Date on Master's Thesis/Doctoral Dissertation
Maurer, Muriel C
Committee Co-Chair (if applicable)
Mueller, Eugene G
Mueller, Eugene G
Wittebort, Richard J
D'Souza, Stanley E
factor xiii; transglutaminase; fibrin clot; oligomerization state; analytical ultracentrifugation; scanning electron microscopy
Factor XIIIA (FXIIIA) is a transglutaminase that crosslinks intra- and extracellular protein substrates. The oligomeric state of active FXIIIA remains controversial, and the present work commenced with addressing this issue. The results of size exclusion chromatography and analytical ultracentrifugation confirmed a dimeric state for zymogen and, for the first time, indicated a monomeric state for the active FXIIIA in solution. Comparing sedimentation properties of proteolytically and nonproteolytically activated FXIIIA suggested conformational and functional differences between the two forms. Those differences were further assessed in a series of catalytic activity studies. Kinetic analysis revealed affinity for the glutamine substrate was higher for proteolytically activated FXIIIA than for FXIIIA activated by high mM Ca2+. FXIIIA proteolytic activation was investigated in a context of fibrin clotting. The V34X FXIIIA variants were generated using site-directed mutagenesis and recombinantly expressed in Escherichia coli. Those variants were ranked in terms of their activation rates by thrombin L34>>V34>F34>>W34. In a series of SDS-PAGE and clot turbidity assays, the proteolytic activation rates of FXIIIA variants were correlated with the extent of covalent fibrin crosslinking. The V34X FXIIIA variants were administered into murine FXIIIA-deficient plasma. Scanning electron microscopy of the whole plasma clots revealed morphological evidence for incorporation of nonfibrous protein into the clot. The magnitude of this incorporation correlated with the FXIIIA activation rate. Overall, the presented research fills in critical gaps in previously identified FXIIIA biochemical attributes and expands the understanding of FXIIIA physiological function.
Anokhin, Boris, "Coagulation Factor XIIIA: biochemical properties underlying physiological function." (2018). Electronic Theses and Dissertations. Paper 3090.