Signaling mechanisms mediated by G-protein coupled receptors in human platelets

Aim: The present study deals with the investigation of mechanisms involved in the synergistic interaction between epinephrine and arachidonic acid (AA).

Methods: Venous blood was taken from healthy human volunteers reported to be free of medications for one week. Platelet aggregation was monitored at 37 degree using Dual-channel Lumi-aggregometer. The resulting aggregation was recorded for 5 min by the measurement of LIGHT transmission as a function of time.

Results: The data show that a synergism in platelet aggregation mediated by subthreshold concentrations of epinephrine (1 micromol/L) and AA (0.2 micromol/L) was inhibited by the alpha2-receptor antagonist (yohimbine, IC50)=0.6 micromol/L) and an inhibitor of AA-cyclooxygenase (COX), indomethacin (IC50=0.25 micromol/L). In examining receptor influence on intraplatelet signalling pathways, it was found that the synergistic effect was inhibited by Calcium Channel blockers, verapamil (IC50=0.4 micromol/L) and diltiazem (IC50=2.5 micromol/L), as well as by low concentrations of inhibitors of Phospholipase C (PLC) (U73122; IC50=0.2 micromol/L) and mitogens activated protein kinase (MAPK) (PD 98059; IC50=3.8 micromol/L). Herbimycin A, a specific inhibitor of tyrosine LIGHT chain kinase (TLCK), showed inhibition at IC50 value of 15 micromol/L, whereas chelerythrine, a protein kinase C (PKC) inhibitor, had no effect up to 20 micromol/L.

Conclusion: These data suggest that synergism between epinephrine and AA in platelet aggregation is triggered through receptors coupled to G-protein, which in turn, activate PLC, COX, and MAP kinase-signaling pathways.