Endothelin-B-receptors-dependent-inhibition of platelet aggregation in the CD-1 mouse

An experimental protocol of adenosine diphosphate-(ADP) induced platelet aggregation in the mouse was designed in order to study the roles played by endothelin-A (ET(A)) and endothelin-B (ET(B)) receptors in the ET-1-induced inhibition of ex vivo platelet aggregation. The pressor effects of ET-1 or IRL-1620 were firstly determined in vivo in anesthetized (ketamine/xylazine) CD-1 mice (males and females; 25-30 g). All agents were administered intravenously (via the jugular vein) and blood samples were collected from the carotid artery into heparinized Eppendorfs (15 U/ml). To obtain platelet-rich plasma (PRP) the blood was immediately centrifuged for 12 min at low speed (1100 g). Platelet-poor plasma (PPP) was then prepared by centrifugation of the whole blood sample at high speed (3700 g) for 30 min. PPP was used to calibrate the aggregometer at 100% transmission. Platelet aggregation was monitored ex vivo as a change in light transmission through PRP following the injection of ADP (5 microM). ET-1 (0.01-2.0 nmol/kg) induced a significant and dose-dependent inhibition of platelet aggregation ex vivo (12-84%). The selective ET(B) agonist, IRL-1620 (0.05-2.0 nmol/kg), also triggered a marked inhibition of platelet aggregation. Indomethacin (10 mg/kg), a nonselective cyclooxygenase inhibitor, abolished the inhibitory effect of ET-1. The selective ET(A) antagonist, BQ-123 (1 nmol/kg), abolished the in vivo pressor effect of exogenous ET-1, without affecting its anti-aggregatory activity. The selective ET(B) antagonist, BQ-788 (0.5 nmol/kg), did not modify the elevation of blood pressure produced by the ET-1; however, it did abrogate dose-dependently the inhibitory effect of ET-1 on platelet aggregation. These results suggest that the anti-aggregatory effect of ET-1, in anesthetized CD-1 mice, relies mainly upon the activation of ET(B) receptors. The mechanism whereby ET-1 exerts this effect, is partially indirect and requires at least the production and the release of prostanoids (possibly PGI2) into the blood stream.