Murine Protein C and Protein S Proof of Principle Research

描述(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。需要对APC和蛋白S的生理和药理作用机制有新的认识。为了在体内建立这些机制的原理证明，该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质。APC可以发挥两种主要的、不同的活性：(1)抗凝活性和(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降低小鼠脓毒症模型的死亡率至关重要。目前对APC细胞信号转导的研究包括内皮蛋白C受体(EPCR)结合APC结合蛋白酶激活受体1(PAR1)的蛋白水解性激活。我们发现，APC还启动了另一条信号通路，包括连接载脂蛋白E受体2(ApoER2)，通过适配子蛋白Dab1和Src家族激酶进行信号传递，并下游激活PI3K-Akt生存通路。小鼠APC和ApoER2突变体的工程将允许询问介导APC：ApoER2相互作用的结合和信号启动的蛋白质表面，并将为体内验证APC在小鼠脓毒症中作用机制的原理研究提供试剂。对ApoER2和Dab1转基因小鼠的研究将确定ApoER2和Dab1是否介导APC在脓毒症中的降低死亡率活动。对S蛋白缺陷小鼠进行血栓刺激，用重组野生型和突变型小鼠蛋白S与抗栓蛋白或其他药物联合治疗，以确定S蛋白依赖于抗栓蛋白和不依赖抗栓蛋白的抗血栓活性的相对有效性。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的基础研究，一种自然产生的血浆蛋白，被转化为现在用于临床的诊断和治疗工具。拟议的关于活化蛋白C的基础研究将为活化蛋白C作用于血细胞以防止可能致命的损害的分子机制提供原理证明和明确的见解。这一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.