In vitro inhibition by endothelins of thrombin-induced aggregation and Ca2+ mobilization in human platelets.

1. The in vitro effects of endothelins (ET-1 and ET-3) on human platelets were investigated by measurement of the aggregatory responses of washed platelets to thrombin and by the determination of cytosolic pH (pHi) and free Ca2+ concentration ([Ca2+]i) determined with the fluorescent indicators, BCECF and Fura-2. 2. ET-1 and ET-3 at concentrations ranging from 10(-10) to 5 x 10(-7) M, did not promote platelet aggregation but inhibited in a dose-dependent manner the aggregation induced by 0.05 u ml-1 thrombin (P less than 0.002 and less than 0.001, respectively) with maximal effects reached at 10(-8) M (17 +/- 3 and 15 +/- 2%, n = 11, P = 0.002 for each). 3. Even at 5 x 10(-7) M, ET-1 and ET-3 did not cause a measurable change in basal [Ca2+]i and pHi. When tested in combination with thrombin, 5 x 10(-7) M ET-1 and ET-3 decreased the transient peak of [Ca2+]i by 17 +/- 7 and 28 +/- 7% (n = 7 and 11, P = 0.03 and P = 0.002). No effect on pHi variations was detected. In the virtual absence of external Ca2+, 5 x 10(-7) M ET-3 inhibited the peak of [Ca2+]i by 18 +/- 6% (n = 6, P = 0.02). 4. The anti-aggregating agents, prostacyclin (PGI2, 10(-8)-10(-7) M) and nitroprusside (NP, 10 ng-50 micrograms l-1) also induced a dose-dependent inhibition of the thrombin-induced [Ca2+]i peak (P = 0.001 for each).A combination of 10-9M PGI2 and 1O ng P' NP augmented the inhibitory effect of each drug(PGI2 alone 52 +/-11, plus NP 90 +/- 2; NP alone 26 +/- 4, plus PGI2 69 +/- 5% inhibition of [Ca2 ], peak, n = 6 for each, P <0.01 and P <0.001, respectively). Platelet preincubation with 5 x 10-7M ET-3 increased by 34+/-11% (n = 6, P = 0.0 14) the inhibitory effect of NP 1O ng without a significant influence on the PGI2 effect.5. In conclusion, endothelins ET-1 and ET-3 can reduce in vitro the aggregating response of human platelets to thrombin by a mechanism that is probably due to decrease Ca2+ mobilization.