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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.

Most wounds to the skin will cause leakage of blood from damaged blood vessels. A stable fibrin matrix is fundamental in wound edge adhesion and wound healing. For topically applied wound treatments to be effective they must penetrate this fibrin clot. Therefore, the objective of the study was to use a fibrin biopolymer as a model wound to investigate drug diffusion from wound dressings. Four compounds known to play an important role in wound healing were utilized. There were apigenin, vitamin E, chlorhexidine digluconate (CHDG) and benzalkonium chloride (BAC). Initial experiments demonstrated that no detectable amounts of apigenin and benzalkonium chloride were found in the provided burn dressings and bandages. As a result, no further attempts were made to study the release of these two compounds from the delivery systems. Release studies from delivery systems containing the other two compounds through the fibrin sealant were continued. The experiments demonstrated that the release data (Q, Q%) from burn dressings containing 4, 6, 10% vitamin E plotted versus time ½ and versus time had linear coefficient from 0.92 to 0.96 and from 0.94 to 0.97. This indicates that the release of vitamin E from burn dressing through fibrin sealant may be matrix controlled or membrane controlled, and the experiment could not distinguish between the two models. There was a linear relationship between the slope 2 of Q- t ½ and initial amount of vitamin E in burn dressing. The in vitro release of chlorhexidine digluconate from sterile and non-sterile bandages was compared. There was no significant difference between these two groups at any cumulative release time interval. The release appears to be a matrix-controlled. The results indicate that fibrin sealant may be potentially a useful model in evaluating release of drugs utilized in treatment of superficial wounds.