Figure 1.
The endocannabinoid anandamide inhibits platelet aggregation.
Washed platelets were exposed to a concentration series of anandamide for 40 minutes at 37°C. Subsequently, they were stimulated with 0.5 or 5 µg/mL collagen (A; representative aggregation curves of 0.5 µg/mL collagen stimulation are shown in Figure S1A); 0.1 or 1 µg/mL CRP-XL (B; representative aggregation curves of 0.1 µg/mL CRP-XL are shown in Figure S1B); 10 µM Arachidonic Acid, (AA) (C; representative aggregation curves are shown in Figure S1C); 10 or 100 µM ADP (D; representative curves of 10 µM are shown in Figure S1D); 1 or 10 µM U46619 (E; representative aggregation curves of 1 µM U46619 are shown in Figure S1E); 1, 2.5, 5 or 10 µM TRAP (F; representative aggregation curves of 2.5 µM are shown in Figure S1F), 0.1 U/mL thrombin or anandamide only (G; representative aggregation curves are shown in Figure S1G). Data were normalized to 100% platelet aggregation obtained with the agonists alone in the presence of vehicle. The time-dependence of anandamide inhibition was investigated by incubating washed platelets with 50 µM anandamide or vehicle (DMSO) at 37°C in a time series and subsequently stimulated with 0.5 µg/mL collagen (H). Data are mean ± SD and show 3 or more individual experiments.
Figure 2.
Anandamide inhibits platelet α-granule secretion and limits platelet aggregate formation.
Platelet α-granule release was studied by flow cytometric analysis of platelet P-selectin externalization. Washed platelets from healthy human donors were pre-exposed to a concentration series of anandamide for 40 min at 37°C and subsequently stimulated with 2.5 µg/mL CRP-XL (A), 2.5 µM U46619 (B) or 5 µM TRAP (C). Data were normalized to the agonist-induced P-selectin expression of vehicle-pretreated control platelets. Reconstituted blood, which was preincubated with anandamide, was perfused over a collagen-coated surface at a shear rate of 1600 s−1 and snapshots were taken after 5 minutes (D). Subsequently, cover slips were rinsed with buffer for 1 minute to investigate aggregate stability (E). The left panel shows vehicle control, the right panel indicates a perfusion in the presence of 50 µM anandamide. Data are shown as mean ± SD and represent 3 or more individual experiments.
Figure 3.
Anandamide reduces glycoprotein IIb/IIIa activation and inhibits platelet spreading.
Washed platelets from healthy human donors were exposed to a concentration series (0–50 µM) of anandamide for 40 minutes at 37°C and subsequently stimulated with 2.5 µg/mL CRP-XL (A), 2.5 µM U46619 (B) or 5 µM TRAP (C). Platelet GPIIb/IIIa activation was studied by flow cytometry. Data were normalized to the agonist-induced GPIIb/IIIa activation of vehicle-pretreated control platelets. Washed platelets were preincubated with 50 µM anandamide or vehicle for 40 minutes at 37°C and perfused over immobilized fibrinogen- at a shear rate of 25 s−1. Subsequently, differential interference contrast microscopy images were taken (D; representative images after 15 minutes of perfusion, scale bars represent 10 µm). Quantification of the spreading behaviour of individual platelets on immobilized fibrinogen (E; spreading was quantified for 3 individual platelets per separate experiment). Quantification of the amount of adhering platelets per field (F). Data represent mean and standard deviation (SD) of 3 or more individual experiments.
Figure 4.
Anandamide inhibits glycoprotein VI-dependent calcium mobilization and Syk-phosphorylation.
Washed platelets were preincubated with 50 µM anandamide or vehicle for 40 minutes at 37°C. Subsequently, they were exposed to 1 µg/mL collagen, 500 ng/mL CRP-XL, 20 µM ADP, 10 µM U46619, 10 µM TRAP or 1 U/mL thrombin and calcium mobilization was monitored. The (residual) calcium mobilization in the presence of anandamide is expressed as a percentage of uninhibited vehicle control in the presence of the same platelet agonist (A). Western blot analysis of collagen-induced Syk phosphorylation in the presence of vehicle, 50 µM anandamide or control Syk phosphorylation inhibitor PP2 (B; upper lanes indicate phosphorylated Syk (Syk-P), the lower lanes indicate total Syk antigen as a loading control (Syk-T)). Densitometric quantification of collagen-induced Syk-phosphorylation in the presence or absence of anandamide or PP2 (C; n = 4: data are expressed as mean +/− SD).
Figure 5.
The inhibitory effect of anandamide on platelet activation is not dependent on platelet preactivation.
Washed platelets were pretreated with 25 µM FAAH-inhibitor URB597. Subsequently, platelet aggregation was induced by various agonists after a suboptimal exposure to anandamide (10 µM, preincubated for 40 minutes). 0.5 µg/mL collagen (A), 0.1 µg/mL CRP-XL (B), 10 µM AA (C), 10 µM ADP (D), 1 µM U46619 (E) or 1 µM TRAP (F). In further experiments, washed platelets were pretreated with 10 µM indomethacin, exposed to anandamide (10 µM, preincubated for 40 minutes) and stimulated with collagen (0.5 µg/mL: G).
Figure 6.
Cannabis sativa consumption limits platelet aggregate formation under flow and reduces platelet responsiveness to collagen.
Whole blood from Cannabis sativa consumers (n = 4; “Consumers”) or healthy control donors (“Controls”) was perfused for 5 minutes over immobilized collagen at a shear rate of 1600 s−1 (A). In further experiments, collagen-induced platelet aggregation was investigated in platelet-rich plasma from these Cannabis sativa consumers and controls (B). Representative aggregation curves are shown in panel C. Finally, collagen-induced platelet aggregation was studied in platelet-rich plasma of three self-reported Cannabis sativa consumers on two separate instances: once after 10 days of daily consumption of Cannabis sativa (“Use”), as well as after a period of 10 days without consumption (“Withdrawal”).