Activation of human platelets by 2-arachidonoylglycerol is enhanced by serotonin

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.