Arachidonate release and phosphatidic acid turnover in stimulated human platelets

We have examined the temporal relationship between arachidonate release and phosphatidic acid (PA) metabolism in human platelets stimulated with thrombin. Within 1 min of stimulation at 37 degrees C, platelets released up to 16 nmol of arachidonate/10(9) cells from membrane phospholipids and exhibited an increase of severalfold in PA mass. At 23 degrees C, arachidonate release was half-maximal by 10-30 s, but PA remained near control levels for 10-30 s, indicating that increased PA is not necessary for release. [3H]Glycerol-labeled platelets synthesized phospholipids from [3H]PA at a rate of 0.08-0.3 nmol/min/10(9) cells at 37 degrees C. This rate of [3H]glycerol-PA turnover was not enhanced by thrombin stimulation; thus, PA did not turn over rapidly enough to be an intermediate in the release of several nanomoles of arachidonate from phosphatidylinositol in the first few seconds of stimulation. When aspirin-treated platelets were prelabeled with [14C]- or [3H]arachidonate, the specific activity of phospholipid-bound arachidonate pools remained constant after thrombin stimulation. Released arachidonate had specific activity intermediate to that in phosphatidylcholine and phosphatidylinositol. [14C]arachidonate in phosphatidylethanolamine had specific activity 6-fold less than released [14C]arachidonate; this phospholipid could therefore contribute only a small fraction of released fatty acid.