EPCR, TAFI as Regulators of PMN/Endothelial Interaction

描述（由申请人提供）：这些研究的重点是内皮蛋白C受体（EPCR）和凝血酶活化纤维蛋白溶解抑制剂（TAFI）作为大肠杆菌诱导的中性粒细胞/内皮相互作用的调节剂。在大肠杆菌脓毒症狒狒模型中研究的所有炎症和止血事件最终导致中性粒细胞/内皮细胞异常相互作用，导致通透性增加和凝血障碍。EPCR和凝血调节蛋白（TM）处于攻击点，因此通过TM/凝血酶复合物激活蛋白C和TAFI以及通过内皮衍生的金属蛋白酶释放可溶性EPCR， EPCR既是这种相互作用的靶标也是调节剂。我们假设TAFla（原羧肽酶β）通过灭活C5a来减弱中性粒细胞的激活，而可溶性EPCR减弱随后中性粒细胞与内皮细胞的紧密结合。EPCR和TAFla与内皮细胞和中性粒细胞的密切联系有利于这些作用。两个一般性问题是：内皮细胞和中性粒细胞之间EPCR t和TAFI的反应和分布是什么？这些信息是否可以用于设计和使用可溶性EPCR和TAFla进行干预，从而提高活化蛋白C的功效？为了研究这些问题，我们采用了螺旋体脓毒症的亚致死模型，因为其他致死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）反应中。该模型允许跟踪和干预这些调控成分的反应在关键的点对E.线圈。具体的问题包括EPCR和TAFI在内皮细胞和中性粒细胞之间的时间和分布与介质（例如，C5a）和中性粒细胞对微血管内皮的粘附有关？在第一阶段是否有涉及这些监管机构的关键事件决定随后的第二阶段事件，以及反应是否成为致命的？APC与sEPCR或TAFI一起使用是否能更好地调节中性粒细胞/内皮细胞对E. coil的反应？干预的时机对决定干预是有益还是有害有多重要？利用免疫组织化学和共聚焦成像技术评估内皮细胞、血管周围组织和中性粒细胞中EPCR、TAFI、蛋白C、血栓调节蛋白、组织因子等的表达和分布变化。这包括FACS分析和中性粒细胞半衰期的测定。elisa检测血浆因子的反应。将遵循标准生理参数（例如，体温、全血细胞计数、血压和整体止血功能测定）。

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)