EPCR, TAFI as Regulators of PMN/Endothelial Interaction

描述(申请人提供)：这些研究集中于内皮蛋白C受体(EPCR)和凝血酶激活的纤溶抑制物(TAFI)在调节由大肠杆菌诱导的中性粒细胞/内皮相互作用中的作用。研究的所有炎症和止血事件都发生在中性粒细胞/内皮细胞的异常相互作用中，导致通透性增加和凝血功能紊乱。EPCR和血栓调节蛋白(TM)处于攻击点，因此通过TM/凝血酶复合体激活蛋白C和TAFI，以及通过内皮衍生金属蛋白酶释放可溶性EPCR，成为这种相互作用的靶标和调节剂。我们推测，TAF1a(原羧基肽酶β)通过灭活C5a来减弱中性粒细胞的激活，而可溶性EPCR则减弱中性粒细胞随后与内皮的紧密结合。EPCR和TAF1a与内皮细胞和中性粒细胞的密切联系有利于这些作用。两个普遍的问题是：EPCRt和TAFI在内皮和中性粒细胞之间的反应和分布是什么？这些信息是否可以用来设计和使用可溶性EPCR和TAF1a进行时间干预，从而提高活化蛋白C的疗效？为了研究这些问题，我们采用了大肠杆菌败血症的亚致死性模型，因为随着时间的推移，否则致命的事件会延伸到初始宿主(阶段1)和缺血再灌注(阶段2)反应。这个模型允许人们跟踪和干预这些调节成分在对E.coil反应的临界点的反应。具体问题包括EPCR和TAFI相对于介质(如C5a)在内皮和中性粒细胞之间的时间和分布，以及中性粒细胞与微血管内皮细胞的黏附？在第一阶段中，是否存在涉及这些监管机构的关键事件，这些事件决定了随后的第二阶段事件，以及反应是否变得致命？APC和sEPCR或TAFI是否可以联合使用来更好地调节中性粒细胞/内皮细胞对E.coil的反应？在决定干预是有益还是有害方面，干预时机的重要性有多大？EPCR、TAFI、蛋白C、血栓调节蛋白、组织因子等的表达和分布变化血管内皮细胞、血管周围组织和中性粒细胞将使用免疫组织化学和共聚焦成像技术进行评估。这包括流式细胞仪分析和中性粒细胞半衰期的测定。血浆因子的反应将用ELISA进行跟踪。将遵循标准的生理参数(例如，体温、血细胞计数、血压和止血功能的整体分析)

DESCRIPTION (provided by applicant): These studies focus on the role of endothelial protein C receptor (EPCR) and thrombin activatable fibrinolytic inhibitor (TAFI) as regulators of the neutrophil/endothelial interaction induced by E. coli. All the inflammatory and hemostatic events studied in the baboon model of E. coil sepsis culminate in an aberrant neutrophil/endothelial interaction leading to increased permeability and coagulation disorders. EPCR and thrombomodulin (TM) are at the point of attack and therefore are both targets and regulators of this interaction through activation of protein C and TAFI by the TM/thrombin complex and through release of soluble EPCR by endothelial-derived metalloproteases. We postulate that TAFla (procarboxypeptidase beta) attenuates neutrophil activation by inactivating C5a, and that soluble EPCR attenuates subsequent tight binding of neutrophils to endothelium. The close association of EPCR and TAFla with the endothelium and neutrophils favor these actions. Two general questions are: What is the response and distribution of EPCR t and TAFI between endothelium and neutrophils? Can the information be used to design and time intervention using soluble EPCR and TAFla that would improve the efficacy of activated protein C? To study such questions we have adopted the sublethal model of E. coil sepsis, because the otherwise lethal events are stretched out over time into an initial host (stage 1) and ischemia reperfusion (stage 2) responses. This model allows one to track and intervene with the responses of these regulatory components at critical points of the response to E. coil. Specific questions include what are the timing and distribution of EPCR and TAFI between endothelium and neutrophils with respect to mediators (e.g., C5a) and adherence of neutrophils to the microvascular endothelium? Are there critical events involving these regulators in stage 1 that determine subsequent stage 2 events and whether the response becomes lethal? Can APC and either sEPCR or TAFI be used together to better regulate the neutrophil/endothelial response to E. coil? How critical is timing of intervention in determining whether it is beneficial or harmful? Changes in the expression and distribution of EPCR, TAFI, protein C, thrombomodulin, tissue factor (etc.) on the endothelium, perivascular tissues and neutrophils will be assessed using immunohistochemical and confocal imaging techniques. This includes FACS analysis, and determination of neutrophil half-life. The responses of plasma factors will be followed with ELISAs. Standard physiological parameters will be followed (e.g., temperature, CBC, blood pressure and global assays of hemostatic function)