Kostyak John C, Mauri Benjamin R, Dangelmaier Carol, Patel Akruti, Zhou Yuhang, Eble Johannes A, Tsygankov Alexander Y, McKenzie Steven E, Kunapuli Satya P
Sol Sherry Thrombosis Research Center, Lewis Katz School of Medicine, Temple University, Philadelphia, Pennsylvania, United States.
Cardeza Foundation for Hematologic Research, Department of Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania, United States.
TH Open. 2018 Dec 5;2(4):e411-e419. doi: 10.1055/s-0038-1676358. eCollection 2018 Oct.
Platelet activation is essential for hemostasis. Central to platelet activation are the signals transmitted through surface receptors such as glycoprotein VI, the protease-activated receptors, and C-type lectin-like receptor 2 (CLEC-2). CLEC-2 is a HemITAM (hem-immunoreceptor tyrosine activation motif)-bearing receptor that binds podoplanin and signals through spleen tyrosine kinase (Syk). T-cell ubiquitin ligand-2 (TULA-2) is a protein tyrosine phosphatase that is highly expressed in platelets and targets phosphorylated Y352 of Syk. We wanted to determine whether TULA-2 regulates Syk phosphorylation and activity downstream of CLEC-2. To that end, we used TULA-2 knockout mice and wild-type (WT) littermate controls. We found that TULA-2 deficiency enhances the aggregation and secretion response following stimulation with an excitatory CLEC-2 antibody or the CLEC-2 agonist rhodocytin. Consistently, Syk phosphorylation of Y346 is enhanced, as well as phosphorylation of the downstream signaling molecule PLCγ2, in TULA-2 knockout platelets treated with either CLEC-2 antibody or rhodocytin, compared with WT control platelets. Furthermore, the kinetics of Syk phosphorylation, as well as that of PLCγ2 and SLP-76, is enhanced in TULA-2 knockout platelets treated with 2.5-μg/mL CLEC-2 antibody compared with WT platelets. Similarly, thromboxane production was enhanced, in both amount and kinetics, in TULA-2 platelets treated with 2.5-μg/mL CLEC-2 antibody. TULA-2 acts as a negative regulator of CLEC-2 signaling by dephosphorylating Syk on Y346 and restraining subsequent Syk-mediated signaling.
血小板活化对于止血至关重要。血小板活化的核心是通过表面受体（如糖蛋白VI、蛋白酶激活受体和C型凝集素样受体2（CLEC-2））传递的信号。CLEC-2是一种带有HemITAM（血免疫受体酪氨酸激活基序）的受体，它与血小板结合蛋白结合并通过脾酪氨酸激酶（Syk）发出信号。T细胞泛素配体-2（TULA-2）是一种蛋白酪氨酸磷酸酶，在血小板中高度表达，并靶向Syk的磷酸化Y352。我们想确定TULA-2是否调节CLEC-2下游的Syk磷酸化和活性。为此，我们使用了TULA-2基因敲除小鼠和野生型（WT）同窝对照。我们发现，在用兴奋性CLEC-2抗体或CLEC-2激动剂红藻氨酸刺激后，TULA-2缺陷增强了聚集和分泌反应。与WT对照血小板相比，在用CLEC-2抗体或红藻氨酸处理的TULA-2基因敲除血小板中，Y346的Syk磷酸化以及下游信号分子PLCγ2的磷酸化均增强。此外，与WT血小板相比，在用2.5μg/mL CLEC-2抗体处理的TULA-2基因敲除血小板中，Syk磷酸化以及PLCγ2和SLP-76的磷酸化动力学均增强。同样，在用2.5μg/mL CLEC-2抗体处理的TULA-2血小板中，血栓素的产生在数量和动力学上均增强。TULA-2通过使Syk的Y346去磷酸化并抑制随后的Syk介导的信号传导，作为CLEC-2信号的负调节因子。
