Distinct pathways regulate Syk protein activation downstream of immune tyrosine activation motif (ITAM) and hemITAM receptors in platelets

Abstract

Tyrosine kinase pathways are known to play an important role in the activation of platelets. In particular, the GPVI and CLEC-2 receptors are known to activate Syk upon tyrosine phosphorylation of an immune tyrosine activation motif (ITAM) and hemITAM, respectively. However, unlike GPVI, the CLEC-2 receptor contains only one tyrosine motif in the intracellular domain. The mechanisms by which this receptor activates Syk are not completely understood. In this study, we identified a novel signaling mechanism in CLEC-2-mediated Syk activation. CLEC-2-mediated, but not GPVI-mediated, platelet activation and Syk phosphorylation were abolished by inhibition of PI3K, which demonstrates that PI3K regulates Syk downstream of CLEC-2. Ibrutinib, a Tec family kinase inhibitor, also completely abolished CLEC-2-mediated aggregation and Syk phosphorylation in human and murine platelets. Furthermore, embryos lacking both Btk and Tec exhibited cutaneous edema associated with blood-filled vessels in a typical lymphatic pattern similar to CLEC-2 or Syk-deficient embryos. Thus, our data show, for the first time, that PI3K and Tec family kinases play a crucial role in the regulation of platelet activation and Syk phosphorylation downstream of the CLEC-2 receptor.

Keywords: Cell Signaling; Platelet; Signal Transduction; Signaling; Thrombosis.

FIGURE 1.

Effect of pan-PI3K inhibitors on CLEC-2-mediated platelet aggregation and secretion in human and murine platelets. Washed human (A) and murine (B) platelets were pretreated with the PP2 (10 μm), LY294002 (25 μm), or wortmannin (100 nm) at 37 °C for 5 min followed by stimulation with convulxin (100 ng/ml) or rhodocytin (30 or 100 nm) or hCLEC-2 Ab (10 μg/ml) or fucoidan (50 μg/ml) under stirred conditions. Platelet aggregation was measured by aggregometry. The tracings are representative of data from at least three independent experiments. Dense granule secretion was assessed by measuring ATP with lumichrome. Graphs represent mean ± S.E. from at least three different experiments (***, p ≤ 0.05).

FIGURE 2.

Effect of specific PI3K isoform inhibitors on CLEC-2-mediated platelet aggregation in human and murine platelets. Washed human (A) and murine (B) platelets were incubated with 25 μm LY294002 (pan-PI3K inhibitor), 100 nm PIK75 (PI3K α inhibitor), 500 nm TGX-221 (PI3K β inhibitor), 2 μm AS-252424 (PI3Kγ inhibitor), or 1 μm IC87114 (PI3K δ inhibitor) and stimulated at 37 °C with rhodocytin (30 nm) under stirred conditions. Platelet aggregation was measured by aggregometry. The tracings are representative of data from at least three independent experiments.

FIGURE 3.

Role of PI3K in CLEC-2 signaling. A, washed human platelets were incubated with 25 μm LY294002 for 5 min followed by stimulation with convulxin (100 ng/ml) or rhodocytin (30 nm) under stirred conditions. Platelet proteins were separated by SDS-PAGE, Western-blotted, and probed for phospho-Syk (Tyr-525/526), LAT (Tyr-191), PLCγ2 (Tyr-759), and Akt (Ser-473). The Western analysis shown is a representative of three experiments. Calcium mobilization was measured by using FURA-2. Data are mean ± S.E. (n = 3); ***, p < 0.05 compared with agonist without inhibitor. B, washed human platelets were incubated with 25 μm LY294002 for 5 min followed by stimulation with convulxin (100 ng/ml) or rhodocytin (30 nm) under stirred conditions. Platelets were lysed in Nonidet P-40 lysis buffer and immunoprecipitated for Syk. Syk kinase assay was performed to measure the activity of Syk by measuring tubulin phosphorylation. C, washed human platelets were incubated with PP2 (10 μm), OXSI-2 (1 μm), Go6976 (1 μm), or LY294002 (25 μm) followed by stimulation with convulxin (100 ng/ml) or rhodocytin (30 nm) under stirred conditions. Platelet proteins were separated by SDS-PAGE, Western-blotted, and probed for phospho-Akt (Ser-473). The results shown are representative of data from at least three independent experiments. D, model representation of role of SFK and Syk in PI3K activation.

