title: Synergism interaction between arachidonic acid by 5-hydroxytryptamine in human platelet aggregation is mediated through multiple signalling pathways.
abstract: OBJECTIVE
  To examine the signalling mechanisms involved in the synergistic interaction of 5-hydroxytryptamine (5-HT) and arachidonic acid (AA) in human platelet aggregation.
  
  
  METHODS
  Blood was obtained from healthy human subjects, mixed with 3.8 % sodium citrate (9:1), and centrifuged to prepare platelet rich plasma (PRP). Aggregation was monitored using a Dual-channel Lumi-aggregometer. The agonist-induced influx of Ca2+ was measured using Fura-2 AM. TXA2 formation was studied using radiochemical method.
  
  
  RESULTS
  Subthreshold concentration of 5-HT (2 micromol/L) potentiated the effect of low dose of AA (0.2 mmol/L) in human platelets. This synergistic effect was blocked by 5-HT2 receptor antagonist (methysergide IC(50)=5.2 nmol/L; cyproheptadine IC(50)=0.6 nmol/L), and thromboxane A2 receptor antagonist (SQ 29 548; IC(50)=30 nmol/L), showing that the effect is receptor-mediated. To examine the down-stream signalling pathways, we found that such an interaction was inhibited by calcium channel blockers (diltiazem; IC(50)=3 micromol/L and verapamil; IC(50)=5 micromol/L), phospholipase C (PLC) inhibitor (U73122; IC50=4 micromol/L), cyclooxygenase inhibitor, (indomethacin; IC(50)=0.2 micromol/L) and mitogen-activated protein (MAP) kinase inhibitor (PD98059; IC(50)=3 micromol/L). The effect was also inhibited by a specific tyrosine light chain kinase (TLCK) inhibitor, herbimycin A with IC(50) value of 5 micromol/L. Pretreatment of platelet with 5-HT and AA induced rise in intracellular calcium and this effect was blocked by verapamil.
  
  
  CONCLUSIONS
  The synergism between 5-HT and AA in platelet aggregation involves activation of PLC/Ca2+, COX, and MAP kinase pathways.
authors.affiliation: Department of Biological and Biomedical Sciences, The Aga Khan University, PO Box 3500, Stadium Road Karachi-748700, Pakistan. arshad.saeed@aku.edu
authors.forename: Sheikh Arshad
authors.initials: SA
authors.lastname: Saeed
authors.forename: Huma
authors.initials: H
authors.lastname: Rasheed
authors.forename: Anwar-ul-Hassan
authors.initials: AU
authors.lastname: Gilani
mesh_terms: D016718:Arachidonic Acid / Q000494:pharmacology*; D019086:Bridged Bicyclo Compounds, Heterocyclic; D002121:Calcium Channel Blockers / Q000494:pharmacology; D003533:Cyproheptadine / Q000494:pharmacology; D004110:Diltiazem / Q000494:pharmacology; D004357:Drug Synergism; D004963:Estrenes / Q000494:pharmacology; D005231:Fatty Acids, Unsaturated; D006801:Humans; D006834:Hydrazines / Q000494:pharmacology; D008784:Methysergide / Q000494:pharmacology; D010974:Platelet Aggregation / Q000187:drug effects*; D011760:Pyrrolidinones / Q000494:pharmacology; D044244:Receptors, Thromboxane A2, Prostaglandin H2 / Q000037:antagonists & inhibitors; D012701:Serotonin / Q000494:pharmacology*; D058830:Serotonin 5-HT2 Receptor Antagonists; D015398:Signal Transduction; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors
chemical_list: D019086:Bridged Bicyclo Compounds, Heterocyclic; D002121:Calcium Channel Blockers; D004963:Estrenes; D005231:Fatty Acids, Unsaturated; D006834:Hydrazines; D011760:Pyrrolidinones; D044244:Receptors, Thromboxane A2, Prostaglandin H2; D058830:Serotonin 5-HT2 Receptor Antagonists; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D016718:Arachidonic Acid; D003533:Cyproheptadine; D012701:Serotonin; C045749:SQ 29548; D010738:Type C Phospholipases; D004110:Diltiazem; D008784:Methysergide