Date on Master's Thesis/Doctoral Dissertation

8-2023

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Chemistry

Degree Program

Chemistry, PhD

Committee Chair

Maurer, Muriel

Committee Co-Chair (if applicable)

Mueller, Eugene

Committee Member

Mueller, Eugene

Committee Member

Wilson, Andrew

Committee Member

Menze, Michael

Author's Keywords

Factor XIII; fibrinogen; mass spectrometry; coagulation; transglutaminase

Abstract

Fibrinogen (Fbg) is a coagulation protein critical for clot formation. Coagulation Factor XIII (FXIII) is a calcium-dependent transglutaminase that crosslinks reactive glutamines (Q) and lysines (K) between fibrin and other anti-fibrinolytic proteins. In the presence of Ca2+, FXIII could be activated non-proteolytically (FXIII-A°), or proteolytically by thrombin (FXIII-A*). Significant increases in clot stability and red blood cell retention are linked to FXIII activity in the fibrinogen αC region (Fbg Aα 221 – 610). This region contains several FXIII-reactive glutamines and lysines, as well as a binding site for FXIII-A* (Fbg αC 389 – 402) that includes a key binding residue, Fbg αC E396. While FXIII-crosslinked clots maintain hemostasis, they also exacerbate the development of deep vein thrombosis (DVT). The work from this research seeks to aid further drug design against DVT by inhibition of FXIII binding and activity on Fbg αC. Fbg αC 233 – 425, a “model” αC system that contains three reactive glutamines and the FXIII binding site (Fbg αC 389 – 402), was recombinantly expressed and purified. A series of mutations were subsequently introduced to the αC FXIII binding site to observe how crosslinking was affected. FXIII activity was monitored through MS-based glycine ethyl ester (GEE) crosslinking and gel-based fluorescence monodansylcadaverine (MDC) crosslinking assays. Fbg αC 389 – 402 was found to selectively enhance Fbg αC crosslinking from FXIII-A* over FXIII-A°. Additionally, Fbg αC 389 – 402 was more instrumental than either αC 403 – 425 or αC 328 – 388 in facilitating FXIII-A* crosslinking. Further work explored αC E395, D390, W391, and F394A as residues within Fbg αC 389 – 402 that could enhance αC FXIII-A* activity by increasing binding affinity. While E395 minimally impacted FXIII-A* activity, αC D390, W391 and F394 were subsequently identified as key residues alongside E396 for promoting FXIII-A* crosslinking. In summary, Fbg αC 389 – 402 was demonstrated to be a major facilitator of FXIII-A* activity on Fbg αC, as well as a potential target for therapeutic inhibition of VTE. Groundwork was laid for future studies through expression and preliminary crosslinking studies on Fbg αC 221 – 425, a new recombinant Fbg αC.

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