Novel targets and agents to treat thrombotic disorders

描述（由申请人提供）： PROWESS III期临床试验表明，活化蛋白C（APC）降低了单纯抗凝剂或单纯抗炎剂均无效的死亡率。因此，APC输注到人类，在动物中，发挥抗凝和抗炎作用。最近的体外和体内研究支持了一种新的范例，其中APC通过与内皮蛋白C受体（EPCR）结合的APC激活蛋白酶激活受体1（PAR 1）的机制对细胞发挥保护性抗炎和抗凋亡的直接作用。研究表明，APC的抗炎和抗凋亡的体内活动可能是由于其改变基因表达谱的能力。因此，我们认为，重组APC是非常有前途的治疗血栓性疾病。我们建议产生和表征重组人和鼠APC突变体作为潜在的治疗剂，并使用鼠模型系统来表征其体内抗血栓形成和抗炎活性。对于体外表征，APC的抗凝活性和APC对细胞的直接作用将在APC抑制星形孢菌素诱导的细胞凋亡和改变内皮细胞基因表达的EPCR依赖性、PAR 1依赖性能力的研究中确定。为了评估APC的体内活性，我们建议使用鼠模型和鼠APC突变体，并确定wt-APC和新的鼠APC突变体的以下内容：1）抗血栓形成活性; 2）抗炎活性;和3）在没有损伤的情况下基因表达的改变。在前期工作中，我们确定了两个APC突变体缺乏抗凝活性与正常的抗凋亡活性。这种APC变体可以提供APC对细胞的有益的EPCR依赖性、PAR 1依赖性直接作用，降低严重出血的风险。由于APC保护性靶向凝血因子和细胞表面受体，该项目的结果可能直接导致开发用于治疗各种血栓性疾病的新型多功能药物。

DESCRIPTION (provided by applicant): The PROWESS phase III clinical trial showed that activated protein C (APC) reduced mortality where neither purely anticoagulant nor purely anti-inflammatory agents were effective. Thus, APC infused into humans, as in animals, exerts both anticoagulant and anti-inflammatory actions. Recent in vitro and in vivo studies support a new paradigm in which APC exerts protective anti-inflammatory and anti-apoptotic direct effects on cells via mechanisms involving activation of Protease Activated Receptor 1 (PAR1) by APC bound to Endothelial Protein C Receptor (EPCR). Studies suggest that APC's anti-inflammatory and anti-apoptotic in vivo activities may result from its ability to alter gene expression profiles. Thus, we suggest that recombinat APC is very promising for treatment of thrombotic disorders. We propose to generate and characterize recombinant human and murine APC mutants as potential therapeutic agents and to use murine model systems to characterize their in vivo antithrombotic and anti-inflammatory activities. For in vitro characterizations, APC's anticoagulant activity and APC's direct effects on cells will be determined in studies of the EPCR-dependent, PAR1 -dependent abilities of APC to inhibit staurosporine-induced apoptosis and to alter endothelial cell gene expression. To assess APC's in vivo activities, we propose to use murine models and murine APC mutants and to determine for wt-APC and novel murine APC mutants the following: 1) antithrombotic activity; 2) anti-inflammatory activity; and 3) alteration of gene expression in the absence of injury. In preliminary work, we identified two APC mutants deficient in anticoagulant activity with normal anti apoptotic activity. Such APC variants may provide APC's beneficial EPCR-dependent, PAR1 -dependent direct effects on cells with reduced risk of serious bleeding. Because APC protectively targets both clotting factors and cell surface receptors, the results of this project may lead directly to development of novel, multifunctional agents for treatment of a variety of thrombotic disorders.