Anti-thrombogenic endothelial cell defense (ATECD) refers to the overall properties that enable the endothelium to prevent circulating blood platelets adhering to, or aggregating on the vascular wall. The basic characteristics of ATECD have been further investigated in cultured arterial endothelial cells (EC). Freshly obtained endothelium cells (ECs) and confluent, quiescent Passage 0 ECs, similarly expressed ATECD, whereas subconfluent dividing ECs and senescent ECs both elicited a markedly lowered ATECD. When ECs were successively exposed to fresh platelets, ATECD was progressively exhausted until a plateau (50% of control ATECD) was reached after the third exposure of ECs to platelets. Similarly, platelet response to the aggregating agent was markedly lowered after the first exposure of ECs to platelets, whereas such an inhibition of platelet activity by ECs was much less pronounced in subsequent exposure of ECs to fresh platelets. Under acute pharmacological circumstances, isosorbide dinitrate (ISDN) antiplatelet activities were found to be profoundly magnified by ECs, thus revealing an EC-mediated antiplatelet activity for ISDN, but not for its mononitrate metabolites, 2-ISMN and 5-ISMN. Long-term exposure of ECs to isosorbide nitrates (ISNs) revealed that ISDN, as well as 2-ISMN elicited an ATECD-stimulation priming effect on ECs, although in the presence of 5-ISMN (a poor antiplatelet agent) the ISN overall effect upon ATECD was less than 50% of what would have been expected by cumulating individual ISN effects. It is concluded that ATECD provides a highly differentiated function for ECs; the ATECD mechanisms involve antiaggregating factors that may be trapped by platelets; and, finally, ATECD may be stimulated by ISDN both under acute and under chronic pharmacological circumstances.