The 2-arachidonoylglycerol effect on myosin light chain phosphorylation in human platelets.

In human platelets the endocannabinoid 2-arachidonoylglycerol (2-AG) stimulates some important pathways leading to thromboxane B2 formation, calcium intracellular elevation, ATP secretion and actin polymerisation. The aim of the present study was to examine the 2-AG effect on myosin light chain (MLC) phosphorylation and to investigate the mechanisms involved. We demonstrated that 2-AG induced a rapid MLC phosphorylation, stimulating both the RhoA kinase (ROCK) and MLC kinase (MLCK) in a dose and time-dependent manner. In addition MLC phosphorylation was strengthened through the MLC phosphatase inhibition. MLC phosphatase inhibition was accomplished through the RhoA/ROCK and protein kinase C mediated phosphorylation of MLC phosphatase inhibiting subunits MYPT1 and CPI-17. The presence of CB1 receptor in human platelets and the involvement of CB1 receptor in MLC phosphorylation and MLC phosphatase inhibition was shown.

Activation of human platelets by 2-arachidonoylglycerol: role of PKC in NO/cGMP pathway modulation.

We demonstrated that the endocannabinoid 2-arachidonoylglycerol (2-AG) activated dose-dependently washed human platelets and increased intracellular calcium levels. Moreover 2-AG activated protein kinase C measured as p47pleckstrin phosphorylation. These parameters were prevented by the tromboxane A2 receptor antagonist SQ29548, by phospholipase C pathway (U73122) and protein kinase C (GF109203X) inhibitors. No effect on 2-AG-induced platelet activation and calcium elevation in the presence of inhibitors of fatty acid amide hydrolase or monoacylglycerol lipase was observed. In addition we have shown that 2-AG dose-dependently increased NO and cGMP levels. These effects were abolished by U73122, GF109203X, EGTA and the intracellular calcium chelator BAPTA/AM. Moreover, 2-AG enhanced eNOS activity through the phosphorylation of its positive regulatory residue ser1177 and by dephosphorylation of the negative one thr495. The eNOS ser1177 phosphorylation was inhibited by U73122 and GF109203X but it was unaffected by the PI3K/AKT pathway inhibitors LY294002 and MK2206. The dephosphorylation of thr495 was reversed by low concentrations of calyculin A. Taken together these data suggest that 2-AG behaves as a true platelet agonist stimulating PKC activation and calcium elevation. Likely 2-AG can modulate platelet activation by increasing NO levels through eNOS activation.

Activation by 2-arachidonoylglycerol of platelet p38MAPK/cPLA2 pathway.

The endogenous cannabinoid 2-arachidonoylglycerol (2-AG) is described as a platelet agonist able to induce aggregation and to increase intracellular calcium. In the present report we have confirmed these data and demonstrated that the inhibitor of p38MAPK SB203580 and the inhibitor of cPLA(2) metabolism ETYA affect both these parameters. Thus, we aimed to define the role of p38MAPK/cytosolic phospholipase A(2) (cPLA(2)) pathway in 2-AG-induced human platelet activation. p38MAPK activation was assayed by phosphorylation. cPLA(2) activation was assayed by phosphorylation and as arachidonic acid release and thromboxane B(2) formation. It was shown that 2-AG in a dose- and time-dependent manner activates p38MAPK peaking at 10 µM after 1 min of incubation. The 2-AG effect on p38MAPK was not impaired by apyrase, indomethacin or RGDS peptide but it was significantly reduced by SR141716, specific inhibitor of type-1 cannabinoid receptor and unaffected by the specific inhibitor of type-2 cannabinoid receptor SR144528. Moreover, the incubation of platelets with 2-AG led to the phosphorylation of cPLA(2) and its activation. Platelet pretreatment with SB203580, inhibitor of p38MAPK, abolished both cPLA(2) phosphorylation and activation. In addition SR141716 strongly impaired cPLA(2) phosphorylation, arachidonic acid release and thromboxane B(2) formation, whereas SR144528 did not change these parameters. Finally platelet stimulation with 2-AG led to an increase in free oxygen radical species. In conclusion, data provide insight into the mechanisms involved in platelet activation by 2-AG, indicating that p38MAPK/cPLA(2) pathway could play a relevant role in this complicated process.

The anandamide effect on NO/cGMP pathway in human platelets.

In this study the effect of the endocannabinoid anandamide on platelet nitric oxide (NO)/cGMP pathway was investigated. Data report that anandamide in a dose-and time-dependent manner increased NO and cGMP levels and stimulated endothelial nitric oxide synthase (eNOS) activity. These parameters were significantly reduced by LY294002, selective inhibitor of PI3K and by MK2206, specific inhibitor of AKT. Moreover anandamide stimulated both eNOSser1177 and AKTser473 phosphorylation. Finally the anandamide effect on NO and cGMP levels, eNOS and AKT phosphorylation/activation were inhibited by SR141716, specific cannabinoid receptor 1 antagonist, supporting the involvement of anandamide binding to this receptor. Overall data of this report indicate that low concentrations of anandamide, through PI3K/AKT pathway activation, stimulates eNOS activity and increases NO levels in human platelets. In such way anandamide contributes to extend platelet survival.

In retinal vein occlusion platelet response to thrombin is increased.

INTRODUCTION: Retinal vein occlusion is a major cause of ocular morbidity. The precise mechanism leading to thrombosis in retinal vein occlusion has not yet been clearly elucidated. Several risk factors have been identified, including hypertension diabetes, history of cardiovascular disease, hypercholesterolemia, hyperhomocysteinaemia, increased ocular pressure and glaucoma. Although thrombus formation in the vein plays a significant role in the onset of retinal vein occlusion, the relationship between platelet aggregation and retinal vein occlusion remains to be clarified. MATERIALS AND METHODS: In the present study the platelet response to thrombin in a selected group of retinal vein occlusion patients was investigated. Retinal vein occlusion patients were compared to a group of healthy subjects matched for age, sex, clinical and metabolic characteristics. In resting and activated platelets of both groups of subjects total protein tyrosine phosphorylation, p38MAPK and cytosolic phospholipase A(2) phosphorylation, arachidonic acid release, intracellular calcium levels, thromboxane B(2) and superoxide anion formation were measured. RESULTS: Results show that platelets of patients were more responsive to thrombin than healthy subjects. In resting or in thrombin stimulated platelets of patients total protein tyrosine phosphorylation, p38MAPK and cytosolic phospholipase A(2) phosphorylation were increased. Also arachidonic acid release, thromboxane B(2) and superoxide anion formation were higher in patients than in healthy subjects. In addition intracellular calcium rise induced by thrombin was increased in patients. CONCLUSIONS: Altogether data suggest that platelet hyperaggregability inducing thrombus formation might be an important factor in the onset and/or development of retinal vein occlusion.