Department of Experimental Medicine, Biochemistry Section, Genoa University, Genoa, Italy.
J Cell Biochem. 2011 Oct;112(10):2794-802. doi: 10.1002/jcb.23194.
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.
内源性大麻素 2-花生四烯酸甘油（2-AG）被描述为一种血小板激动剂，能够诱导聚集并增加细胞内钙。在本报告中，我们证实了这些数据，并表明 p38MAPK 的抑制剂 SB203580 和 cPLA2 代谢抑制剂 ETYA 均影响这两个参数。因此，我们旨在确定 p38MAPK/胞质型 PLA2（cPLA2）途径在 2-AG 诱导的人血小板激活中的作用。通过磷酸化来测定 p38MAPK 的激活。通过磷酸化以及花生四烯酸释放和血栓素 B2 形成来测定 cPLA2 的激活。结果表明，2-AG 以剂量和时间依赖性方式激活 p38MAPK，在孵育 1 分钟后达到 10 μM 的峰值。APYRASE、吲哚美辛或 RGDS 肽并不能削弱 2-AG 对 p38MAPK 的作用，但特异性 1 型大麻素受体抑制剂 SR141716 显著降低了该作用，2 型大麻素受体抑制剂 SR144528 则对其没有影响。此外，2-AG 孵育导致 cPLA2 的磷酸化和激活。p38MAPK 抑制剂 SB203580 预处理血小板可消除 cPLA2 的磷酸化和激活。此外，SR141716 强烈抑制 cPLA2 的磷酸化、花生四烯酸释放和血栓素 B2 形成，而 SR144528 则不改变这些参数。最后，2-AG 刺激血小板导致氧自由基的增加。总之，这些数据为 2-AG 诱导血小板激活的机制提供了深入的了解，表明 p38MAPK/cPLA2 途径可能在这个复杂的过程中发挥重要作用。
