Effect of Endothelin on Human Platelet Aggregation and Prostacyclin Generation from Cultured Human Vascular Endothelial Cells

The effects of endothelin (ET-1) on cultured human umbilical vein endothelial cells (HUVEC) and platelet aggregation were investigated with its mode of action. Platelet aggregation (PRP: 30×104 platelets/μl) induced by ADP (2×10-6M) was measured using aggregometer. PGI2, TXB2 and cAMP concentration ([cAMP]i) were assayed by radioimmunoassay. And cytosolic free Ca++ concentration ([Ca++]i) was measured using Fura-2/AM. The results were as follows: The PGI2 generation was enhanced by ET-1 (2×10-7, 5×10-8, 5×10-9M) without change of [cAMP]i or [Ca++]i, and when HUVEC were pretreated with acetylsalicylic acid (ASA), its enhancement was completely inhibited, although the inhibited platelet aggregation by addition of conditioned media of HUVEC incubated with ET-1 was not completely abolished. The addition of conditioned media of HUVEC incubated with ET-1 decreased platelet aggregation and TXB2 generation, but increased [cAMP]i, however the addition of ET-1 alone had no effect on them. These decreased TXB2 generation and increased [cAMP]i in the platelet were suppressed by the pretreatment of HUVEC with ASA. These results suggested that ET-1 attenuated platelet aggregation through its decreased TXB2 generation and increased [cAMP]i via the increased release of PGI2 and the other mediators from HUVEC.