Delayed accumulation of diacylglycerol in platelets as a mechanism for regulation of onset of aggregation and secretion

An important mechanism of platelet regulation is the formation of the second messenger diacylglycerol (DAG) and the activation of protein kinase C (PKC). Our previous studies suggested that the DAG/PKC pathway plays an important role in the induction of secretion and secondary aggregation rather than the earlier events of shape change and primary aggregation. We therefore examined the hypothesis that the delayed effects of PKC on platelets may result from delayed accumulation of DAG. The kinetics of DAG formation in human platelets were determined. When platelets were stimulated with gamma-thrombin, the largest phase of DAG accumulation was delayed for 0.6 to 0.8 minutes and DAG mass levels remained elevated for at least 2 minutes. In platelets stimulated with collagen, DAG accumulation was delayed for 1.0 to 1.2 minutes and DAG mass levels remained elevated for at least 3 minutes after stimulation. Sustained DAG production was also associated with sustained activation of PKC as monitored by phosphorylation of the 40- Kd substrate of PKC. The physiologic significance of delayed DAG accumulation was investigated using the cell-permeable DAG analog, dioctanoylglycerol (diC8). In synergy with subthreshold gamma-thrombin or collagen, exogenous diC8 reconstituted platelet activation. The optimal timing of addition of diC8 was 0.5 minutes after stimulation with gamma-thrombin or collagen. These kinetics were similar to those of endogenous DAG accumulation. These studies underscore the importance of a delayed accumulative phase of DAG generation as a mechanism controlling the onset of platelet secretion and irreversible aggregation.