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In vitro inhibition by endothelins of thrombin‐induced aggregation and Ca 2+ mobilization in human platelets
Author(s) -
AstarieDequeker Catherine,
Iouzalen Lahcen,
DavidDufilho Monique,
Devynck MarieAude
Publication year - 1992
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/j.1476-5381.1992.tb14443.x
Subject(s) - thrombin , platelet , chemistry , endothelins , biophysics , cytosol , prostacyclin , fura 2 , calcium , endothelin 1 , biochemistry , medicine , receptor , biology , enzyme , organic chemistry
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 Ca 2+ concentration ([Ca 2+ ] i ) determined with the fluorescent indicators, BCECF and Fura‐2. 2 ET‐1 and ET‐3 at concentrations ranging from 10 −10 to 5 × 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 < 0.002 and < 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 × 10 −7 m , ET‐1 and ET‐3 did not cause a measurable change in basal [Ca 2+ ] i and pHi. When tested in combination with thrombin, 5 × 10 −7 m ET‐1 and ET‐3 decreased the transient peak of [Ca 2+ ] 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 Ca 2+ , 5 × 10 −7 m ET‐3 inhibited the peak of [Ca 2+ ] i by 18 ± 6% ( n = 6, P = 0.02). 4 The anti‐aggregating agents, prostacyclin (PGI 2 , 10 −8 −10 −7 m ) and nitroprusside (NP, 10 ng‐50 μg l −1 ) also induced a dose‐dependent inhibition of the thrombin‐induced [Ca 2+ ] i peak ( P = 0.001 for each). A combination of 10 −9 m PGI 2 and 10 ng l −1 NP augmented the inhibitory effect of each drug (PGI 2 alone 52 ± 11, plus NP 90 ± 2; NP alone 26 ± 4, plus PGI 2 69 ± 5% inhibition of [Ca 2+ ] i peak, n = 6 for each, P < 0.01 and P < 0.001, respectively). Platelet preincubation with 5 × 10 −7 m ET‐3 increased by 34 ± 11% ( n = 6, P = 0.014) the inhibitory effect of NP 10 ng l −1 without a significant influence on the PGI 2 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 Ca 2+ mobilization.