Quantitative analysis of diacylglycerol second messengers in human platelets: Correlation with aggregation and secretion

The action of many agonists results in rapid production of sn-1,2-diacylglycerol (DAG). Using platelets as a model system, we previously identified a delayed phase of DAG accumulation that is temporally associated with secondary aggregation and secretion. In the present study, we examined the quantitative relationship between this delayed DAG accumulation and platelet aggregation and secretion. To quantitate the low levels of DAG in platelets, we used the sensitive DAG kinase assay and simultaneously compared DAG levels with aggregation and ATP secretion. In platelets stimulated by γ-thrombin or collagen, there was a dose response between concentration of agonist and DAG accumulation. Significantly, a dose response was observed between DAG accumulation and extent of aggregation and secretion in platelets stimulated by either agonist. A concentration of either γ-thrombin or collagen that caused secondary aggregation and secretion was associated with DAG accumulation above 0.2 pmol of DAG/nmol of phospholipid. Subthreshold concentrations of γ-thrombin or collagen resulted in DAG levels less than 0.2 pmol/nmol of phospholipid. Thus, these data suggest that a threshold level of DAG must be achieved for platelet activation to occur. Moreover, secretion was blocked when DAG production was blocked with aspirin or when protein kinase C was inhibited with sphingosine. We conclude that endogenously formed DAG plays a critical role in regulating secondary aggregation and secretion and, therefore, represents an important target for future antiplatelet agents.