Grants and Contributions:

Grant or Award spanning more than one fiscal year. (2017-2018 to 2022-2023)

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a protein found in blood plasma that regulates blood clot breakdown, a process known as fibrinolysis. Our proposal seeks to investigate the mechanisms of how TAFI is activated and how its activity is turned off. Together, these events control the function of TAFI in normal physiology (such as prevention of bleeding) and in disease (such as the development of heart attack and strokes).

TAFI is the precursor of an enzyme, and requires cleavage by another enzyme to form activated TAFI (TAFIa). The enzymes that are capable of activating TAFI are thrombin or plasmin, although both of these enzymes are very inefficient in this role. However, when thrombin interacts with a cell-surface cofactor called thrombomodulin (TM), the efficiency with which it activates TAFI increases by more than a thousand fold. While previous studies have defined particular parts of TM that are necessary to accelerate TAFI activation, it is not known how these parts contribute to the remarkable degree of acceleration that is observed. We believe that some parts of TM interact with thrombin, and other parts interact with TAFI. We plan to focus on each of these parts of TM. We will make mutations in both TM and in TAFI to define the exact amino acid residues in the respective proteins participate in the TAFI-TM interaction. We will assess the effect of the mutations on the rate of TAFI activation and on the ability of TM to bind to TAFI. In addition, we will mutate a key residue in TM that influences how TM interacts with thrombin and that appears to discriminate between the two substrates of thrombin-TM: TAFI and Protein C. These structure-function studies will be allied with molecular modeling analyses in order to corroborate the roles of the various amino acid residues we believe to be important.

Most enzymes formed in the blood have matching inhibitors that bind rapidly and tightly to control the amount of enzyme that remains. TAFIa is unusual in that it lacks such an inhibitor. Instead, TAFIa activity rapidly decays with a half-life of only 8 – 15 minutes at body temperature. Previous findings have shown that TAFIa undergoes a large structural change as it loses activity. Some regions of the protein that may participate in this structural change have been identified, but their exact role remains a mystery. In addition, there are other regions that likely contribute, and the details of the structural change remain to be determined. We will identify all of the amino acids in TAFIa that control the rate of its inactivation by mutating particular amino acids or groups of amino acids. We will use a technique known as hydrogen-deuterium exchange (a form of structural mass spectrometry) in order to track, in real time, structural changes in TAFIa as it loses activity. These highly innovative investigations will establish new paradigms for the understanding of the structural basis for enzyme instability.