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When platelets are stimulated by activating agents, arachidonic acid (AA) is released from phospholipids and rapidly transformed to metabolites including prostaglandins and hydroxy eicosatetraenoic acids (HETEs). Most of AA metabolites are hydrophilic and the accurate recovery of these metabolites was not obtained by usual lipid extraction methods, such as the Bligh-Dyer method. Two methods are used in this study to evaluate the AA liberation in [3H]AA-labeled cells; the centrifugation method and the method employing an AA metabolism inhibitor, BW 755C.In the centrifugation method, the reaction was terminated by ice-cold buffer containing 10mM EDTA and centrifuged rapidly by microfuge, and the radioactivity in the resulting supernatant was counted. However, the supernatant fraction contained considerable amount of phospholipids as well as AA and its metabolites. Unmetabolized AA was mainly remained in the pellet fraction.When BW 755C, an inhibitor of cyclooxygenase and lipoxygenase activities, was employed, the released AA was almost recovered in the lipid fraction. However, in the thrombin-stimulated platelets the radioactivity in the AA fraction reached its peak within 1min after stimulation and gradually decreased. Concomitantly, the radioactivity in phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol fell within 1min and showed a progressive increase thereafter. Thus, in the presence of metabolic inhibitor, once released AA was considered to be reincorporated into phospholipids.

Evaluation of arachidonic acid release in activated human platelets - Comparative study of the centrifugation method and the method employing BW775C.