BACKGROUND The glycoprotein (GP) Ib-IX-V complex is a unique platelet plasma membrane receptor, which is essential for platelet adhesion and thrombus formation. GPIbα, part of the GPIb-IX-V complex, has several physiological ligands such as von Willebrand factor (vWF), thrombospondin and distinct coagulation factors, which trigger platelet activation. Despite having an important role, intracellular GPIb-IX-V signaling and its regulation by other pathways are not well defined. Our aim was to establish the intracellular signaling response of selective GPIbα activation in human platelets, in particular the role of the tyrosine kinase Syk and its regulation by cAMP/PKA and cGMP/PKG pathways, respectively. We addressed this using echicetin beads (EB), which selectively bind to GPIbα and induce platelet aggregation. METHODS Purified echicetin from snake Echis carinatus venom was validated by mass spectrometry. Washed human platelets were incubated with EB, in the presence or absence of echicetin monomers (EM), Src family kinase (SFK) inhibitors, Syk inhibitors and the cAMP- and cGMP-elevating agents iloprost and riociguat, respectively. Platelet aggregation was analyzed by light transmission aggregometry, protein phosphorylation by immunoblotting. Intracellular messengers inositolmonophosphate (InsP1) and Ca2+i were measured by ELISA and Fluo-3 AM/FACS, respectively. RESULTS EB-induced platelet aggregation was dependent on integrin αIIbβ3 and secondary mediators ADP and TxA2, and was antagonized by EM. EB stimulated Syk tyrosine phosphorylation at Y352, which was SFK-dependent and Syk-independent, whereas Y525/526 phosphorylation was SFK-dependent and partially Syk-dependent. Furthermore, phosphorylation of both Syk Y352 and Y525/526 was completely integrin αIIbβ3-independent but, in the case of Y525/526, was partially ADP/TxA2-dependent. Syk activation, observed as Y352/ Y525/Y526 phosphorylation, led to the phosphorylation of direct substrates (LAT Y191, PLCγ2 Y759) and additional targets (Akt S473). PKA/PKG pathways inhibited EB-induced platelet aggregation and Akt phosphorylation but, surprisingly, enhanced Syk and LAT/PLCγ2 tyrosine phosphorylation. A similar PKA/PKG effect was confirmed with convulxin-/GPVI-stimulated platelets. EB-induced InsP1 accumulation/InsP3 production and Ca2+-release were Syk-dependent, but only partially inhibited by PKA/PKG pathways. CONCLUSION EB and EM are specific agonists and antagonists, respectively, of GPIbα-mediated Syk activation leading to platelet aggregation. The cAMP/PKA and cGMP/PKG pathways do not inhibit but enhance GPIbα-/GPVI-initiated, SFK-dependent Syk activation, but strongly inhibit further downstream responses including aggregation. These data establish an important intracellular regulatory network induced by GPIbα.

中文翻译：

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cAMP和cGMP升高剂抑制人血小板中GPIbα介导的聚集，但不抑制GPIbα刺激的Syk活化。

背景技术糖蛋白（GP）Ib-IX-V复合物是独特的血小板质膜受体，对于血小板粘附和血栓形成至关重要。GPIb-IX-V复合物的一部分GPIbα具有几个生理配体，例如von​​ Willebrand因子（vWF），血小板反应蛋白和独特的凝血因子，可触发血小板活化。尽管起重要作用，但细胞内GPIb-IX-V信号传导及其通过其他途径的调控尚未明确。我们的目标是建立选择性GPIbα活化在人血小板中的细胞内信号传导应答，特别是酪氨酸激酶Syk的作用及其分别通过cAMP / PKA和cGMP / PKG途径的调节。我们使用echicetin珠（EB）解决了这一问题，该蛋白选择性结合GPIbα并诱导血小板聚集。方法采用质谱法验证蛇毒棘皮蛇毒中纯化的蛇菊素。将洗涤过的人血小板在存在或不存在echicetin单体（EM），Src家族激酶（SFK）抑制剂，Syk抑制剂以及cAMP-和cGMP升高剂iloprost和riociguat的存在或不存在下与EB孵育。通过光透射聚集法分析血小板聚集，通过免疫印迹分析蛋白质磷酸化。分别通过ELISA和Fluo-3 AM / FACS测量细胞内信使肌醇单磷酸酯（InsP1）和Ca2 + i。结果EB诱导的血小板聚集依赖于整联蛋白αIIbβ3以及次要介质ADP和TxA2，并被EM拮抗。EB刺激了Y352的Syk酪氨酸磷酸化，这是SFK依赖性和Syk依赖性的，而Y525 / 526的磷酸化是SFK依赖的，部分是Syk依赖的。此外，Syk Y352和Y525 / 526的磷酸化完全不依赖整合素αIIbβ3，但对于Y525 / 526，则部分依赖ADP / TxA2。Syk活化（观察为Y352 / Y525 / Y526磷酸化）导致直接底物（LAT Y191，PLCγ2Y759）和其他靶标（Akt S473）的磷酸化。PKA / PKG途径抑制EB诱导的血小板聚集和Akt磷酸化，但令人惊讶的是，增强了Syk和LAT /PLCγ2酪氨酸的磷酸化。惊厥蛋白/ GPVI刺激的血小板证实了类似的PKA / PKG效应。EB诱导的InsP1积累/ InsP3产生和Ca2 +释放是Syk依赖性的，但仅部分受到PKA / PKG途径的抑制。结论EB和EM分别是特异性激动剂和拮抗剂，GPIbα介导的Syk活化导致血小板聚集。cAMP / PKA和cGMP / PKG途径不抑制但增强GPIbα-/ GPVI引发的SFK依赖性Syk活化，但强烈抑制进一步的下游反应，包括聚集。这些数据建立了由GPIbα诱导的重要的细胞内调节网络。