Gq pathway regulates proximal C-type lectin-like receptor-2 (CLEC-2) signaling in platelets

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 G_q inhibitor, UBO-QIC, or G_q knock-out murine platelets, we demonstrate that G_q 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 G_q 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 G_q-PLCβ-PKCα-mediated regulation of proximal CLEC-2 signaling by G_q-coupled receptors.

Keywords: CLEC-2 receptor; G-protein-coupled receptor (GPCR); Gq signaling; cell signaling; platelet; platelets; rhodocytin; signal transduction; snake venom.

Figure 1.

Non-aspirinated platelets respond more robustly to rhodocytin than aspirin-treated platelets. A, washed non-aspirin or aspirin (1 mm) or indomethacin (10 μm)-treated human platelets were stimulated with 30 nm rhodocytin for 5 min at 37 °C under stirred conditions in a lumi-aggregometer. The tracings are representative of data from at least three independent experiments. B, washed non-aspirin (panel i) or aspirin-treated human platelets (panel ii) were stimulated with 30 nm rhodocytin for the indicated time points, and the reaction was stopped by using 6.6 n perchloric acid. Platelet proteins were separated by SDS-PAGE, Western-blotted, and probed for phospho-Syk (Tyr-525/526) and -PLCγ2 (Tyr-759). Statistical analysis of phospho-Syk (panel iii) and phospho-PLCγ2 (panel iv) of Western blottings are from panels i and ii. C, washed aspirin-treated or non-aspirin human platelets pre-treated with indomethacin (10 μm) were stimulated with rhodocytin (30 nm) for 3 min under stirred conditions. Platelet proteins were separated by SDS-PAGE, Western-blotted, and probed for phospho-Syk (Tyr-525/526), Syk (Tyr-352), PLCγ2 (Tyr-759), and PLCγ2 (Tyr-1217) in panel i and statistical analysis of the Western blots in panel ii. D, panel i, washed murine wild-type platelets were stimulated with rhodocytin (5 nm) under stirred conditions in the presence or absence of indomethacin (10 μm). Platelet aggregation was measured by aggregometry. The tracings are representative of data from at least three independent experiments. Panel ii, washed murine platelets were stimulated with 5 nm rhodocytin for 1 min. Platelet proteins were separated by SDS-PAGE, Western-blotted, and probed for phospho-Syk (Tyr-525/526) and PLCγ2 (Tyr-759). Panel iii, statistical analysis of the Western blottings in panel ii. All the Western blot analyses shown are representative of three independent experiments. **, p <0.01.

Figure 1.

Non-aspirinated platelets respond more robustly to rhodocytin than aspirin-treated platelets. A, washed non-aspirin or aspirin (1 mm) or indomethacin (10 μm)-treated human platelets were stimulated with 30 nm rhodocytin for 5 min at 37 °C under stirred conditions in a lumi-aggregometer. The tracings are representative of data from at least three independent experiments. B, washed non-aspirin (panel i) or aspirin-treated human platelets (panel ii) were stimulated with 30 nm rhodocytin for the indicated time points, and the reaction was stopped by using 6.6 n perchloric acid. Platelet proteins were separated by SDS-PAGE, Western-blotted, and probed for phospho-Syk (Tyr-525/526) and -PLCγ2 (Tyr-759). Statistical analysis of phospho-Syk (panel iii) and phospho-PLCγ2 (panel iv) of Western blottings are from panels i and ii. C, washed aspirin-treated or non-aspirin human platelets pre-treated with indomethacin (10 μm) were stimulated with rhodocytin (30 nm) for 3 min under stirred conditions. Platelet proteins were separated by SDS-PAGE, Western-blotted, and probed for phospho-Syk (Tyr-525/526), Syk (Tyr-352), PLCγ2 (Tyr-759), and PLCγ2 (Tyr-1217) in panel i and statistical analysis of the Western blots in panel ii. D, panel i, washed murine wild-type platelets were stimulated with rhodocytin (5 nm) under stirred conditions in the presence or absence of indomethacin (10 μm). Platelet aggregation was measured by aggregometry. The tracings are representative of data from at least three independent experiments. Panel ii, washed murine platelets were stimulated with 5 nm rhodocytin for 1 min. Platelet proteins were separated by SDS-PAGE, Western-blotted, and probed for phospho-Syk (Tyr-525/526) and PLCγ2 (Tyr-759). Panel iii, statistical analysis of the Western blottings in panel ii. All the Western blot analyses shown are representative of three independent experiments. **, p <0.01.

