Molecular mechanisms of von Willebrand-factor (VWF)-stimulated, NO-independent acitivation of soluble guanylyl cyclase (sGC) in human platelets

人血小板中血管性血友病因子 (VWF) 刺激、不依赖 NO 激活可溶性鸟苷酸环化酶 (sGC) 的分子机制

• 批准号: 111697699
• 负责人: Dr. Stephan Gambaryan
• 金额: --
• 依托单位: Centrum für Thrombose und Hämostase (CTH)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2009
• 资助国家: 德国
• 起止时间: 2008-12-31 至 2011-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/111697699?language=en
• 关键词: Molecular; mechanisms; von; Willebrand; factor

Platelets, specialized adhesive cells, play a key role in normal and pathological haemostasis through their ability to rapidly adhere to subendothelial matrix proteins (adhesion) and to other activated platelets (aggregation), functions inhibited by NO/cGMP/PKG signalling pathways. Recently, we addressed a controversial issue in this field. We discovered that human and murine platelets do not express any type of functional NOS proteins and that platelet soluble guanylyl cyclase (sGC) is NOS-independently activated by von Willebrand factor (VWF). This may represent a new mechanism of feedback inhibition. The main goal of this new project is to prove our hypothesis that NO-independent sGC activation by VWF and GPIb-IX-V signalling represents a novel inhibitory platelet feedback mechanism. We also want to elucidate the molecular mechanisms and posttranslational modifications involved in platelet sGC regulation. Using our recently established quantitative phosphoproteomic approach and immunoprecipitation (IP) of sGC we will elucidate the phosphorylation status of sGC and characterize the sGC protein complexes upon platelet stimulation by VWF. Generation of GC mutants will help to analyze the role of specific phosphorylation sites in regulation of sGC activity in model systems. Finally, we will use different flow models to establish conditions for maximal NO-independent activation of platelet sGC.

血小板是一种特殊的黏附细胞，通过与内皮下基质蛋白(黏附)和其他激活的血小板(聚集)快速黏附，在正常和病理性止血中发挥关键作用，这些功能被NO/cGMP/PKG信号通路抑制。最近，我们讨论了这一领域中的一个有争议的问题。我们发现，人和小鼠的血小板不表达任何类型的功能性一氧化氮合酶蛋白，而血小板可溶性鸟苷酸环化酶(SGC)是由von Willebrand因子(VWF)激活的。这可能代表了一种新的反馈抑制机制。这个新项目的主要目标是证明我们的假设，即通过VWF和GPIB-IX-V信号激活sGC不是独立的，这代表了一种新的抑制血小板反馈的机制。我们还想阐明参与血小板sGC调节的分子机制和翻译后修饰。利用我们最近建立的定量磷酸蛋白质组学方法和sGC的免疫沉淀(IP)，我们将阐明sGC的磷酸化状态，并表征sGC蛋白复合体在VWF刺激血小板时的特性。GC突变体的产生将有助于分析特定的磷酸化位点在模型系统中调节sGC活性中的作用。最后，我们将使用不同的流动模型来建立最大限度地激活血小板sGC的条件。

项目成果

The thrombin inhibitors hirudin and Refludan® activate the soluble guanylyl cyclase and the cGMP pathway in washed human platelets

• DOI: 10.1160/th11-07-0461
• 发表时间: 2012-03-01
• 期刊: THROMBOSIS AND HAEMOSTASIS
• 影响因子: 6.7
• 作者: Kobsar, Anna;Koessler, Juergen;Walter, Ulrich
• 通讯作者: Walter, Ulrich