The endocannabinoid 2-arachidonoylglycerol activates human platelets through non-CB1/CB2 receptors

Summary. Background:  The endocannabinoid 2-arachidonoylglycerol (2-AG) is an endogenous lipid that acts through the activation of G-protein-coupled cannabinoid receptors and plays essential roles in many physiological contexts. In the cardiovascular system 2-AG is generated by both activated endothelial cells and platelets, and participates in the regulation of inflammation and thrombosis. Although human platelets actively metabolize endocannabinoids, 2-AG also binds to platelet surface and leads to cell activation. Objective: To investigate the biological consequence of 2-AG interactions with human platelets and to clarify the role of cannabinoid receptors. Methods: Gel-filtered platelets were stimulated with 2-AG in the presence or absence of various inhibitors. Platelet aggregation and secretion were measured in a lumiaggregometer. Calcium ion movements were measured in FURA-2 loaded platelets. Thromboxane A2 (TxA2) generation was evaluated as Thromboxane B2 accumulation with a commercial EIA assay. Results: 2-AG induced platelet shape change, aggregation and secretion with a dose-dependent mechanism that required engagement of platelet TxA2 receptors. 2-AG caused also cytosolic calcium increase; however, it was totally dependent on availability of TxA2. Indeed 2-AG was able to induce a robust generation of TxA2 through the cyclooxygenase pathway. Treatment of platelets with inhibitors of monoacylglycerol lipase and fatty acid amide hydrolase did not affect the activation induced by 2-AG. Moreover, neither CB1 and CB2 proteins nor CB1/CB2 mRNAs were detected in platelets. Conclusions: 2-AG can be considered a new physiologic platelet agonist able to induce full platelet activation and aggregation with a non-CB1/CB2 receptor-mediated mechanism.