Tissue factor (TF) is a transmembrane glycoprotein that regulates hemostasis and is thought to play a critical role in mediating arterial thrombosis. In animal models, TF also mediates the intimal response to arterial injury and the extent of damage resulting from ischemia-reperfusion injury. TF was thought to exist as a single form whose activity was dependent upon its insertion in the cell membrane. TF has recently been found in microparticles that are present in tissues and in the circulation. In addition, an alternatively spliced form of TF (asTF) has been identified that excludes exon 5 and contains a frame shift that generates a unique C-terminus without a transmembrane domain. asTF activity requires exposure to lipids, but not insertion into a lipid bilayer. asTF has been isolated from blood and is a major component of ex vivo and in vivo thrombi. The investigators hypothesize that asTF plays critical roles in regulating arterial thrombosis, in mediating normal cardiac hemostasis, and in mediating intimal hyperplasia and the response to ischemia-reperfusion. This proposal will examine the regulation and biologic role(s) of asTF and full-length TF (flTF) in cell culture and in genetically engineered mice.
Aim 1 will establish the time course of asTF expression in smooth muscle cells, macrophages, endothelial cells, and cardiocytes and determine whether alternative splicing exhibits agonist, tissue and developmental specificity. It will also determine whether, as a soluble molecule, asTF binds specifically to vascular cells and exhibits agonist properties distinct from its ability to initiate coagulation.
Aim 2 will establish the role of asTF in hemostasis, thrombosis, and in TF-mediated responses to injury. Mice will be generated in which one or both forms of TF are regulated by the endogenous TF promoter. The investigators will test the hypotheses that: 1) flTF is critical for embryonic survival; 2) asTF is critical for generating significant thrombosis at sites of arterial injury; 3) asTF plays a pivotal role in regulating the intimal response to arterial injury; 4) flTF plays the dominant role in mediating normal hemostasis, but asTF is important in mediating hemostasis at the level of the myocardial capillaries; 5) asTF and flTF play complementary roles in mediating ischemia-reperfusion injury; and 6) asTF mediates normal cell functions, including growth, migration, and adhesion. These studies will provide insights into a novel form of circulating TF that may play a key role in cardiovascular disease.
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