Murine Protein C and Protein S Proof of Principle Research

描述（由申请人提供）：蛋白C和蛋白S缺乏导致人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对APC和蛋白S作用的生理学和药理学机制有新的认识是主要的需要。为了建立这种机制的体内原理证明，该项目使用转基因小鼠，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活性：（1）抗凝活性和（2）对细胞的直接有益作用，包括多种细胞保护作用。后一种活性对于APC在鼠脓毒症模型中降低死亡率至关重要。APC细胞信号传导的当前范例涉及APC通过内皮蛋白C受体（EPCR）结合蛋白酶激活受体-1（PAR 1）蛋白水解激活。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（apoER 2）的连接，通过衔接蛋白、Dab 1和Src家族激酶的信号传导以及PI 3 K-Akt存活通路的下游激活。鼠APC和apoER 2突变体的工程化将允许询问介导APC：apoER 2相互作用的结合和信号起始的蛋白质表面，并将为APC在鼠脓毒症中的作用机制的体内原理研究证据提供试剂。对apoER 2和Dab 1基因修饰小鼠的研究将确定apoER 2和Dab 1是否介导APC在脓毒症中的死亡率降低活性。蛋白S缺陷小鼠将经受血栓形成激发，并用重组野生型和突变鼠蛋白S和APC或其它试剂的组合治疗，以确定蛋白S抗血栓形成活性的相对功效，所述蛋白S抗血栓形成活性依赖于APC或不依赖于APC。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。 公共卫生相关性：以前对血浆蛋白C（一种天然存在的血浆蛋白）的基础研究被转化为现在临床上使用的诊断和治疗工具。拟议的活化蛋白C基础研究将提供原理证明和对活化蛋白C作用于血细胞以防止可能致命的损伤的分子机制的明确见解。这些发现很可能转化为未来的临床进展。

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.