Multiphasic generation of diacylglycerol in thrombin-activated human platelets

Abstract

The diacylglycerol (DAG)/protein kinase C pathway plays an important role in platelet aggregation and granule secretion. In this study, we examined the detailed kinetics of DAG formation in response to platelet stimulation. Both alpha- and gamma-thrombin caused multiphasic generation of DAG mass, with DAG production reaching peaks at 0.3-0.6 min intervals. A sub-threshold concentration of gamma-thrombin (1.5 nM) produced oscillations of DAG, but peak DAG levels rapidly returned to baseline (unstimulated) values. Intermediate concentrations of gamma-thrombin (8-30 nM) resulted in prominent phases of DAG production whose troughs became significantly elevated compared with baseline levels. This delayed accumulation of DAG coincided in time with the onset of secretion and irreversible aggregation. In contrast, stimulation of platelets with collagen resulted in delayed single-phase DAG production. The kinetics of DAG production in stimulated platelets may control both the timing and the degree of DAG accumulation. This may ensure that protein kinase C is activated optimally at the onset of secondary aggregation and secretion. This is the first report of oscillating DAG production in a biological system.