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 (CBPT). Here, washed human platelets were used to characterize the binding of [H-3]2-AG to CBPT, 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 CBI and CB2 cannabinoid receptors inhibited this binding, which was enhanced up to similar to230% over the controls by 1 muM serotonin (5-hydroxytryptamine, 5-HT). Human platelets were also able to bind [H-3]5-HT (Kd = 79 +/- 17 nM, Bmax = 14.6 +/- 1.3 pmol.mg protein(-1)), and 1 muM 2-AG enhanced this binding up to similar to150%. Moreover, they were able to take Up [3H]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 muM 2-AG led to increased intracellular inositol-1,4,5-trisphosphate (up to similar to300%) and decreased cyclic AMP (down to similar to50%). Furthermore, treatment of pre-loaded platelets with 1 muM 2-AG induced a similar to300% increase in [H-3]2-AG release, according to a CBPT-dependent mechanism. Also, 1 muM 5-HT enhanced the effect of 2-AG on inositol-1,4,5-trisphosphate (similar to500% of the controls), cyclic AMP (similar to20%) and [H-3]2-AG release (similar to570%), and the latter process was shown to be partly (similar to50%) 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.