We have previously shown that aspirin-treated endothelial cells synthesize prostacyclin (PGI₂) from the purified prostaglandin endoperoxide PGH₂ (1978. J. Biol. Chem.253: 7138). To ascertain whether aspirin-treated endothelial cells produce PGI₂ from endoperoxides released by stimulated platelets, [³H]arachidonic acid-prelabeled platelets were reacted in aggregometer cuvettes with the calcium ionophore A 23187, thrombin, or collagen in the presence of aspirin-treated endothelial cell suspensions. This procedure permitted thin-layer radiochromatographic quantitation of [³H]PGI₂ as [³H]6-keto-PGF_1α and [³H]thromboxane A₂ (TXA₂) as [³H]TXB₂, as well as analysis of platelet aggregation responses in the same sample. In the presence of aspirin-treated endothelial cells, platelet aggregation in response to all three agents was inhibited. [³H]6-keto-PGF_1α was recovered from the supernates of the combined cell suspensions after stimulation by all three agents. The order of PGI₂ production initiated by the stimuli was ionophore > thrombin > collagen. The amounts of platelet [³H]TXB₂ recovered were markedly reduced by the addition of aspirin-treated endothelial cells. In separate experiments, 6-keto-PGF_1α and TXB₂ were quantitated by radioimmunoassay; the results paralleled those obtained with the use of radiolabeling. The quantity of 6-keto-PGF_1α measured by radioimmunoassay represented amounts of PGI₂ sufficient to inhibit platelet aggregation. These results were obtained when 200,000 platelets/μl were combined with 3,000-6,000 aspirin-treated endothelial cells/μl. At higher platelet levels the proportion of 6-keto-PGF_1α to TXB₂ decreased and platelet aggregation occurred. Control studies indicated that aspirin-treated endothelial cells could not synthesize PGI₂ from exogenous radioactive or endogenous arachidonate when stimulated with thrombin. Therefore the endothelial cell suspensions could only have used endoperoxides from stimulated platelets.

Thus, under our experimental conditions, production by endothelial cells of PGI₂ from endoperoxides derived from activated platelets could be demonstrated by two independent methods. These experimental conditions included: (a) enhanced platelet-endothelial cell proximity, as attainable in stirred cell suspensions; (b) use of increased endothelial cell/platelet ratios; and (c) utilization of arachidonate of high specific activity in radiolabeling experiments. Furthermore, when a mixture of platelets and endothelial cells that were not treated with aspirin was stimulated with thrombin, more than twice as much 6-keto-PGF_1α was formed than when endothelial cells were stimulated alone. These results indicate that endothelial cells can utilize platelet endoperoxides for PGI₂ formation to a significant extent.