Doctoral Dissertations

Date of Award

5-2000

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Cynthia B. Peterson

Committee Members

Elizabeth Howell, Salil Niyogi, Barry Bruce

Abstract

Vitronectin is a plasma protein involved in the maintenance of hemostasis, the balance between coagulation and fibrinolysis, as well as cellular adhesion. This multifunctional glycoprotein is present in the circulation as well as in the extracellular matrix. Vitronectin has a proposed domain structure that may explain why it is capable of binding s number of diverse ligands. The amino-terminal 44 amino acids are termed the somatomedin B domain. This domain is reported to be the primary high affinity binding site for two ligands, plasminogen activator inhibitor-1 (PAI-1) and the urokinase receptor (uPAR). However, ligand binding sites for these proteins have also been identified in the heparin binding domain near the carboxy-terminus. The goal of this research was two fold: to determine if the high affinity binding site for PAI-1 was contained within the somatomedin B domain and to evaluate the potential for using gene transfer to confer an adhesive phenotype to endothelial cells. To achieve the first goal, the eukaryotic baculovirus expression system was utilized to express a somatomedin B deletion mutant. This recombinant protein was characterized functionally for ligand binding abilities, with special attention focused on PAI-1 binding. The second goal was pursued using retroviral mediated gene transfer of PAI-1 and vitronectin into human umbilical vein endothelial cells. The cells were characterized in terms of protein expression levels, integrity of integrins and ability to withstand physiological shear stress. Results of these studies indicate there are multiple PAI-1 binding sites, with the high affinity site residing in the somatomedin B domain. In addition, over-expression of PAI-1 or vitronectin by endothelial cells had no deleterious effects on cellular adhesion.

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