Changes In Arachidonate-Containing Complex Lipid During Platelet Stimulation

We have examined changes in the ³H-arachidonic acid (³HC20:4) content of phospholipid following stimulation of human platelets by thrombin, ionophore A23187, or collagen. Resolution of lipids and lipid metabolites was achieved using HPLC and thin-layer chromatography after extraction of platelets by a modified Bligh and Dyer technique. The most rapid changes, leading to formation of TXB₂, occur in response to thrombin and A23187. Both stimuli induce marked losses of ³HC20:4 from phosphatidylcholine (PC) and phosphatidylinositol (PI) within 30 sec. Collagen also induces losses in PI and PC and formation of diglyceride (DG), which become appreciable only after 1-3 min. Whereas thrombin and A23187 exhibit similarities in terms of the rate of response, these agents differ with regard to the phospholipases activated. Exposure of platelets to A23187 causes some activation of Ca⁺²-dependent PI-specific phospholipase C (PI-PLC); however, A23187 is an inefficient stimulus for this enzyme. Ionophore induces the formation of 1/4 to 1/6 as much PI- PLC-derived DG as does thrombin when comparable amounts of PI are hydrolyzed. A similar discrepancy is found with respect to generation of phosphatidic acid. Simultaneous addition of A23187 and thrombin does not impair the formation and metabolism of DG promoted by thrombin alone. Further, whereas indcmethacin (1-100 μg/ml) exerts no inhibitory effects on PLC or the formation of DG in stimulated platelets, indomethacin does inhibit 1) the loss of ³HC20:4 from PC in response to thrombin and 2) the loss of ³HC20:4 from PC and PI in response to A23187. Indomethacin has been reported to inhibit platelet phospholipase A₂. It is clear from these studies that the activation of PI-PLC is not triggered merely by a general Ca⁺² flux. In contrast, a shift in platelet Ca⁺² stores appears to be a sufficient stimulus for phospholipase A.