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The action of phosphatidic acid generated from membrane phospholipids on phospholipase A2 activation in rabbit platelets was investigated. When [3H]arachidonic acid-labelled platelets were treated with phorbol 12-myristate 13-acetate (PMA) and the membranes isolated from the cells incubated at 37 degrees C with 50 microM CaCl2 and 50 microM guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), both phosphatidic acid production and arachidonic acid liberation increased in PMA- and GTP gamma S-concentration-dependent manners. Ethanol dose-dependently inhibited these responses, accompanied by the formation of phosphatidylethanol. Since propranolol, an inhibitor of phosphatidic acid phosphohydrolase, had no influence on the production of phosphatidic acid, the arachidonic acid liberated does not appear to be derived from diacylglycerol which may be produced from phosphatidic acid through the action of this enzyme. In another approach, treatment of [3H]arachidonic acid-labelled membranes with phospholipase D from Streptomyces chromofuscus induced arachidonic acid liberation as well as phosphatidic acid formation in time- and dose-dependent manners. The former response was suppressed by p-bromophenacyl bromide, a phospholipase A2 inhibitor. These results suggest that phosphatidic acid derived from membrane phospholipids potentiates phospholipase A2 activation and contributes to the amplification of platelet activation.
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