Moroi A J, Watson S P
Centre for Cardiovascular Science, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK.
J Thromb Haemost. 2015 Jun;13(6):1139-50. doi: 10.1111/jth.12954. Epub 2015 May 9.
The C-type lectin-like receptor 2 (CLEC-2) and the collagen receptor glycoprotein (GP)VI activate platelets through Src and Syk tyrosine kinases, and phospholipase Cγ2. The initial events in the two signaling cascades, however, are distinct, and there are quantitative differences in the roles of proteins downstream of Syk activation. The activation of Akt and mitogen-activated protein kinases (MAPKs) has been shown to enhance platelet activation by GPVI, but their role in CLEC-2 signaling is not known.
We sought to investigate the role of the Akt and MAPK pathways in platelet activation by CLEC-2.
The CLEC-2 agonist rhodocytin stimulated phosphorylation of Akt and p38 and extracellular signal-related kinase (ERK) MAPKs, but with a delay relative to Syk. Phosphorylation of these proteins was markedly inhibited in the combined presence of apyrase and indomethacin, consistent with the reported feedback action of ADP and thromboxane A2 in CLEC-2 signaling. Phosphorylation of Akt and phosphorylation of ERK were blocked by the phosphoinositide 3-kinase (PI3K) inhibitor wortmannin and the protein kinase C (PKC) inhibitor Ro31-8220, respectively, whereas Syk phosphorylation was not altered. On the other hand, both inhibitors reduced phosphorylation of the Akt substrate glycogen synthase kinase 3α/β (GSK3α/β). Phosphorylation of GSK3α/β was also blocked by the Akt inhibitor MK2206, and reduced at late, but not early, times by the MEK inhibitor PD0325901. MK2206 and PD0325901 inhibited aggregation and secretion in response to a low concentration of rhodocytin, which was restored by GSK3α/β inhibitors.
These results demonstrate that CLEC-2 regulates Akt and MAPK downstream of PI3K and PKC, leading to phosphorylation and inhibition of GSK3α/β, and enhanced platelet aggregation and secretion.
C型凝集素样受体2（CLEC-2）和胶原受体糖蛋白（GP）VI通过Src和Syk酪氨酸激酶以及磷脂酶Cγ2激活血小板。然而，这两个信号级联反应的初始事件是不同的，并且在Syk激活下游的蛋白质作用方面存在定量差异。已证明Akt和丝裂原活化蛋白激酶（MAPK）的激活可增强GPVI介导的血小板激活，但它们在CLEC-2信号传导中的作用尚不清楚。
我们试图研究Akt和MAPK途径在CLEC-2介导的血小板激活中的作用。
CLEC-2激动剂红豆细胞凝集素刺激Akt、p38和细胞外信号调节激酶（ERK）MAPK的磷酸化，但相对于Syk存在延迟。在同时存在腺苷双磷酸酶和吲哚美辛的情况下，这些蛋白质的磷酸化受到明显抑制，这与报道的ADP和血栓素A2在CLEC-2信号传导中的反馈作用一致。Akt的磷酸化和ERK的磷酸化分别被磷脂酰肌醇3激酶（PI3K）抑制剂渥曼青霉素和蛋白激酶C（PKC）抑制剂Ro31-8220阻断，而Syk的磷酸化未改变。另一方面，两种抑制剂均降低了Akt底物糖原合酶激酶3α/β（GSK3α/β）的磷酸化。GSK3α/β的磷酸化也被Akt抑制剂MK2206阻断，并且在晚期（而非早期）被MEK抑制剂PD0325901降低。MK2206和PD0325901抑制了对低浓度红豆细胞凝集素的聚集和分泌反应，而GSK3α/β抑制剂可恢复这种反应。
这些结果表明，CLEC-2在PI3K和PKC下游调节Akt和MAPK，导致GSK3α/β的磷酸化和抑制，并增强血小板聚集和分泌。
