Exogenous diacylglycerols synergize with PAF with human platelets, but inhibit PAF-induced responses of rabbit platelets

Abstract

To investigate whether diacylglycerol (DAG) has a role in reversible platelet aggregation induced by low concentrations of platelet-activating 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 [³H]PAF to human and rabbit platelets and to rabbit platelet membranes. We then investigated whether exogenous, cell-penetrating DAGs (1,2-dihexanoyl-sn-glycerol (DHG) and 1-oleoyl-2-acetyl-sn-glycerol (OAG)) act synergistically with low concentrations of PAF that alone induce only reversible aggregation. Platelets were isolated and labeled with [¹⁴C]serotonin. DHG (25–75 μM) caused slow, weak aggregation and some release of [¹⁴C]serotonin with human, but not rabbit, platelets. OAG (25–75 μM) did not aggregate either species' platelets. Phosphorylation of pleckstrin by DHG was more transient in rabbit platelets than previously observed with human platelets. Both DHG and OAG synergistically potentiated PAF-induced aggregation of human platelets, but, paradoxically, concurrently inhibited the PAF-induced increase in intracellular Ca²⁺ ([Ca²⁺]_i); potentiation decreased upon incubation with DAGs before PAF addition. In contrast, DHG strongly inhibited PAF-induced aggregation of rabbit platlets; inhibition decreased upon preincubation. OAG, added with PAF, slightly potentiated aggregation of rabbit platelets; upon preincubation, 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 stimulation of protein kinase C by DAGs, and then metabolism of DAGs and/or negative feedback by DAGs, but results with rabbit platelets indicate both an unexpected species difference and a difference between the effects of DHG and OAG on PAF-induced platelet aggregation.