2-arachidonyl glycerol activates platelets via conversion to arachidonic acid and not by direct activation of cannabinoid receptors

Abstract

What is already known about this subject: There are conflicting views in the literature as to whether cannabinoids have an impact on platelet activity and to what extent cannabinoid receptors are involved. This is an important issue to resolve because platelet effects of putative therapeutic cannabinoid inhibitors and stimulators will have an impact on their potential benefits and safety.

What this paper adds: The data presented in this manuscript clearly show that the endocannabinoid 2-arrachidonyl glycerol can activate platelet activity, but that the effects are mediated through an aspirin-sensitive pathway that is not affected by cannabinoid receptor antagonists or FAAH inhibition, but is abolished by MAGL inhibition. The findings question the role of cannabinoid receptors in platelet function and suggest that platelet function is unlikely to be directly affected by cannabinoid receptor antagonists, at least in the acute phase.

Aims: Cannabinoid receptor-1 (CB(1)) antagonists suppress appetite and induce weight loss. Direct antagonism of CB(1) receptors on platelets might be an additional benefit for CB(1) antagonists, but the role of CB(1) receptors in platelets is controversial. We tested the hypothesis that the endocannabinoid, 2-arachidonyl glycerol (2-AG), induces platelet aggregation by a COX-mediated mechanism rather than through CB(1) receptor activation, in blood obtained from healthy volunteers and patients with coronary artery disease receiving low dose aspirin.

Methods: Aggregatory responses to the cannabinoids 2-AG and Delta(9)-THC were examined in blood sampled from healthy volunteers (n= 8) and patients (n= 12) with coronary artery disease receiving aspirin using whole blood aggregometry. The effects of CB(1) (AM251) and CB(2) (AM630) antagonists, as well as fatty acid amide hydrolase (FAAH) and monoacyl glycerol lipase (MAGL) inhibitors and aspirin on 2-AG-induced aggregation were also assessed.

Results: AM251 (100 nm-30 microm) had no effect on platelet aggregation induced by either ADP (P= 0.90) or thrombin (P= 0.86). 2-AG, but not Delta(9)-THC, induced aggregation. 2-AG-induced aggregation was unaffected by AM251 and AM630 but was abolished by aspirin (P < 0.001) and by the MAGL inhibitor, URB602 (P < 0.001). Moreover, the aggregatory response to 2-AG was depressed (by >75%, P < 0.001) in blood from patients with coronary artery disease receiving aspirin compared with that from healthy volunteers.

Conclusions: 2-AG-mediated activation of platelets is via metabolism to arachidonic acid by MAGL, and not through direct action on CB(1) or CB(2) receptors, at least in the acute phase.

Figure 2

The aggregatory effect of (A) ADP (20 µm) and (B) thrombin (0.25 U ml⁻¹) on their own (control) and after pre-incubation with AM251 (100 nM–30 µm), in whole blood samples from healthy human volunteers (n= 6). It is important to note that data were standardized by subtracting vehicle-induced aggregation in each case and are expressed as the mean ± SEM; statistical analysis was performed using a one-way anova and no significance was found [(A) P= 0.90; (B) P= 0.86]

Figure 1

The aggregatory effect of 2-AG (200 µm) on its own (control) and after pre-incubation with AM251 (100 nm–30 µm), in whole blood samples from healthy human volunteers (n= 6). It is important to note that data were standardized by subtracting vehicle-induced aggregation in each case and are expressed as the mean ± SEM; statistical analysis was performed using a one-way anova and no significance was found (P= 0.78)

Figure 3

The aggregatory effect of 2-AG on its own (control) and after incubation with (A) AM251 (0.3, 3, 30 µm) and (B) AM630 (0.3, 3, 30 µm) at different time points (10–120 min), in whole blood samples from healthy human volunteers (n= 8; solid lines) and patients (n= 12; dotted lines). Data were standardized by subtracting vehicle-induced aggregation in each case and are expressed as the mean ± SEM; statistical analysis was performed using a two-way anova. Statistical significance was observed (***P < 0.001) in the difference between the average 2-AG-induced aggregation of healthy volunteers compared with patients for both drugs at all three concentrations of AM251 or AM630, as well as with the control. (A) Control (); AM251 30 µm (); AM251 3 µm (); AM251 300 nm (); (B) Control (); AM630 30 µm (); AM630 3 µm (); AM630 300 nm ()

Figure 4

The inhibitory effect of aspirin (100 µm) on (A) 2-AG (30, 100 and 200 µm) and (B) AA (100, 300 and 750 µm) induced aggregation (n= 8). Data were standardized by subtracting vehicle-induced aggregation in each case and are expressed as the mean ± SEM; statistical analysis was performed using a two-way anova. 2-AG-induced aggregation was significantly inhibited by aspirin at concentrations of 100 µm (***P < 0.001) and 200 µm (*P < 0.05). AA-induced aggregation was significantly inhibited at all three concentrations, 100 µm (*P < 0.05), 300 µm (***P < 0.001) and 750 µm (**P < 0.01). Control (); + aspirin 100 µm ()

Figure 5

The inhibitory effect of URB597 (300 nm), URB602 (300 µm) and aspirin (100 µm) on 2-AG (200 µm)-induced aggregation in whole blood and platelet rich plasma. 2-AG-induced aggregation was inhibited by URB602, [URB597 + URB602] and aspirin in both whole blood and platelet rich plasma. ***P < 0.001 (two-way anova with Bonferoni post-test; n= 5). whole blood (); PRP (□)