Endothelin-1 evoked an increase and oscillations in cytosolic calcium concentration in adherent single human platelets and increased GMP-140 (P-selectin) in platelet suspension

Abstract

The effect of endothelin-1 (ET-1) on cytosolic calcium ion concentration ([Ca 2+]i) in adherent single human blood platelet was determined by fluorescence digital imaging microscopy using Fura-2 as calcium probe. A dose dependent increase and oscillatory changes in [Ca 2+]i in single platelets were evoked by ET-1 as with thrombin. Half and 1 μM of ET-1 increased (p < 0.01) the [Ca 2+]i in single platelets from a resting level of 83 ± 3.4 nM to 120 ± 13 nM and 240 ± 20 nM respectively. The ET-1 induced increase in [Ca 2+]i was suppressed by 1 mM EGTA, a calcium chelating agent in the medium. ET-1 increased the production of IP 3 (quantitified by IP 3 3H-radioreceptor assay kit) in platelets significantly (p < 0.05) in a dose dependent way. We measured the GMP-140 (P-selectin) level in the supernatant of human platelet suspensions incubated with thrombin and ET-1. Both thrombin and ET-1 increased the secretion of soluble GMP-140 in the supernatant of platelet suspensions. Therefore, we suggested that ET-1 increased [Ca 2+]i in platelets by both calcium influx and IP 3 mediated Ca 2+ release resulting in activation and release of GMP-140.