Figure 2.

Thromboxane potentiates the rhodocytin-induced platelet activation in the aspirin treated platelets. A, washed aspirin-treated human platelets were co-stimulated with rhodocytin (30 nm) and U46619 (10 μm) (panel i) or U46619 (10 μm) alone (panel ii) for the indicated time points under stirred conditions, and the reaction was stopped by using 6.6 n perchloric acid. Platelet proteins were separated by SDS-PAGE, Western-blotted, and probed for phospho-Syk (Tyr-525/526) and PLCγ2 (Tyr-759). Statistical analysis of phospho-Syk (panel iii) and phospho-PLCγ2 (panel iv) of Western blottings from panels i and ii. B, Western blotting (panel i) and statistical analysis (panel ii) of washed murine platelets stimulated with 5 nm rhodocytin for 1 min in the presence or absence of indomethacin (10 μm). C, panel i, washed human platelets without aspirin were pre-treated with either indomethacin (10 μm) or TP receptor antagonist, BAY u3405 (10 μm) for 5 min and stimulated with rhodocytin (30 nm) for 1 min. Also, aspirin-treated platelets were stimulated with rhodocytin or co-stimulated with rhodocytin and U46619 (10 μm) for 1 min in the presence or absence of BAY u3405 (10 μm); panel ii shows statistical analysis of the Western blots. D, panel i, washed human platelets were pre-incubated with αIIbβ₃ antagonist, GR 144053 (GR) (200 nm) for 5 min followed by stimulation with rhodocytin (30 nm) and U46619 (10 μm) for 1 min under stirred conditions; panel ii shows statistical analysis of the Western blots. Platelet proteins were separated by SDS-PAGE, Western-blotted, and probed for phospho-Syk (Tyr-525/526) and PLCγ2 (Tyr-759). N.S., not significant. **, p <0.01. All the Western blot analyses shown are representative of three independent experiments.

Figure 2.

Thromboxane potentiates the rhodocytin-induced platelet activation in the aspirin treated platelets. A, washed aspirin-treated human platelets were co-stimulated with rhodocytin (30 nm) and U46619 (10 μm) (panel i) or U46619 (10 μm) alone (panel ii) for the indicated time points under stirred conditions, and the reaction was stopped by using 6.6 n perchloric acid. Platelet proteins were separated by SDS-PAGE, Western-blotted, and probed for phospho-Syk (Tyr-525/526) and PLCγ2 (Tyr-759). Statistical analysis of phospho-Syk (panel iii) and phospho-PLCγ2 (panel iv) of Western blottings from panels i and ii. B, Western blotting (panel i) and statistical analysis (panel ii) of washed murine platelets stimulated with 5 nm rhodocytin for 1 min in the presence or absence of indomethacin (10 μm). C, panel i, washed human platelets without aspirin were pre-treated with either indomethacin (10 μm) or TP receptor antagonist, BAY u3405 (10 μm) for 5 min and stimulated with rhodocytin (30 nm) for 1 min. Also, aspirin-treated platelets were stimulated with rhodocytin or co-stimulated with rhodocytin and U46619 (10 μm) for 1 min in the presence or absence of BAY u3405 (10 μm); panel ii shows statistical analysis of the Western blots. D, panel i, washed human platelets were pre-incubated with αIIbβ₃ antagonist, GR 144053 (GR) (200 nm) for 5 min followed by stimulation with rhodocytin (30 nm) and U46619 (10 μm) for 1 min under stirred conditions; panel ii shows statistical analysis of the Western blots. Platelet proteins were separated by SDS-PAGE, Western-blotted, and probed for phospho-Syk (Tyr-525/526) and PLCγ2 (Tyr-759). N.S., not significant. **, p <0.01. All the Western blot analyses shown are representative of three independent experiments.

