Murine Protein C and Protein S Proof of Principle Research

描述（由申请人提供）：蛋白质C和蛋白质S缺乏导致男性和小鼠的发病率和死亡率。重组活化蛋白C （APC）治疗可降低成人严重脓毒症患者的死亡率。目前迫切需要对APC和s蛋白作用的生理和药理学机制有新的认识。为了建立这种机制的体内原理证明，本项目使用了转基因小鼠、小鼠损伤模型和新型重组小鼠蛋白。APC可以发挥两种主要的、不同的活性：(1)抗凝血活性和(2)对细胞的直接有益作用，包括各种细胞保护作用。后一种活性对于APC在小鼠败血症模型中降低死亡率至关重要。目前APC的细胞信号传导模式包括内皮蛋白C受体（EPCR）结合蛋白酶激活受体-1 （PAR1）蛋白水解激活APC。我们发现APC启动的另一个信号通路涉及载脂蛋白E受体2 （apoER2）的连接，通过接头蛋白、Dab1和src家族激酶发出信号，下游激活PI3K-Akt存活通路。小鼠APC和apoER2突变体的工程设计将允许对介导APC与apoER2相互作用的结合和信号启动的蛋白质表面进行调查，并将为APC在小鼠败血症中的作用机制的体内原理研究提供试剂。对apoER2和Dab1基因修饰小鼠的研究将确定apoER2和Dab1是否介导APC在脓毒症中的降低死亡率活性。蛋白S缺陷小鼠将受到血栓性刺激，并用重组野生型和突变型小鼠蛋白S与APC或其他药物联合治疗，以确定依赖于APC或不依赖于APC的蛋白S抗血栓活性的相对疗效。通过这些临床前动物模型研究建立的新原理可能最终转化为涉及蛋白C和蛋白S系统的诊断或治疗进展。

DESCRIPTION (provided by applicant): Protein C and protein S deficiencies contribute to morbidity and mortality in men and mice. Recombinant activated protein C (APC) therapy reduces mortality in adult severe sepsis patients. There is a major need for new insights into the physiologic and pharmacologic mechanisms of action of APC and protein S. To establish in vivo proof of principle for such mechanisms, this project uses genetically modified mice, murine injury models, and novel recombinant murine proteins. APC can exert two major, distinct activities: (1) anticoagulant activity and (2) direct beneficial effects on cells comprising a variety of cytoprotective actions. This latter activity is critical for mortality reduction by APC in murine sepsis models. The current paradigm for APC's cell signaling involves binding of APC by endothelial protein C receptor (EPCR) combined with protease activated receptor-1 (PAR1) proteolytic activation. We found that there is another signaling pathway initiated by APC that involves ligation of apolipoprotein E Receptor 2 (apoER2), signaling via the adaptor protein, Dab1, and Src-family kinases with downstream activation of the PI3K-Akt survival pathway. Engineering of murine APC and apoER2 mutants will allow interrogation of the protein surfaces that mediate binding and signal initiation by APC:apoER2 interactions and will provide reagents for in vivo proof of principle studies for mechanisms of APC's action in murine sepsis. Studies of mice genetically modified in apoER2 and Dab1 will establish whether apoER2 and Dab1 mediate APC's mortality reduction activities in sepsis. Protein S deficient mice will be subjected to thrombotic provocation and treated with combinations of recombinant wild type and mutant murine protein S and APC or other agents to define the relative efficacies for protein S antithrombotic activity that is either dependent on APC or independent of APC. Novel Principles that are established by these preclinical animal model studies may ultimately be translated into diagnostic or therapeutic advances involving the protein C and protein S systems. PUBLIC HEALTH RELEVANCE: Previous basic research on plasma Protein C, a naturally occurring plasma protein, was translated into diagnostic and therapeutic tools now used in the clinic. The proposed basic research studies on activated protein C will provide proof of principle and definitive insights into molecular mechanisms by which activated protein C acts on blood cells to prevent damage that can be fatal. The findings may well be translatable into future clinical advances.