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

血小板是一种特殊的粘附细胞，通过其快速粘附内皮下基质蛋白（粘附）和其他活化血小板（聚集）的能力，在正常和病理止血中发挥关键作用，其功能受到NO/cGMP/PKG信号通路的抑制。最近，我们讨论了这个领域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NOS蛋白，血小板可溶性观酰基环化酶（sGC）被冯氏血友病因子（VWF）独立激活。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no独立的sGC代表了一种新的抑制血小板反馈机制。我们还希望阐明参与血小板sGC调控的分子机制和翻译后修饰。利用我们最近建立的定量磷酸化蛋白质组学方法和sGC的免疫沉淀（IP），我们将阐明sGC的磷酸化状态，并表征VWF刺激血小板时sGC蛋白复合物的特征。GC突变体的产生将有助于分析特定磷酸化位点在模型系统中调控sGC活性的作用。最后，我们将使用不同的流动模型来建立最大的no独立激活血小板sGC的条件。

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.