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

Background; Factor XIII (FXIII) is composed of an activation peptide segment, a β‐sandwich domain, a catalytic core, and finally β‐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 β‐barrels 1 and 2 in FXIII, we expressed and characterised the full‐length FXIII‐A subunit (FXIII‐A) and variants truncated to residue 628 [truncated to β‐barrel 1 (TB1)], 515 [truncated to catalytic core (TCC)] and 184 [truncated to β‐sandwich (TBS)]. Methods; Proteins were analysed by gel electrophoresis, circular dichroism, fluorometric assays, colorimetric activity assays, clot structure by turbidity measurements, confocal microscopy, and clot formation by Chandler Loop. Results and Conclusions; Circular dichroism spectroscopy and tryptophan fluorometry indicate that full length FXIII‐A and the truncations TCC and TB1 retain their secondary and tertiary structure. Removal of β‐barrel 2 (TB1) resulted in total loss of transglutaminase activity, whilst the additional removal of β‐barrel 1 (TCC) restored enzymatic activity to approximately 30% of full length FXIII‐A. These activity trends were observed with physiological substrates and smaller model substrates. Our data suggest that the β‐barrel 1 domain protects the active site cysteine in the FXIII protransglutaminase while the β‐barrel 2 domain is necessary for exposure of the active site cysteine during activation. This study demonstrates the importance of individual β‐barrel domains in modulating access to the FXIII active site region.