Novel Lyn-Mediated Regulation of CLEC-2 Signaling By G_q-Coupled Receptors in Platelets

The CLEC-2 agonist, rhodocytin, elicits powerful platelet activation signals in conjunction with Src family kinases, Syk, and PLCg2. Previous reports have shown that rhodocytin-induced aggregation is dependent on secondary mediators. However, the role of secondary mediators in CLEC-2 signaling is not defined. In this study we report that CLEC-2-induced Syk activation is aspirin-sensitive and that TxA2 plays an important role in the most proximal events such as CLEC-2 phosphorylation and Syk activation. We also show that the activation of other GPCRs, such as the ADP receptors and PARs, can also potentiate CLEC-2 signaling. By using the G_q-inhibitor, UBO-QIC, or P2Y1/P2Y₁₂ antagonists, we show that the G_q-signaling, but not G_12/13 or G_i, is essential for GPCR-induced Syk phosphorylation downstream of CLEC-2. We further elucidated the signaling involved in G_q-mediated Syk phosphorylation and identified an important role for PKCs downstream of PLCb regulating SFK activation (Figure 1A). Using Lyn-knock out murine platelets we identified a potential role for Lyn downstream of the G_q-pathway in potentiating CLEC-2 signaling by using. We suggest that, at low concentration of CLEC-2 agonist, the unclustered CLEC-2 receptors are phosphorylated by the G_q-activated Lyn resulting in the potentiation of the initial CLEC-2 signal (Figure 1B). However, G_q receptors by themselves cannot phosphorylate CLEC-2 receptors and require minimal levels of ITAM-containing receptor stimulation in order to initiate unclustered CLEC-2 receptor phosphorylation. Together, these results provide evidence for a novel Lyn-mediated regulation of CLEC-2 signaling by G_q-coupled receptors thereby leading to potentiation of CLEC-2-induced signaling (Figure 1C).

Figure 1

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A) Western blot showingeffect G_q-pathway inhibitors on CLEC-2 signaling. B) Effect of low concentration of CLEC-2 agonist in Lyn-knock out murine platelets. C) Model showing role of G_q-activated Lyn in CLEC-2 signaling.

Figure 1

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A) Western blot showingeffect G_q-pathway inhibitors on CLEC-2 signaling. B) Effect of low concentration of CLEC-2 agonist in Lyn-knock out murine platelets. C) Model showing role of G_q-activated Lyn in CLEC-2 signaling.

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