Initiation and Regulation of Blood Coagulation

描述（由申请人提供）：凝血级联是通过凝血因子VII(a)与其细胞表面受体组织因子(TF)的结合而启动的。 TF-VIIa复合物的形成不仅触发凝血级联的激活，而且还诱导其他细胞反应。 TF 的异常表达是与各种疾病相关的血栓性疾病的主要原因。它还有助于各种疾病的发病机制。因此，TF-VIIa表达的适当调节对于维持止血平衡和总体健康至关重要。我们最近的研究表明，受体介导的内吞作用可以作为调节细胞表面 TF-VIIa 表达的额外调节机制。这些研究还揭示了新的信息，即内化的VIIa与细胞骨架蛋白相关并且内化的VIIa和活性位点抑制的VIIa的细胞内分布存在显着差异。目前，TF和VIIa的细胞内运输途径以及它们在TF-VIIa调节中的精确作用尚不清楚。类似地，VIIa与细胞骨架相互作用的性质及其潜在意义仍有待确定。本申请致力于解决这些重要问题。目标 1 定义了 TF 的亚细胞定位，这对于阐明 TF 和 VIIa 的细胞内运输至关重要。对于这些实验，成纤维细胞将用 TF-GFP 融合构建体转染以定位 TF。目标 2 通过使用完善的内吞作用测定和荧光共聚焦显微镜分析组织因子途径抑制剂如何影响 TF-VIIa 内化模式及其细胞内运输途径。目标 3 重点研究 VIla 与肌动蛋白相互作用在调节肌动蛋白动力学中的功能相关性，并定义 VIIa-肌动蛋白相互作用的分子基础。最近购买的最先进的 Biacore 仪器和大量 Vlla 突变体的可用性将促进这些研究。拟议研究的数据将为理解 TF-VIIa 表达如何在细胞表面上调节以及 VIIa 的潜在细胞内效应提供新的见解。该知识将有助于设计针对出血性和血栓性疾病的更好的治疗策略，并且将为TF-VIIa的异常表达促成发病机制的疾病的治疗干预提供新的策略。

DESCRIPTION (provided by applicant): The coagulation cascade is initiated by the binding of coagulation factor VII(a) to its cell surface receptor tissue factor (TF). The formation of TF-Vlla complexes not only triggers activation of the coagulation cascade but also induces other cellular responses. An aberrant expression of TF is the primary reason for thrombotic disorders associated with various diseases. It also contributes to the pathogenesis of various diseases. Thus, a proper regulation of TF-Vlla expression is critical for the maintenance of hemostatic balance and health in general. Our recent studies suggest that the receptor-mediated endocytosis could serve as an additional regulatory mechanism in modulation TF-Vlla expression on cell surfaces. These studies also reveal novel information, i.e., the internalized Vlla associates with cytoskeletal proteins and substantial differences exist in intracellular distribution of the internalized Vlla and active site-inhibited Vlla. At present, intracellular trafficking pathways of TF and Vlla, and their precise role in the regulation of TF-Vlla are unknown. Similarly, the nature of Vlla interaction with the cytoskeleton and its potential significance remain to be established. The present application focuses on resolving these important issues. Aim 1 defines subcellular localization of TF, which is essential for elucidating intracellular trafficking of TF and Vlla. For these experiments, fibroblasts will be transfected with TF-GFP fusion construct to localize TF. Aim 2 analyzes how tissue factor pathway inhibitor affects the mode of TF-Vlla internalization and its intracellular trafficking pathways by using well-established endocytosis assays and fluorescence confocal microscopy. Aim 3 focuses on investigating the functional relevance of VIla interaction with actin in modulating actin dynamics, and defining the molecular basis for Vlla-actin interaction. Recent acquisition of a state-of-the-art Biacore instrument and availability of a large panel of Vlla mutants will facilitate these studies. Data from the proposed studies will provide new insights towards understanding how TF-Vlla expression is regulated on cell surfaces and potential intracellular effects of Vlla. This knowledge will be useful in designing better treatment strategies for hemorrhagic and thrombotic diseases, and would provide novel strategies for therapeutic interventions in diseases where aberrant expression of TF-Vlla contributes to the pathogenesis.