Our previous studies have shown that subthreshold concentrations of two platelet agonists exert synergistic effects on platelet aggregation. Here we studied the mechanism of synergistic interaction of 5-hydroxytryptamine (5-HT) and epinephrine mediated platelet aggregation. We show that 5-HT had no or little effect on aggregation but it did potentiate the aggregation response of epinephrine. The synergistic interaction of 5-HT (1-5 microM) and epinephrine (0.5-2 microM) was inhibited by alpha2-adrenoceptor blocker (yohimbine; IC50= 0.4 microM), calcium channel blockers (verapamil and diltiazem with IC50 of 10 and 48 mM, respectively), PLC inhibitor (U73122; IC50=6 microM) and nitric oxide (NO) donor, SNAP (IC50=1.6 microM)). The data suggest that synergistic effects of platelet agonists are receptor-mediated and occur through multiple signalling pathways including the activation PLC/Ca2+ signalling cascades.
Blotting, Western ; Calcium Channel Blockers/pharmacology ; Calcium Signaling* ; Drug Synergism ; Enzyme Activation ; Enzyme Inhibitors/pharmacology ; Epinephrine/pharmacology ; G-Protein, Inhibitory Gi/metabolism* ; GTP-Binding Proteins/metabolism* ; Human ; Phospholipase C/metabolism* ; Phospholipase C/antagonists & inhibitors ; Platelet Aggregation/physiology ; Platelet Aggregation/drug effects* ; Serotonin/pharmacology ; Signal Transduction

Our recent studies have shown that co-activation of Gq and Gi proteins by 5-hydroxytryptamine (5-HT) and adrenaline show synergism in human platelet aggregation. This study was conducted to examine the mechanism(s) of synergistic interaction of 5-HT and platelet activating factor (PAF) in human platelets. We show that PAF, but not 5-HT, increased platelet aggregation in a concentration-dependent manner. However, low concentrations of 5-HT (2 microM) potentiated platelet aggregation induced by subthreshold concentration of PAF (40 nM) indicating a synergistic interaction between the two agonists and this synergism was blocked by receptor antagonists to either 5-HT or PAF. 5-HT also potentiated the effect of PAF on thromboxane A2 (TXA2) formation and phosphorylation of extracellularly regulated mitogen-activated protein kinases (ERK1/2). The synergism of 5-HT and PAF in platelet aggregation was inhibited by calcium (Ca2+) channel blockers, verapamil and diltiazem, phospholipase C (PLC) inhibitor, U73122, cyclooxygenase (COX) inhibitor, indomethacin, and MEK inhibitor, PD98059. These data suggest that synergistic effect of 5-HT and PAF on human platelet aggregation involves activation of PLC/Ca2+, COX and MAP kinase pathways.
Diltiazem/pharmacology ; Dose-Response Relationship, Drug ; Drug Synergism ; Estrenes/pharmacology ; Flavones/pharmacology ; Human ; In Vitro ; Indomethacin/pharmacology ; Kinetics ; Mitogen-Activated Protein Kinases/metabolism ; Phosphorylation/drug effects ; Platelet Activating Factor/*pharmacology ; Platelet Activation/drug effects ; Platelet Aggregation/*drug effects/physiology ; Pyrrolidinones/pharmacology ; Serotonin/*pharmacology ; Thromboxane A2/biosynthesis ; Verapamil/pharmacology

Our recent studies have shown that co-activation of Gq and Gi proteins by 5-hydroxytryptamine (5-HT) and adrenaline show synergism in human platelet aggregation. This study was conducted to examine the mechanism(s) of synergistic interaction of 5-HT and platelet activating factor (PAF) in human platelets. We show that PAF, but not 5-HT, increased platelet aggregation in a concentration-dependent manner. However, low concentrations of 5-HT (2 microM) potentiated platelet aggregation induced by subthreshold concentration of PAF (40 nM) indicating a synergistic interaction between the two agonists and this synergism was blocked by receptor antagonists to either 5-HT or PAF. 5-HT also potentiated the effect of PAF on thromboxane A2 (TXA2) formation and phosphorylation of extracellularly regulated mitogen-activated protein kinases (ERK1/2). The synergism of 5-HT and PAF in platelet aggregation was inhibited by calcium (Ca2+) channel blockers, verapamil and diltiazem, phospholipase C (PLC) inhibitor, U73122, cyclooxygenase (COX) inhibitor, indomethacin, and MEK inhibitor, PD98059. These data suggest that synergistic effect of 5-HT and PAF on human platelet aggregation involves activation of PLC/Ca2+, COX and MAP kinase pathways.
Diltiazem/pharmacology ; Dose-Response Relationship, Drug ; Drug Synergism ; Estrenes/pharmacology ; Flavones/pharmacology ; Human ; In Vitro ; Indomethacin/pharmacology ; Kinetics ; Mitogen-Activated Protein Kinases/metabolism ; Phosphorylation/drug effects ; Platelet Activating Factor/*pharmacology ; Platelet Activation/drug effects ; Platelet Aggregation/*drug effects/physiology ; Pyrrolidinones/pharmacology ; Serotonin/*pharmacology ; Thromboxane A2/biosynthesis ; Verapamil/pharmacology