Morphological evidence that 2-arachidonoylglycerol is a true agonist of human platelets

The endocannabinoids anandamide and 2-arachidonoylglycerol (2AG) have important pathophysiological roles in the cardiovascular system (1-3). Human platelets have the tools to bind and metabolize endocannabinoids that have been proposed as physiological (co-) agonists of these cells (4). In fact, physiological concentrations of 2AG have been shown to activate human platelets in platelet-rich plasma through a “platelet-type” cannabinoid (CBPT) receptor (5). Conversely, some of the observed signs of platelet agglutination or the increase in optical density have been interpreted either as markers of a true aggregation or as symptoms of apoptosis (6, 7). Therefore, we have undertaken an ultrastructural study in order to get conclusive morphological evidence that 2AG is a true agonist of human platelets. Treatment of human platelet-rich plasma with a physiological concentration (200 μM) of 2AG (5) led to the following morphological changes: i) At just 6 min after drug exposure, remarkable alterations of the cell surface were detectable by scanning electron microscopy. These were mainly represented by long and thin cell protrusions that represent typical signs of an “activation” state (Fig. 1A, B); ii) Quantitative morphometric evaluation of the phenomenon (8) clearly indicated that soon after administration of 2AG (6 min) a significant increase of platelets showing protrusions at least 2-fold longer than the cell body was detectable (Fig. 1C). This was paralleled by a significant rearrangement in the main cytoskeletal component involved in cell shape maintenance, i.e. the actin cytoskeleton. In fact, intensified charge-coupled device video microscopy (IVM) analyses (Fig. 1D, E) clearly showed the presence of long actin-positive protrusions (inset in Fig. 1E) and a marked marginalization and patching of actin network at the cell periphery in 2AG-treated cells (Fig. 1E), with respect to control cells (Fig. 1D). This “qualitative” remodeling was accompanied by changes in the G/F (i.e., monomeric/polymeric) actin ratio within platelet cytoplasm. In fact, a significantly lower content of G-actin (Fig. 1F) was detectable in 2AG-treated platelets, while a significant increase of F-actin (Fig. 1G) was observed. However, the total amount of actin molecules, as detected by using anti-actin monoclonal antibodies, remained substantially unchanged (Fig. 1H). This means that an imbalance of monomeric/polymeric actin in favour of the latter clearly occurs in the presence of 2AG, leading to an increased formation of actin filaments and cell protrusions. Instead, actin depolymerization without formation of filopodia and cell surface protrusions should be expected in platelets undergoing apoptosis (6, 7). Therefore, on the basis of reported data, a proapoptotic activity of 2AG on human platelets can be excluded, and 2AG can be considered a true agonist of these cells. Finally, 2AG did not change platelet morphology in the presence of CBPT receptor antagonists SR141716 and SR144528 (5), used at a concentration (10 μM) known to prevent the binding of 200 μM 2AG to the same receptor (not shown). Thus, CBPT receptors are engaged by 2AG in order to activate platelets. In conclusion, this ultrastructural study presents conclusive morphological evidence that 2AG is a true agonist of human platelets.