Endocannabinoids and platelet aggregation: effects of 2-arachidonylglycerol in rat whole blood

Jennifer Shearer, Kathleen Kane, Hilary Carswell, Susan Coker. University of Strathclyde, Glasgow, United Kingdom.

Activated human platelets release the endocannabinoid, 2-arachidonylglycerol (2 AG), which can modulate platelet aggregation. There is controversy, however, about the involvement of cannabinoid receptors and thromboxane A2, in the response to 2 AG (Maccarrone et al., 2001; Baldassarri et al., 2008). The aim of the present work was therefore to characterise the effects of 2 AG on rat platelet aggregation, investigate interactions with other aggregating agents and explore possible mechanisms. Male Sprague Dawley rats (300-500g) were anaesthetized with isoflurane and a carotid artery was cannulated to allow blood withdrawal. Impedence aggregometry was used to study the effects of 2 AG, adenosine diphosphate (ADP) and the interactions among 2 AG, ADP and 5-hydroxytryptamine (5-HT) in heparinised rat whole blood which was diluted 50:50 with normal saline. Data are expressed as mean ± s.e. mean (n = 6-7) and compared with an unpaired Student’s t-test or a repeated measure 2-way ANOVA (*P<0.05). 2 AG caused slowly developing aggregation that peaked at 10 min in contrast to the response to ADP which peaked at 2 min. Maximal responses to 75, 150 and 300 µM 2 AG were 9.8 ± 3.4, 13.2 ± 0.9 and 14.0 ± 0.7Ω. The aggregation induced by 150 µM 2 AG was reduced to 2.0 ± 1.7*Ω by 1 µM AM251 (CB1 antagonist) and to 0.6 ± 0.5*Ω by 1 µM AM630 (CB2 antagonist). 5-HT did not cause platelet aggregation but it potentiated the response to 75 µM 2 AG at 5 min from 5.1 ± 2.8 to 12.4 ± 0.7*Ω. In a further set of experiments, the cyclooxygenase inhibitor indomethacin (3 µM) reduced aggregation induced by 150 µM 2 AG from 9.3 ± 2.4 to 3.2 ± 1.9*Ω and the TP antagonist ICI 192,605 (1 µM) abolished the 2 AG response (-0.5 ± 0.1*Ω). 2 AG prolonged and enhanced the aggregatory response to ADP. At 10 min the responses induced by 2 AG (75 µM), ADP (1 µM) and 2 AG + ADP were 1.8 ± 1.8, 2.2 ± 0.9 and 13.6 ± 1.1*Ω respectively. This potentiation was attenuated by ICI 192,605 to 9.4 ± 1.8*Ω but modestly enhanced by indomethacin to 16.1 ± 0.8*Ω. These results demonstrate that the endocannabinoid, 2 AG, caused platelet aggregation in rat whole blood and that responses to 2 AG can be potentiated by 5-HT and ADP. The experiments on the possible mechanisms suggest that the effects of 2 AG on rat platelet aggregation measured in whole blood involve CB1 and CB2 receptors and the production of thromboxane A2.

Maccarrone et al., (2001) Eur J Biochem 268: 819-825.
Baldassarri et al., (2008) J Thromb Haemostas 6: 1772-1779.