The antithrombogenic endothelial cell defense (ATECD) describes the properties that enable the endothelium to prevent circulating blood platelets from adhering to, or aggregating on, the vascular wall. ATECD was investigated in an experimental model in which bovine passage 0 cultured endothelial cells (EC) were incubated with aggregating platelets and autologous plasma in a computer-operated aggregometer-like device. A maximal platelet aggregation required 150 ± 10-6 M adenosine diphosphate (ADP) to be present in ECs. A 5-minute coincubation for ECs and platelets was found to be adequate in evaluating the maximal ATECD value. By increasing the EC number in the aggregation suspension, platelet aggregation was progressively inhibited through a sigmoid curve (50% inhibition of aggregation required 2 ± 104 EC). Pharmacologic modulations of ATECD by isosorbide dinitrate (ISDN) + 2-isosorbide mononitrate (2-ISMN) + 5-isosorbide mononitrate (5-ISMN) were investigated under experimental conditions reflecting either an acute nitrate effect (platelet + control ECs + drug + ADP) or a chronic effect (platelet + 5-day nitrate-treated ECs + ADP). Under acute circumstances, ISDN antiplatelet activities were profoundly magnified by ECs. Aggregation was fully arrested with 5 ± 10-5 M ISDN and an EC number of 2 ± 104, whereas the same ISDN concentration alone induced 30% inhibition of control aggregation. In contrast, there were no significant changes in platelet aggregation whether incubation was done in the presence or absence of 2-ISMN or 5-ISMN, ISDN metabolites. Long-term exposure of ECs to isosorbide nitrates (ISN) resulted in increased acquired EC changes in ATECD. 5-ISMN was a poor antiplatelet agent but was capable of counteracting ISDN effects on ATECD. Under chronic circumstances, the overall ISN effect was a stimulation of ATECD, but the final effect was lower than that expected from the summation of individual ISN effects. Such an endothelium-dependent ISDN (and perhaps 2-ISMN) antiplatelet activity is likely to explain why ISDN inhibits platelet activity in vivo, while in vitro, ISDN fails to elicit such platelet aggregation inhibition unless suprapharmacologic ISDN concentrations are used. It is suggested that pharmacologic modulation of ATECD may be a new antithrombotic therapy.