A role for cyclooxygenase products in the formation of phosphatidic acid in stimulated human platelets. Differential mechanisms of action of thrombin and collagen

Abstract

Human platelets prelabeled with (32P)orthophosphate or [14C]arachidonic acid (AA) were stimulated with collagen or thrombin, and platelet activation (shape change, aggregation, and release of serotonin) was determined in parallel to the formation of 32P- or 14C-labeled phosphatidic acid (PA). The results show a close correlation between the degree of platelet activation and the amount of PA formed. Activation of platelets and formation of PA induced by collagen (2 to 20 micrograms/ml) was blocked by pretreatment of platelets with trifluoperazine, indomethacin, aspirin, or N-methylimidazole. This suggests that the formation of AA by phospholipase A2 and its subsequent metabolism by cyclooxygenase and thromboxane synthetase are required for the collagen-induced formation of PA. Endoperoxide analog U-44069 induces formation of PA in human platelets that have been pretreated with or without aspirin. The action of thrombin does not follow the same pattern of collagen. Low concentrations of thrombin (0.05 units/ml) induce only platelet shape change and a small stimulation of PA, changes which are only minimally inhibited by indomethacin. However, a small increase in the thrombin concentration (to 0.1 unit/ml) induces platelet aggregation, release of serotonin, and a sharp increase in PA accumulation which are effectively inhibited by indomethacin. Even higher thrombin concentrations (0.4 to 0.8 units/ml), however, result in a further stimulation of PA formation, platelet aggregation, and release of serotonin which are insensitive to inhibition by indomethacin. The data show that cyclooxygenase metabolites of AA, produced after platelet activation, may be differentially involved in the formation of PA in platelets stimulated with collagen or thrombin. Formation of PA following collagen or intermediate concentrations of thrombin (0.1 to 0.2 units/ml) is dependent on the cyclooxygenase pathway. However, formation of PA by very low or by high concentrations of thrombin is not mediated by cyclooxygenase metabolites of AA.