EPCR, TAFI as Regulators of PMN/Endothelial Interaction

描述（由申请人提供）：这些研究集中于内皮蛋白C受体（EPCR）和凝血酶激活的纤维蛋白溶解抑制剂（TAFI）作为E.杆菌在E.狒狒模型中研究的所有炎症和止血事件。线圈脓毒症最终导致异常的中性粒细胞/内皮细胞相互作用，导致渗透性增加和凝血障碍。EPCR和血栓调节蛋白（TM）处于攻击点，因此通过TM/凝血酶复合物激活蛋白C和TAFI以及通过内皮源性金属蛋白酶释放可溶性EPCR，两者都是这种相互作用的靶标和调节剂。我们假设TAFla（羧肽酶原β）通过灭活C5 a来减弱中性粒细胞活化，并且可溶性EPCR减弱中性粒细胞与内皮的随后紧密结合。EPCR和TAFla与内皮细胞和中性粒细胞的密切关联有利于这些作用。两个普遍的问题是：什么是反应和分布的EPCR t和TAFI之间的内皮细胞和中性粒细胞？这些信息能否用于设计和时间干预，使用可溶性EPCR和TAFla，将提高活性蛋白C的疗效？为了研究这些问题，我们采用了大肠杆菌亚致死模型。线圈脓毒症，因为否则致命的事件随着时间的推移延伸到初始宿主（阶段1）和缺血再灌注（阶段2）反应。该模型允许人们在对E的反应的关键点跟踪和干预这些调节成分的反应。线圈具体的问题包括内皮细胞和中性粒细胞之间EPCR和TAFI的时间和分布（例如，C5 a）和中性粒细胞粘附微血管内皮细胞？是否存在涉及第1阶段中的这些调节因子的关键事件，这些事件决定了随后的第2阶段事件以及反应是否变得致命？APC和sEPCR或TAFI是否可以一起使用以更好地调节中性粒细胞/内皮细胞对E.线圈？在确定干预是有益的还是有害的方面，干预的时机有多重要？EPCR、TAFI、蛋白C、血栓调节蛋白、组织因子等表达和分布的变化将使用免疫组织化学和共聚焦成像技术评估内皮、血管周围组织和中性粒细胞上的细胞毒性。这包括FACS分析和中性粒细胞半衰期的测定。将使用ELISA跟踪血浆因子的响应。将遵循标准生理参数（例如，体温、CBC、血压和止血功能的总体测定）

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)