Initiation and Regulation of Blood Coagulation

描述(申请人提供)：凝血级联由凝血因子VII(A)与其细胞表面受体组织因子(Tf)结合而启动。Tf-V11a复合体的形成不仅能激活凝血级联反应，还能诱导其他细胞反应。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。它还与各种疾病的发病机制有关。因此，适当地调节TF-V11a的表达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能是调节细胞表面Tf-V11a表达的另一种调节机制。这些研究还揭示了新的信息，即内化的V11a与细胞骨架蛋白有关，内化的V11a和活性位点抑制的V11a在细胞内的分布存在实质性差异。目前，Tf和V11a在细胞内的转运途径以及它们在Tf-V11a调控中的确切作用尚不清楚。同样，V11a与细胞骨架相互作用的性质及其潜在意义仍有待确定。本申请侧重于解决这些重要问题。目的1定义Tf的亚细胞定位，这对于阐明Tf和V11a在细胞内的转运是必不可少的。在这些实验中，将用Tf-GFP融合构建体转染成纤维细胞以定位Tf。目的2利用已有的内吞实验和荧光共聚焦显微镜分析组织因子途径抑制物对Tf-Vlla内化方式及其细胞内转运途径的影响。目的研究Vla与肌动蛋白相互作用在肌动蛋白动力学调控中的功能相关性，明确Vla与肌动蛋白相互作用的分子基础。最近收购了一台最先进的Biacore仪器，并提供了一大批Vlla突变体，这将促进这些研究。来自拟议研究的数据将为理解Tf-V11a在细胞表面的表达是如何调节的以及V11a在细胞内的潜在作用提供新的见解。这些知识将有助于设计更好的出血性和血栓性疾病的治疗策略，并将为Tf-V11a异常表达参与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.