in reversible p latelet aggregation induced by low concentrations of platelet-activati ng factor (PAF), we attempted to use the DAG kinase inhibitor, R59022, to prevent rapid conversion of DAG to phosphatidic acid. However, we found that R59022 inhibited the binding of [H-3]PAF to human and rabbi t platelets and to rabbit platelet membranes. We then investigated whe ther exogenous, cell-penetrating DAGs (1,2-dihexanoyl-sn-glycerol (DHG ) and 1-oleoyl-2-acetyl-sn-glycerol (OAG)) act synergistically with lo w concentrations of PAF that alone induce only reversible aggregation. Platelets were isolated and labeled with [C-14]serotonin. DHG (25-75 mu M) caused slow, weak aggregation and some release of [C-14]serotoni n with human, but not rabbit, platelets. OAG (25-75 mu M) did not aggr egate either species' platelets. Phosphorylation of pleckstrin by DHG was more transient in rabbit platelets than previously observed with h uman platelets. Both DHG and OAG synergistically potentiated PAF-induc ed aggregation of human platelets, but, paradoxically, concurrently in hibited the PAF-induced increase in intracellular Ca2+ ([Ca2+](i)); po tentiation decreased upon incubation with DAGs before PAF addition. In contrast, DHG strongly inhibited PAF-induced aggregation of rabbit pl atlets; inhibition decreased upon preincubation. OAG, added with PAF, slightly potentiated aggregation of rabbit platelets; upon preincubati on, OAG progressively inhibited. Effects of DHG and OAG on PAF-induced increases in [Ca2+](i) in rabbit platlets followed a similar pattern; thus, with rabbit platelets. inhibition of the [Ca2+](i) increase may at least partially account for inhibition of PAF-induced aggregation by exogenous DAGs. Results with human platelets are consistent with st imulation of protein kinase C by DAGs, and then metabolism of DAGs and /or negative feedback by DAGs, but results with rabbit platelets indic ate both an unexpected species difference and a difference between the effects of DHG and OAG on PAF-induced platelet aggregation.