The role of barrels 1 and 2 in the enzymatic activity of factor XIII-A.

Background: Factor XIII is composed of an activation peptide segment, a beta-sandwich domain, a catalytic core, and, finally, beta-barrels 1 and 2. FXIII is activated following cleavage of its A-subunits by thrombin. The resultant transglutaminase activity leads to increased resistance of fibrin clots to fibrinolysis. Objectives: To assess the functional roles of beta-barrels 1 and 2 in FXIII, we expressed and characterized the full-length FXIII A-subunit (FXIII-A) and variants truncated to residue 628 (truncated to beta-barrel 1 [TB1]), residue 515 (truncated to catalytic core [TCC]), and residue 184 (truncated to beta-sandwich). Methods: Proteins were analyzed by gel electrophoresis, circular dichroism, fluorometric assays, and colorimetric activity assays, clot structure was analyzed by turbidity measurements and confocal microscopy, and clot formation was analyzed with a Chandler loop system. Results and Conclusions: Circular dichroism spectroscopy and tryptophan fluorometry indicated that full-length FXIII-A and the truncation variants TCC and TB1 retain their secondary and tertiary structure. Removal of beta-barrel 2 (TB1) resulted in total loss of transglutaminase activity, whereas the additional removal of beta-barrel 1 (TCC) restored enzymatic activity to similar to 30% of that of full-length FXIII-A. These activity trends were observed with physiological substrates and smaller model substrates. Our data suggest that the beta-barrel 1 domain protects the active site cysteine in the FXIII protransglutaminase, whereas the beta-barrel 2 domain is necessary for exposure of the active site cysteine during activation. This study demonstrates the importance of individual beta-barrel domains in modulating access to the FXIII active site region.