Figure 3.

G-protein-coupled receptors potentiate CLEC-2 signaling through G_q pathways. A, panel i, washed aspirin-treated human platelets were co-stimulated with rhodocytin (30 nm) and 2MeSADP (100 nm) or AYPGKF (500 μm) or U46619 (10 μm) or all the agonists alone for 1 min at 37 °C under stirred conditions in lumi-aggregometer. Platelet proteins were separated by SDS-PAGE, Western-blotted, and probed for phospho-Syk (Tyr-525/526), and PLCγ2 (Tyr-759); panel ii statistical analysis of the Western blots. B, panel i, washed non-aspirin human platelets, incubated with specific G_q inhibitor, UBO-QIC (100 nm), were stimulated with rhodocytin (30 nm). Western blottings of the separated proteins by SDS-PAGE were probed for phospho-Syk (Tyr-525/526) and PLCγ2 (Tyr-759); panel ii statistical analysis of the Western blots. C, panel i, washed aspirin-treated human platelets, incubated with specific G_q inhibitor, UBO-QIC (100 nm), were co-stimulated with rhodocytin (30 nm) and U46619 (10 μm) or D, panel i, AYPGKF (500 μm) for 1 min under stirred conditions. C, panel ii, and D, panel ii, statistical analysis of the respective Western blots. E, panel i, washed aspirin-treated human platelets pre-incubated for 5 min with either P2Y1 antagonist, MRS2179 (100 μm), or P2Y₁₂ antagonist, ARC 69931MX (100 nm), were co-stimulated with rhodocytin (30 nm) and 2MeSADP (100 nm); panel ii, statistical analysis of the Western blots. F, panel i, washed wild-type (WT) and Gα_q-deficient (knock-out (KO)) murine platelets were stimulated at 37 °C for 1 min with rhodocytin (5 nm). Western blottings of the proteins separated by SDS-PAGE were probed for phospho-Syk (Tyr-525/526) and phospho-PLCγ2 (Tyr-759); panel ii, statistical analysis of the Western blots. All the Western blot analysis shown is a representative of three independent experiments. N.S., not significant. **, p <0.01.

Figure 3.

G-protein-coupled receptors potentiate CLEC-2 signaling through G_q pathways. A, panel i, washed aspirin-treated human platelets were co-stimulated with rhodocytin (30 nm) and 2MeSADP (100 nm) or AYPGKF (500 μm) or U46619 (10 μm) or all the agonists alone for 1 min at 37 °C under stirred conditions in lumi-aggregometer. Platelet proteins were separated by SDS-PAGE, Western-blotted, and probed for phospho-Syk (Tyr-525/526), and PLCγ2 (Tyr-759); panel ii statistical analysis of the Western blots. B, panel i, washed non-aspirin human platelets, incubated with specific G_q inhibitor, UBO-QIC (100 nm), were stimulated with rhodocytin (30 nm). Western blottings of the separated proteins by SDS-PAGE were probed for phospho-Syk (Tyr-525/526) and PLCγ2 (Tyr-759); panel ii statistical analysis of the Western blots. C, panel i, washed aspirin-treated human platelets, incubated with specific G_q inhibitor, UBO-QIC (100 nm), were co-stimulated with rhodocytin (30 nm) and U46619 (10 μm) or D, panel i, AYPGKF (500 μm) for 1 min under stirred conditions. C, panel ii, and D, panel ii, statistical analysis of the respective Western blots. E, panel i, washed aspirin-treated human platelets pre-incubated for 5 min with either P2Y1 antagonist, MRS2179 (100 μm), or P2Y₁₂ antagonist, ARC 69931MX (100 nm), were co-stimulated with rhodocytin (30 nm) and 2MeSADP (100 nm); panel ii, statistical analysis of the Western blots. F, panel i, washed wild-type (WT) and Gα_q-deficient (knock-out (KO)) murine platelets were stimulated at 37 °C for 1 min with rhodocytin (5 nm). Western blottings of the proteins separated by SDS-PAGE were probed for phospho-Syk (Tyr-525/526) and phospho-PLCγ2 (Tyr-759); panel ii, statistical analysis of the Western blots. All the Western blot analysis shown is a representative of three independent experiments. N.S., not significant. **, p <0.01.

