Structure-function of cytoprotective coagulation proteases and their receptors

描述(申请人提供)：本申请旨在提高对凝血酶对细胞的细胞保护作用的分子机制的基本理解。可用于治疗血管、血栓和炎症性疾病的解决方案有限，死亡率仍然高得令人无法接受。激活的蛋白C(APC)对细胞的有利作用与其他凝血酶(如凝血酶和Xa因子)对细胞的促炎作用形成对比，并开启了对细胞内受体介导的串扰的复杂网络的新视角。要提高治疗的成功率，需要对相关的分子机制有更深入的了解。这一建议以内皮蛋白C受体为中心，它是一种关键的细胞保护受体。这项应用的长期目标是通过基础研究和翻译研究促进知识的发展，从而促进血管、血栓和炎症性疾病的诊断和治疗进展。这项应用的目的是获得对EPCR介导的细胞保护作用的分子机制的新见解。这一应用的主要焦点是EPCR的结构-功能关系，这些关系是EPCR在临床相关的APC介导的细胞保护效应的转导中的辅因子作用，并将这些信息转化为改进的血栓性炎症性疾病的治疗策略。新的假说将使用生化和细胞生物学方法进行检验。其具体目标是：1)产生具有独特性能的工程EPCR变体，以增强其介导细胞保护效应的能力；2)表征依赖于EPCR的PAR-1激活的结构-功能决定因素，这些决定因素负责APC介导的细胞保护效应；以及3)确定与肝素诱导的针对PF4-EPCR和PF4-血栓调节蛋白(TM)复合体的“非靶标”肝素诱导的血小板减少(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、血栓和炎症性疾病的认识和理解，并可能为开发各种疾病的新治疗策略提供平台，在这些疾病中，血栓形成、细胞凋亡和炎症参与了发病机制。 公共卫生相关性：血管、血栓和炎症性疾病，如败血症、心脏病发作或中风，将在人生的某个阶段影响我们大多数人，并对此后的生活质量和持续时间产生深远影响。可用的治疗方案有限，死亡率仍然高得令人无法接受。在重组活化蛋白C在脓毒症和中风中令人鼓舞的有益作用的指导下，拟议的研究将确定新的分子机制，并为翻译研究和潜在的更安全和更有效的治疗应用创造工程化的分子变体。

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.