Laura L. Neese, Ph.D.

Dissertation research performed under the guidence of Dr. Frank C. Church

ABSTRACT
    Protein C Inhibitor (PCI) is a heparin binding serine protease inhibitor (serpin) that regulates proteases involved in coagulation and fibrinolysis. This study localized PCI in blood vessels and investigated how its activity is modulated by vessel wall components. H helix and D helix recombinant PCI (rPCI) mutants were generated to further define its heparin binding site. An R278A rPCI mutant and three variable region 1 (VR1) recombinant APC (rAPC) mutants were evaluated to investigate putative interactions between PCI and APC. Finally, PCI expression in prostate cancer was evaluated. Antithrombin-heparin, thrombomodulin, vitronectin and leukocyte elastase were each found to alter PCI-thrombin and PCI-APC interactions. PCI was localized by immunohistochemistry to the intima of the vessel wall. To further define the heparin binding site of PCI, H and D helix rPCI mutants were generated based on a rPCI mutant with decreased heparin activity (R269A:K270A). The H helix rPCI mutants demonstrated that K266 and K273 are also critical for heparin-enhanced inhibition of APC and thrombin. In contrast, the D helix mutants indicated that K82, K86, and R90 are not required for heparin enhanced protease inhibition. R278A rPCI three rAPC VR1 (K37-39) mutants (EEE, DED, and GGG) were generated to investigate intermolecular interactions between PCI and APC. In the absence of heparin, inhibition of wild-type rAPC by R278A rPCI and inhibition of the VR1 rAPC mutants by wild-type PCI increased. Heparin accelerated inhibition by PCI and heparin binding of each VR1 rAPC were both decreased, indicating that the VR1 basic residues participate in heparin binding. PCI expression was found in malignant human prostate cancer, a nude mouse xenograft prostate cancer model, and two prostate carcinoma cell lines. PCI expression increased in the xenograft model, especially post-castration and in the androgen-independent recurrent tumor. PCI expression was also found to be androgen regulated in the LNCaP cell line.