Structure-function of cytoprotective coagulation proteases and their receptors

描述（由申请人提供）：本申请旨在提高对介导凝血蛋白酶对细胞的细胞保护作用的分子机制的基本理解。血管、血栓和炎症性疾病的可用治疗方案有限，死亡率仍然高得令人无法接受。活化蛋白C （APC）对细胞的有益作用与其他凝血蛋白酶（如凝血酶和Xa因子）对细胞的促炎作用形成对比，并对细胞中受体介导的串扰的复杂网络提出了新的观点。提高治疗成功率需要对所涉及的分子机制有更深入的了解。这个建议是围绕内皮蛋白C受体（EPCR），一个关键的细胞保护受体。本应用程序的长期目标是通过基础和转化研究推进知识，促进血管、血栓形成和炎症性疾病的诊断和治疗进展。本应用的目的是获得EPCR介导的细胞保护作用的分子机制的新见解。本应用的主要焦点是EPCR的结构-功能关系，该关系是EPCR在临床相关apc介导的细胞保护作用转导中的辅助因子作用的基础，并将该信息转化为改善血栓性炎症疾病的治疗策略。新的假设将使用生化和细胞生物学方法进行测试。具体目标是：1)生成具有独特特性的工程EPCR变体，增强其介导细胞保护作用的能力；2)表征EPCR依赖的PAR-1激活的结构-功能决定因素，该激活负责apc介导的细胞保护作用；3)定义与PF4-EPCR和pf4 -血栓调节素（TM）复合物的“脱靶”肝素诱导的血小板减少症（HIT）抗体相关的血栓并发症。这些研究的成功完成将增加我们对血管性、血栓性和炎症性疾病的认识和理解，并可能为开发新的治疗策略提供平台，以治疗血栓、细胞凋亡和炎症导致的各种疾病。

DESCRIPTION (provided by applicant): This application seeks to improve basic understanding of molecular mechanisms that mediate cytoprotective actions of coagulation proteases on cells. Available therapeutic solutions for vascular, thrombotic, and inflammatory diseases are limited and mortality rates remain unacceptably high. Beneficial effects of activated protein C (APC) on cells contrast with pro-inflammatory effects of other coagulation proteases (e.g. thrombin and factor Xa) on cells and initiated novel perspectives on the intricate complex networks of receptor-mediated cross talk in cells. Improving therapeutic success requires a more thorough understanding of the molecular mechanisms involved. This proposal is centered on the endothelial protein C receptor (EPCR), a key cytoprotective receptor. The long-term objectives of this application are to contribute to diagnostic and therapeutic progress for vascular, thrombotic, and inflammatory diseases by advancing knowledge through both basic and translational research. The goal of this application is to gain novel insights into the molecular mechanisms of cytoprotective actions mediated by EPCR. The major focus of this application is on structure- function relationships for EPCR that underlie EPCR's cofactor role in the transduction of clinically relevant APC-mediated cytoprotective effects and on translation of this information into improved therapeutic strategies for thrombotic inflammatory diseases. Novel hypotheses will be tested using biochemical and cellular biology methods. The specific aims are: 1) To generate engineered EPCR variants with unique properties that will enhance its ability to mediate cytoprotective effects, 2) To characterize the structure-function determinants for EPCR-dependent PAR-1 activation that are responsible for APC-mediated cytoprotective effects on cells, and 3) To define the thrombotic complications associated with "off-target" Heparin-Induced Thrombocytopenia (HIT) antibodies against PF4-EPCR and PF4-thrombomodulin (TM) complexes. Successful completion of the proposed studies will increase our knowledge and understanding of vascular, thrombotic, and inflammatory diseases and may provide a platform for the development of novel therapeutic strategies for a variety of disorders in which thrombosis, apoptosis and inflammation contribute to pathogenesis. PUBLIC HEALTH RELEVANCE: Vascular, thrombotic, and inflammatory diseases, such as sepsis, heart attack or stroke, will affect most of us at some point in life, with a profound impact on the quality and duration of life thereafter. Available therapeutic solutions are limited and mortality rates remain unacceptably high. Guided by the encouraging beneficial effects of recombinant activated protein C in sepsis and stroke, the proposed studies will identify novel molecular mechanisms and create engineered molecular variants for translational research and potential safer and more effective therapeutic applications.