Master of Science
Boffa, Michael B.
Thrombin-activatable fibrinolysis inhibitor (TAFI) is a key regulator of hemostasis and inflammation. TAFI is activated by thrombin proteolysis, most effectively when in complex with cofactor thrombomodulin (TM). Soluble TM, encompassing EGF-like domains 3 through 6 is sufficient to promote TAFI activation, though the role of EGF 4 has not been assessed. Through rearrangement of the soluble TM domains, we determined that EGF 4 was acting as a spacer to promote TAFI activation. Activated TAFI (TAFIa) is a metastable enzyme, with a half-life of only 8-15 min at 37oC. The mobile loop of TAFIa was expected to modulate stability; however, mutations in this region mimicking the stable pancreatic carboxypeptidase B did not increase half-life. To identify regions responsible for the instability of TAFIa, Trp-to-Phe mutations were made to monitor the decay in intrinsic fluorescence of TAFIa associated with the loss of enzymatic activity. No Trp residues were deemed independently responsible for the decay.
Summary for Lay Audience
Maintaining proper blood flow is essential to sustain life. This process involves the formation and breakdown of blood clots. An imbalance in this system resulting in excessive clotting can lead to heart attacks or strokes, while excessive breakdown can lead to bleeding as seen in hemophilia. Activated thrombin-activatable fibrinolysis inhibitor (TAFIa) is formed during the clotting process and acts to stabilize the blood clot. Once TAFIa is no longer needed, it must cease activity to maintain proper blood flow. The mechanics of TAFI activation and TAFIa inactivation are quite unique: once activated, the protein appears to rapidly unfold on its own with a half-life of approximately 10 min at body temperature, after which, activity is lost. We studied a region of the cofactor which helps effectively activate TAFI to elucidate the necessary domains for sufficient activation. We determined that not all components of the cofactor are required for TAFI to be activated. Within the protein, we looked at a region known as the mobile loop, whose mobility may be acting as a trigger to unfolding. Surprisingly, mutations to this region did not impact the rate at which the TAFI variants unfolded or impact the ability of the TAFI variants to maintain a blood clot under laboratory settings. We next looked at several fluorescent residues of the protein to monitor how their fluorescence changed over time, as TAFIa is inactivated. Upon initial TAFI activation, fluorescence is quite high, however it degrades over time corresponding with the loss of TAFIa activity. Of the residues we monitored, none appeared to be independently responsible for the mobility and decrease in fluorescence leading to the massive TAFIa unfolding event. These studies serve to better understand the regulation of TAFIa and can help to modify the concentration of circulating TAFIa by modifying TAFI activation, or modulating the rate at which TAFIa is inactivated.
Marier, Haley D N, "The inner workings of thrombin-activatable fibrinolysis inhibitor: a study of the TM-mediated activation of TAFI and inactivation of TAFIa" (2023). Electronic Thesis and Dissertation Repository. 9490.