Extracellular matrix (ECM) is a complex mixture of proteoglycans that function in cell adhesion, migration, growth and differentiation. In addition, the ECM serves as a reservoir for a number of growth factors, proteinases and proteinase inhibitors essential for ECM homeostasis. Recent new information from the applicant's laboratory has shown that one of the serine proteinase inhibitors found in the ECM is a 32kDa glycoprotein consisting of three tandemly-arranged Kunitz-type proteinase inhibitor domains homologous to tissue factor pathway inhibitor, an important negative regulator of the extrinsic pathway of blood coagulation. This novel inhibitor, designated as type-2 tissue factor pathway inhibitor, or TFPI-2, is a potent inhibitor of trypsin, plasmin, kallikrein and factor XIa amidolytic activity in the test tube. A variety of human endothelial cells synthesize TFPI-2, and 60-90 percent of the synthesized TFPI-2 is secreted by the endothelial cells into their ECM. Inflammatory mediators upregulate endothelial cell TFPI-2 synthesis 2-14 fold. In addition, evidence was obtained that recombinant TFPI-2 inhibited the plasmin-mediated invasion and degradation of Matrigel by a high invasive fibrosarcoma cell line in a dose-dependent manner. Furthermore, treatment of confluent endothelial cell monolayers with anti-TFPI-2 IgG detached the cells from the substrata in a time and IgG concentration dependent manner, suggesting an important role for TFPI-2 in maintaining the integrity of the ECM essential for cell attachment. In spite of these findings several questions concerning the physiological role of TFPI-2 remain unanswered, such as (1) which Kurutz domain is responsible for its inhibitory activity, (2) what proteinase(s) are regulated by ECM-bound TFPI-2, and (3) what are the regulatory mechanisms of TFPI-2 expression. The objectives of this proposal are to accomplish a comprehensive characterization of TFPI-2 in order to gain further insight into its biological role in normal and pathological ECM turnover.
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