Abstract
Platelets play a key role in the physiological hemostasis or pathological process of thrombosis. Rhodocytin, an agonist of the C-type lectin-like receptor-2 (CLEC-2), elicits powerful platelet activation signals in conjunction with Src family kinases (SFKs), spleen tyrosine kinase (Syk), and phospholipase γ2 (PLCγ2). Previous reports have shown that rhodocytin-induced platelet aggregation depends on secondary mediators such as thromboxane A2 (TxA2) and ADP, which are agonists for G-protein-coupled receptors (GPCRs) on platelets. How the secondary mediators regulate CLEC-2-mediated platelet activation in terms of signaling is not clearly defined. In this study, we report that CLEC-2-induced Syk and PLCγ2 phosphorylation is potentiated by TxA2 and that TxA2 plays a critical role in the most proximal event of CLEC-2 signaling, i.e. the CLEC-2 receptor tyrosine phosphorylation. We show that the activation of other GPCRs, such as the ADP receptors and protease-activated receptors, can also potentiate CLEC-2 signaling. By using the specific Gq inhibitor, UBO-QIC, or Gq knock-out murine platelets, we demonstrate that Gq signaling, but not other G-proteins, is essential for GPCR-induced potentiation of Syk phosphorylation downstream of CLEC-2. We further elucidated the signaling downstream of Gq and identified an important role for the PLCβ-PKCα pathway, possibly regulating activation of SFKs, which are crucial for initiation of CLEC-2 signaling. Together, these results provide evidence for novel Gq-PLCβ-PKCα-mediated regulation of proximal CLEC-2 signaling by Gq-coupled receptors.
Highlights
Platelets play a key role in the physiological hemostasis or pathological process of thrombosis
We report that C-type lectin-like receptor-2 (CLEC-2)-induced spleen tyrosine kinase (Syk) and phospholipase ␥2 (PLC␥2) phosphorylation is potentiated by thromboxane A2 (TxA2) and that TxA2 plays a critical role in the most proximal event of CLEC-2 signaling, i.e. the CLEC-2 receptor tyrosine phosphorylation
We propose to investigate the mechanism of cross-talk between G-protein-coupled receptors and CLEC-2 during platelet activation induced by rhodocytin
Summary
Non-aspirinated platelets respond more robustly to rhodocytin than aspirin-treated platelets. In Gq knock-out murine platelets, there was no Syk/PLC␥2 phosphorylation observed when stimulated with rhodocytin (5 nM) for 1 min as compared with wild types (Fig. 3F) Together, these results suggest that the Gq pathways downstream of different GPCRs play an essential role in regulating Syk phosphorylation upon CLEC-2 receptor activation. There is enhanced phosphorylation of SFKs by U46619 over control This phosphorylation is significantly inhibited in the presence of either UBO-QIC or GF109203X, suggesting that the Gq-PKC pathway downstream of GPCRs regulates the SFKs. to study the effect of enhanced SFK activation on the CLEC-2 tyrosine phosphorylation, we co-stimulated platelets with rhodocytin and U46619, and looked at the CLEC-2 tyrosine phosphorylation, which is mediated by SFKs. Fig. The activity of the inhibitor was tested using known controls (data not shown)
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