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.
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