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，激活的蛋白C(APC)降低了死亡率，而单纯的抗凝剂或纯粹的抗炎剂都没有效果。因此，像在动物体内一样，注射到人类体内的APC既有抗凝作用，又有抗炎作用。最近的体外和体内研究支持一种新的范式，即APC通过与内皮蛋白C受体(EPCR)结合的APC激活蛋白激活受体1(PAR1)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在体内的抗炎和抗凋亡活性可能源于其改变基因表达谱的能力。因此，我们认为重组APC在治疗血栓性疾病方面是非常有前途的。我们建议制备和鉴定重组人和小鼠APC突变体作为潜在的治疗剂，并使用小鼠模型系统来表征它们在体内的抗血栓和抗炎活性。对于体外鉴定，APC的抗凝血活性和APC对细胞的直接影响将在对APC抑制星状孢子素诱导的细胞凋亡和改变内皮细胞基因表达的能力的研究中确定。为了评估APC的体内活性，我们建议使用小鼠模型和小鼠APC突变体，并对wt-APC和新的小鼠APC突变体进行以下检测：1)抗血栓活性；2)抗炎活性；3)在没有损伤的情况下基因表达的改变。在初步工作中，我们鉴定了两个抗凝血活性缺乏但抗细胞凋亡活性正常的APC突变体。这样的APC变异体可能提供APC有益的EPCR依赖、PAR1依赖的对细胞的直接作用，并降低严重出血的风险。由于APC保护性地针对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.