ROLE OF PROTEIN-KINASE-C IN THE ANTI-AGGREGATORY EFFECTS OF ENDOTHELIN-L ON HUMAN PLATELETS

1. Endothelin-1 has anti-aggregatory properties, but the mechanism underlying this inhibitory action is unknown. This in vitro study investigates effects of endothelin-1 on thrombin-stimulated aggregation and intracellular free calcium concentration in human platelets and assesses the role of protein kinase C in the interactions between endothelin-1 and thrombin. Aggregation was measured turbidometrically and the intracellular free calcium concentration was determined with the fluorescent indicator fura 2-acetoxymethyl ester. 2. Endothelin-1 at concentrations from 10(-11) to 10(-6) mol/l had no effect on platelet aggregation or intracellular free calcium concentration but inhibited in a dose-dependent manner aggregation induced by 0.05 unit/ml thrombin (pD(2) for inhibition by endothelin = 8.1 +/- 0.12). 3. Endothelin-1 at 10(-9) mol/l significantly decreased (P < 0.01) thrombin-stimulated aggregation from 81.4 +/- 1.5% (in the absence of endothelin-1) to 53.5 +/- 1.1% (in the presence of endothelin) and thrombin-stimulated intracellular free calcium concentration from 179 +/- 1.7 nmol/l to 140 +/- 1.8 nmol/l. 4. Preincubation of platelets with 10(-7) mol/l staurosporine (protein kinase C inhibitor), calphostin C (highly selective protein kinase C inhibitor) or 5-(N,N-hexamethylene) amiloride (highly selective Na+-H+ exchange blocker) significantly inhibited (P < 0.01) thrombin-stimulated platelet responses and suppressed the inhibitory effect of endothelin-1 on thrombin-induced aggregation and intracellular free calcium concentration. 5. In conclusion, endothelin-1 decreases the aggregatory response of human platelets to thrombin by mechanisms that probably involve protein kinase C and Na+-H+ linked pathways.