Endocannabinoid hydrolases are differentially distributed in human blood fractions and differentially influenced by thrombin

Abstract

Plasma levels of the endocannabinoids anandamide and 2‐arachidonoylglycerol have been reported in a number of studies using both humans and laboratory animals, with typically much higher levels of 2‐arachidonoylglycerol than anandamide. In this investigation, we sought to characterize activities of the two primary endocannabinoid hydrolases, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) responsible for anandamide and 2‐arachidonoylglycerol hydrolysis, respectively, in different fractions from human blood.Following approval from the School of Life Sciences Ethics Committee, University of Nottingham, overnight fasting blood samples were taken in the morning from otherwise healthy volunteers. Blood fractions were isolated, including platelet‐rich plasma, packed erythrocytes (RBC), washed platelets, plasma and serum. Thrombin was employed to trigger clot formation, particularly with RBC and platelet‐rich plasma. Following the generation of a clot, the supernatant layer from a low speed centrifugation, equivalent to serum, was harvested and assayed for endocannabinoid hydrolase activity. The activities of FAAH and MAGL were quantified using both radiometric and ABPP assays.Of the blood fractions examined, MAGL activity was highest in platelets (92 % of the MAGL activity in whole blood), while FAAH activity was highest in RBC. Intriguingly, expressing the FAAH activity of various fractions relative to whole blood summated to 854 %, with RBC constituting 824 % of the whole blood activity. There were no differences between male and female volunteers in the levels and pattern of enzyme activities in the different blood fractions. Assessing enzyme activities from individual donors in these fractions on three visits separated by two week intervals showed no significant differences. The level of FAAH and MAGL activities was relatively stable in washed platelet and RBC samples after being stored in liquid nitrogen, while more than 50 % of their activities were lost after 3 days of storage at −80 °C.Interestingly, FAAH activity was low but detectable, while MAGL was essentially undetectable in serum. Thrombin addition to a washed platelet or RBC fraction caused clot formation within 5 min. FAAH activity in the supernatant fraction following thrombin exposure was much higher from RBC relative to platelets, in accordance with the activities in those fractions. In contrast, the activity of MAGL was significantly reduced in RBC and platelet fractions induced by thrombin compared to nonactivated erythrocytes and platelets, with negligible activities present in the releasate.In summary, we have identified a differential distribution of endocannabinoid hydrolases across different fractions of human blood, and identified the potential for FAAH, but not MAGL, release from RBC and platelets during thrombus formation.Support or Funding InformationFunded by the Saudi Cultural Bureau