FIGURE 4.

Effect of ibrutinib on CLEC-2-mediated platelet activation and signaling in humans. A, washed human platelets were incubated with ibrutinib (10 nm) for 5 min followed by stimulation with convulxin (100 ng/ml) or rhodocytin (30 nm) under stirred conditions, and aggregation was measured. *** represents significance of p < 0.05. B, washed human platelets were incubated with ibrutinib (5 nm) for 5 min followed by stimulation with convulxin (100 ng/ml) or rhodocytin (30 nm) under stirred conditions. Platelet proteins were separated by SDS-PAGE, Western-blotted, and probed for phospho-Syk (Tyr-525/526) and PLCγ2 (Tyr-759). β-Actin was used as a lane loading control. C, washed human platelets were incubated with ibrutinib (10 nm) for 5 min followed by stimulation with convulxin (100 ng/ml) or rhodocytin (30 nm) under stirred conditions. Platelets were lysed in Nonidet P-40 lysis buffer and immunoprecipitated for Syk. Syk kinase assay was performed to measure the activity of Syk by measuring tubulin phosphorylation. The results shown are representative of data from at least three independent experiments.

FIGURE 5.

Role of PI3K and Syk in membrane translocation of Tec family kinases downstream of CLEC-2. Washed human platelets, incubated with LY294002 (25 μm) or OXSI-2 (1 μm), were stimulated with convulxin (100 ng/ml) or rhodocytin (30 nm), and the translocation of Btk to the membrane was measured. β₃-Integrin was used as lane loading control. All the Western blot analysis shown is a representative of at least three independent experiments.

FIGURE 6.

Effect of ibrutinib and LY294002 on human platelet activation at high concentrations of CLEC-2 agonist. A, washed human platelets, incubated with ibrutinib (10 nm) and LY294002 (25 μm), were stimulated with 100 and 300 nm rhodocytin. Platelet aggregation was measured by aggregometry. The tracings are representative of data from at least three independent experiments. B, platelet proteins were separated by SDS-PAGE, Western-blotted, and probed for phospho-Syk (Tyr-525/526) and PLCγ2 (Tyr-759 and Tyr-1217). All the Western blot analyses shown are a representative of at least three independent experiments.

FIGURE 7.

Effect of ibrutinib on murine platelet activation downstream of CLEC-2 receptor. A, washed murine wild-type platelets were incubated with DMSO or ibrutinib (10 nm) for 5 min followed by stimulation with convulxin (30 ng/ml) or rhodocytin (10 nm) or mouse CLEC-2 ab (3 μg/ml) under stirred conditions. Platelet aggregation was measured by aggregometry. The tracings are representative of data from at least three independent experiments. B, platelet proteins were separated by SDS-PAGE, Western-blotted, and probed for phospho-Syk (Tyr-525/526), Btk (Tyr-223), and PLCγ2 (Tyr-759). β-Actin was used as a lane loading control. C, washed Xid (Btk-defective) and wild-type murine platelets were incubated with DMSO or ibrutinib (5 nm) for 5 min followed by stimulation with mouse CLEC-2 Ab (3 μg/ml) under stirred conditions. Platelet aggregation was measured by aggregometry. The tracings are representative of data from at least three independent experiments.

FIGURE 8.

Btk and Tec are essential for CLEC-2 function in vivo. A and B, E15.5 embryos lacking both Btk and Tec exhibit cutaneous edema and a pattern of blood-filled cutaneous vessels consistent with lymphatic vessels like those observed in CLEC-2-deficient embryos. C and D, immunostaining for the lymphatic endothelial marker LYVE1 demonstrates blood-filled lymphatic vessels in the mesentery (C) and skin (D). These results are representative of at least three independent experiments.

FIGURE 9.

Model for PI3K/Btk axis regulating Syk activation downstream of CLEC-2. We propose the following: 1) upon activation of CLEC-2 in platelets, SFKs become activated and phosphorylates the hemITAM. 2) SFK activates PI3K. 3) Once activated, PI3K generates PIP₃ that is important for the recruitment of Tec family kinases to the membrane, where it is phosphorylated and activated by SFKs. 4) Activated Tec family kinases then phosphorylates and activates Syk that is already bound to phosphorylated CLEC-2 hemITAM.