Date on Master's Thesis/Doctoral Dissertation

12-2011

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Chemistry

Committee Chair

Maurer, Muriel C.

Author's Keywords

Factor XIII; Coagulation; Hydrogen-deuterium exchange; Mass spectrometry; Transglutaminase

Subject

Blood--Coagulation

Abstract

One of the last events that occurs during blood coagulation, a process taken for granted on a daily basis, involves Factor XIII (FXIII) cross-linking fibrin monomers to form an insoluble clot. In plasma, FXIII-A2 is not active and exists as the heterotetramer FXIII-A2B2. Through the utilization of hydrogen - deuterium exchange (HDX) coupled with Matrix Assisted Laser Desorption ionization - Time of Flight - Mass Spectrometry (MALDI-TOF-MS), it was determined that FXIII-A2 becomes nearly uniformly protected when bound to FXIII-B2 and the FXIII-A2 ß-barrels play a major role in heterotetramer formation. After dissociation from FXIII-B2, FXIII-A2 has the ability to become activated in the presence of Ca2+. The regions/residues of FXIII-A2 Ca2+ affects during activation were identified using HDX. It is debated whether FXIII-A2 undergoes an open conformation during activation. Transglutaminase 2 (TG2) has been observed crystallographically in an open conformation. HDX was utilized to compare the conformational dynamics of Transglutaminase 2 in solution to that of FXIII-A2. The increase in exposure between the catalytic core and ß-barrels of TG2 yields evidence of an open conformation. A structural comparison of FXIII-A2 and TG2 identified steric hinderance within the A2 dimer that could thwart a similar conformational change. Once activated physiologically, FXIII-A2 is solely responsible for forming the cross-links between fibrin monomers. The aC (233 - 425) region of fibrin contains three reactive Gin residues and acts as a substrate for FXIII. Fibrin aC (233 - 425) was expressed and its structure investigated via 15N-HSQC when in solution with FXIII-A2. The integral role of FXIII-A2 in the coagulation cascade leads to a dire need for investigating its conformational dynamics during activation. The research herein provides a stronger knowledge of FXIII-A2 structural changes during activation and outlines FXIII-A2 interactions with 82 and the fibrin aC domain. This progress in understanding FXIII-A2 dynamics could lead to improved treatments for excessive bleeding and thrombosis.

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