title: Activation of human platelets by 2-arachidonoylglycerol is enhanced by serotonin.
abstract: The endocannabinoid 2-arachidonoylglycerol (2-AG) has been shown to activate human platelets in platelet-rich plasma, by binding to a "platelet-type" cannabinoid receptor (CB(PT)). Here, washed human platelets were used to characterize the binding of [(3)H]2-AG to CB(PT), showing a dissociation constant (Kd) of 140 +/- 31 nM and a maximum binding (Bmax) of 122 +/- 10 pmol.mg protein(-1). Selective antagonists of both CB1 and CB2 cannabinoid receptors inhibited this binding, which was enhanced up to approximately 230% over the controls by 1 micro M serotonin (5-hydroxytryptamine, 5-HT). Human platelets were also able to bind [(3)H]5-HT (Kd = 79 +/- 17 nM, Bmax = 14.6 +/- 1.3 pmol.mg protein(-1)), and 1 micro M 2-AG enhanced this binding up to approximately 150%. Moreover, they were able to take up [(3)H]5-HT through a high affinity transporter (Michaelis-Menten constant = 22 +/- 2 nM, maximum velocity = 344 +/- 15 pmol.min(-1).mg protein(-1)), which was not affected by 2-AG. Interestingly, 5-HT did not affect the activity of the 2-AG transporter of human platelets. Treatment of washed platelets with 1 micro M 2-AG led to increased intracellular inositol-1,4,5-trisphosphate (up to approximately 300%) and decreased cyclic AMP (down to approximately 50%). Furthermore, treatment of pre-loaded platelets with 1 micro M 2-AG induced a approximately 300% increase in [(3)H]2-AG release, according to a CBPT-dependent mechanism. Also, 1 micro M 5-HT enhanced the effect of 2-AG on inositol-1,4,5-trisphosphate ( approximately 500% of the controls), cyclic AMP ( approximately 20%) and [(3)H]2-AG release ( approximately 570%), and the latter process was shown to be partly ( approximately 50%) involved in the 5-HT-dependent platelet activation. Taken together, reported findings represent the first demonstration that 2-AG and 5-HT can mutually reinforce their receptor binding on platelet surface, which might have therapeutic implications.
authors.affiliation: Department of Biomedical Sciences, University of Teramo, Teramo, Italy. Maccarrone@vet.unite.it
authors.forename: Mauro
authors.initials: M
authors.lastname: Maccarrone
authors.forename: Monica
authors.initials: M
authors.lastname: Bari
authors.forename: Domenico
authors.initials: D
authors.lastname: Del Principe
authors.forename: Alessandro
authors.initials: A
authors.lastname: Finazzi-Agrò
mesh_terms: D000328:Adult; D001095:Arachidonic Acids; D001792:Blood Platelets / Q000378:metabolism; D063385:Cannabinoid Receptor Modulators; D002352:Carrier Proteins / Q000378:metabolism; D000242:Cyclic AMP / Q000096:biosynthesis; D004357:Drug Synergism; D063388:Endocannabinoids; D005231:Fatty Acids, Unsaturated / Q000502:physiology; D005989:Glycerides / Q000494:pharmacology*; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000096:biosynthesis; D007700:Kinetics; D008297:Male; D008562:Membrane Glycoproteins / Q000378:metabolism; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D015539:Platelet Activation / Q000187:drug effects*; D043882:Receptors, Cannabinoid; D011955:Receptors, Drug / Q000187:drug effects / Q000502:physiology; D015290:Second Messenger Systems / Q000187:drug effects; D012701:Serotonin / Q000494:pharmacology*; D050486:Serotonin Plasma Membrane Transport Proteins
chemical_list: D001095:Arachidonic Acids; D063385:Cannabinoid Receptor Modulators; D002352:Carrier Proteins; D063388:Endocannabinoids; D005231:Fatty Acids, Unsaturated; D005989:Glycerides; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D043882:Receptors, Cannabinoid; D011955:Receptors, Drug; C493230:SLC6A4 protein, human; D050486:Serotonin Plasma Membrane Transport Proteins; D012701:Serotonin; D015544:Inositol 1,4,5-Trisphosphate; C094503:glyceryl 2-arachidonate; D000242:Cyclic AMP