Figure 4.

G_q potentiates CLEC-2 signaling through PLCβ-PKC pathway. A, washed aspirin-treated human platelets were pre-incubated with either G_q inhibitor, UBO-QIC (100 nm), or PLCβ inhibitor, U73122 (5 μm), or GF 109203X (5 μm) for 5 min followed by stimulation with rhodocytin (30 nm) and U46619 (10 μm) for 1 min under stirred conditions. Western blot (panel i) and statistical analysis (panel ii) of the proteins were obtained after subjecting platelet lysates to separation by SDS-PAGE. B, washed aspirin-treated human platelets were pre-incubated with GF 109203X (5 μm), or rottlerin (10 μm), or LY33531 (10 μm), or PKCβ inhibitor (10 μm), or PKCδ/θ inhibitor (200 nm) followed by co-stimulation with rhodocytin (30 nm) and U46619 (10 μm) for 1 min. Western blot (panel i) and statistical analysis (panel ii) of the same are shown. In all the cases, platelet proteins were separated by SDS-PAGE, Western-blotted, and probed for phospho-Syk (Tyr-525/526) and PLCγ2 (Tyr-759). Western blot analysis shown is a representative of three independent experiments. N.S., not significant. **, p <0.01.

Figure 5.

PKC activation by PMA potentiates CLEC-2 signaling. A, washed non-aspirin human platelets were pre-incubated with DMSO or G_q inhibitor, UBO-QIC (100 nm), for 5 min followed by stimulation with rhodocytin (30 nm) and PMA (0.5 μm) for 2 min under stirred conditions. Western blot (panel i) and statistical analysis (panel ii) of the proteins obtained after subjecting platelet lysates to separation by SDS-PAGE are shown. B, washed wild-type murine platelets were stimulated for 2 min with rhodocytin (5 nm) and PMA (0.5 μm) in the presence or absence of UBO-QIC (100 nm) or stimulated with rhodocytin or PMA alone. Western blots (panel i) and statistical analysis (panel ii) of the proteins separated by SDS-PAGE were probed for phospho-Syk (Tyr-525/526) and phospho-PLCγ2 (Tyr-759). All the Western blot analyses shown are representative of three independent experiments. **, p <0.01.

Figure 6.

SFK activation by PKC downstream of G_q regulates CLEC-2 tyrosine phosphorylation and CLEC-2 signaling. A, panel i, washed non-aspirin and aspirin-treated human platelets were stimulated with rhodocytin (30 nm) alone or co-stimulated with rhodocytin (30 nm) and U46619 (10 μm) for 1 min under stirred conditions. Platelet proteins were separated by SDS-PAGE, Western-blotted, and probed for phospho-Btk (Tyr-223); panel ii, statistical analysis of the Western blots. B, panel i, washed aspirin-treated human platelets were pre-incubated with either UBO-QIC (100 nm) or GF 109203X (5 μm) and stimulated with U46619 (10 μm) for 1 min. Platelet proteins were separated by SDS-PAGE, Western-blotted, and probed for phospho-Src family kinases (Tyr-416). β-Actin was used as the lane loading control; panel ii, statistical analysis of the Western blots. C, panel i, washed aspirin-treated human platelets were pre-incubated with either UBO-QIC (100 nm) or GF 109203X (5 μm) followed by co-stimulation with rhodocytin (30 nm) and U46619 (10 μm). Platelet lysates were immunoprecipitated with agarose-conjugated Tyr(P) 4G10 antibody, and samples were probed for immunoprecipitating CLEC-2 using anti-CLEC-2 goat antibody; panel ii, statistical analysis of the Western blots obtained. Washed murine platelets from Lyn-deficient mice (Lyn^−/−) (D) or Fyn-deficient mice (Fyn^−/−) and wild-type controls (Lyn^+/+ or Fyn^+/+) (E) were either stimulated with rhodocytin (5 nm) alone in the presence or in the absence of indomethacin (10 μm) or co-stimulated with rhodocytin (5 nm) and U46619 (10 μm) in the presence of indomethacin for 1 min. Panel i, Western blot and statistical analysis (panel ii) of the respective figures. F, washed human platelets were pre-incubated with Pyk2 inhibitor, AG-17 (1 μm), or DMSO for 5 min followed by stimulation with rhodocytin (30 nm) and U46619 (10 μm) for 1 min under stirred conditions. In all the cases, platelet proteins were separated by SDS-PAGE, Western-blotted, and probed for phospho-Syk (Tyr-525/526) and PLCγ2 (Tyr-759). Panel i, Western blot, and panel ii, statistical analysis of the same. Western blot analysis shown is a representative of three independent experiments. N.S., not significant. **, p <0.01.

