Date of Award

2-18-2016

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Chemistry and Biochemistry

First Advisor

Boffa, Michael

Keywords

Carboxypeptidase B, Enzyme Kinetics, Fibrinolysis, Glycosaminoglycans, TAFI, Thrombomodulin

Rights

CC BY-NC-ND 4.0

Abstract

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a plasma zymogen that provides a molecular link between the coagulation and fibrinolytic systems. TAFI is activated through proteolytic cleavage by thrombin and plasmin although they are relatively weak activators. However, thrombin in complex with the endothelial cell cofactor thrombomodulin (TM) or plasmin in the presence of glycosaminoglycans (GAGs), such as heparin, are able to greatly accelerate the activation of TAFI to generate the enzyme activated TAFI (TAFIa). Additionally, the presence of GAGs has been shown to stabilize enzyme TAFIa increasing its half-life. TAFIa possesses basic carboxypeptidase activity which down-regulates fibrin clot lysis by removing carboxyl-terminal lysine and arginine residues from partially degraded fibrin thereby attenuating the positive feedback in the fibrinolytic cascade that is essential for plasminogen activation. The work in this dissertation is focused on understanding of the regulation of TAFI activation and evaluating TAFIa activity. Through construction of mutant variants of TAFI in putative interaction regions with TM and GAGs, we have identified important regions on the TAFI structure that mediate the accelerated activation by the thrombin-TM complex and the acceleration of plasmin-mediated TAFI activation by GAGs and the stabilization of the TAFIa enzyme. We found that the region on TAFI that showed the strongest TM dependence was focused around the activation domain of TAFI, which would be near where the c-loop of EGF-3 of TM would contact TAFI. We also identified residues on TAFI that could be part of a heparin binding site that allows GAGs to mediate the acceleration of plasmin-mediated TAFI activation and stabilization of the TAFIa enzyme. As well, we have designed a novel fluorescence based assay for the measurement of the basic carboxypeptidase activity of TAFIa which has significantly increased sensitivity compared to the currently available carboxypeptidase B (CPB) substrates. Taken together, the results of the studies described will aid in further propelling the determination of the mechanism of TAFI activation by the respective activators as well as provide a sensitive tool for monitoring the carboxypeptidase activity of TAFIa.

Available for download on Sunday, December 31, 2017

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