Aspirin, an inhibitor of cyclooxygenase, inhibits platelet aggregation in response to many stimuli. Previous studies suggested an important and necessary role for protein kinase C (PKC) in platelet aggregation and secretion. Therefore, the effects of aspirin on sn-1,2-diacylglycerol (DAG), the endogenous activator of PKC, were investigated. Specifically, we sought to determine whether inhibition of DAG production is critical for aspirin action on platelets. Total DAG mass was measured using the DAG kinase assay. At low doses of gamma-thrombin (4 nM), aspirin (5 mM) completely inhibited secondary aggregation; this inhibition was associated with near-complete inhibition of DAG production. Inhibition of collagen-induced aggregation by aspirin (50 microM) was also associated with complete inhibition of collagen-stimulated DAG production and secondary aggregation. Concomitantly, aspirin reduced phosphorylation of the 40-kDa protein, a specific PKC substrate strongly suggesting inhibition of PKC in response to aspirin. To determine the physiologic significance of the inhibition of DAG production by aspirin, reconstitution studies were conducted with dioctanoylglycerol (diC8), a cell-permeable DAG. Under conditions in which aspirin completely inhibited secondary aggregation induced by gamma-thrombin, collagen, or arachidonic acid, diC8 overcame aspirin inhibition of agonist action and reconstituted secondary aggregation. DiC8 exerted these effects at low concentrations (2-3 microM), which caused minimal aggregation of control platelets. Phorbol 12,13-dibutyrate, a phorbol ester that directly activates PKC, mimicked the effects of diC8 in overcoming aspirin inhibition of collagen-induced platelet activation. However, subthreshold concentrations of the calcium ionophore ionomycin, arachidonic acid, or gamma-thrombin were unable to overcome aspirin inhibition of collagen-induced platelet aggregation, suggesting that the ability to overcome aspirin inhibition is not shared by other second messengers and is not due to nonspecific synergy. These studies constitute evidence that inhibition of DAG production and subsequent PKC activation are crucial to the antiaggregatory effects of aspirin. They also support the hypothesis that DAG production and PKC activation may be the final common pathway for induction of secondary aggregation.