Figure 6.

SFK activation by PKC downstream of G_q regulates CLEC-2 tyrosine phosphorylation and CLEC-2 signaling. A, panel i, washed non-aspirin and aspirin-treated human platelets were stimulated with rhodocytin (30 nm) alone or co-stimulated with rhodocytin (30 nm) and U46619 (10 μm) for 1 min under stirred conditions. Platelet proteins were separated by SDS-PAGE, Western-blotted, and probed for phospho-Btk (Tyr-223); panel ii, statistical analysis of the Western blots. B, panel i, washed aspirin-treated human platelets were pre-incubated with either UBO-QIC (100 nm) or GF 109203X (5 μm) and stimulated with U46619 (10 μm) for 1 min. Platelet proteins were separated by SDS-PAGE, Western-blotted, and probed for phospho-Src family kinases (Tyr-416). β-Actin was used as the lane loading control; panel ii, statistical analysis of the Western blots. C, panel i, washed aspirin-treated human platelets were pre-incubated with either UBO-QIC (100 nm) or GF 109203X (5 μm) followed by co-stimulation with rhodocytin (30 nm) and U46619 (10 μm). Platelet lysates were immunoprecipitated with agarose-conjugated Tyr(P) 4G10 antibody, and samples were probed for immunoprecipitating CLEC-2 using anti-CLEC-2 goat antibody; panel ii, statistical analysis of the Western blots obtained. Washed murine platelets from Lyn-deficient mice (Lyn^−/−) (D) or Fyn-deficient mice (Fyn^−/−) and wild-type controls (Lyn^+/+ or Fyn^+/+) (E) were either stimulated with rhodocytin (5 nm) alone in the presence or in the absence of indomethacin (10 μm) or co-stimulated with rhodocytin (5 nm) and U46619 (10 μm) in the presence of indomethacin for 1 min. Panel i, Western blot and statistical analysis (panel ii) of the respective figures. F, washed human platelets were pre-incubated with Pyk2 inhibitor, AG-17 (1 μm), or DMSO for 5 min followed by stimulation with rhodocytin (30 nm) and U46619 (10 μm) for 1 min under stirred conditions. In all the cases, platelet proteins were separated by SDS-PAGE, Western-blotted, and probed for phospho-Syk (Tyr-525/526) and PLCγ2 (Tyr-759). Panel i, Western blot, and panel ii, statistical analysis of the same. Western blot analysis shown is a representative of three independent experiments. N.S., not significant. **, p <0.01.

Figure 7.

Proposed model depicting cross-talk between G_q-coupled GPCRs and CLEC-2 pathways. CLEC-2 activation by rhodocytin leads to initial signaling downstream of CLEC-2 resulting in thromboxane generation and secretion of ADP. TxA2 and ADP activate the G_q pathway, resulting in the activation of PKCα, which in turn can activate SFKs possibly through direct phosphorylation at Ser-12. PKCα can also regulate Syk directly, via phosphorylation on Ser-297 site, or indirectly, via activation of DUSP3 and/or inhibition of TULA-2 phosphatase. The activated SFKs or Syk can contribute and thereby potentiate the initial CLEC-2 signaling. The green arrows represent positive regulation, and red lines represent negative regulation.