Iloprost antagonizes the increase in internal calcium concentration induced by α-thrombin in human platelets: A study of desensitization

We studied the interaction between the synthetic prostacyclin analog iloprost and the aggregating agent a-thrombin by measuring the internal calcium ion concentration ([Ca²⁺]i) of human fura-2-loaded platelets. Iloprost (0.003–100 µg/l) did not modify the resting calcium level; when added 2 minutes before exposure of the platelets to a submaximally active concentration of a-thrombin (10 U/l), iloprost dose-dependently antagonized the increase in [Ca²⁺]i. To evaluate if iloprost retained this antagonistic effect even after a prolonged contact, which is well known to cause a “desensitization” phenomenon, platelets were prein-cubated with iloprost (35 µg/l) for 3 hours. After washout, the effect of newly added iloprost (0.01–100 µg/l) on the a-thrombin-induced increase in [Ca²⁺]i was tested. Iloprost was still able to antagonize the increase in [Ca²⁺]i induced by a-thrombin in “desensitized” platelets; however, the dose-inhibitory response curve was significantly shifted to the right when compared with that obtained in control platelets (i.e., platelets preincubated for 3 hours with iloprost's solvent), and the resulting IC₅₀ was significantly higher: 1.78 versus 0.2 µg/l (p<0.001). Since the maximal inhibitory effect of iloprost could also be reached under these experimental conditions, we conclude that iloprost retains its ability to antagonize the increase in [Ca²⁺]i induced by a-thrombin in desensitized platelets.