Factor XIII\nActivation Peptide Residues Play Important Roles in Stability, Activation,\nand Transglutaminase Activity

A subunit of factor XIII (FXIII-A)\ncontains a unique\nactivation\npeptide (AP) that protects the catalytic triad and prevents degradation.\nIn plasma, FXIII is activated proteolytically (FXIII-A*) by thrombin\nand Ca<sup>2+</sup> cleaving AP, while in cytoplasm, it is activated\nnonproteolytically (FXIII-A°) with increased Ca<sup>2+</sup> concentrations.\nThis study aimed to elucidate the role of individual parts of the\nFXIII-A AP in protein stability, thrombin activation, and transglutaminase\nactivity. Recombinant FXIII-A AP variants were expressed, and SDS-PAGE\nwas used to monitor thrombin hydrolysis at the AP cleavage sites R37–G38.\nTransglutaminase activities were assessed by cross-linking lysine\nmimics to Fbg αC (233–425, glutamine–substrate)\nand monitoring reactions by mass spectrometry and in-gel fluorescence\nassays. FXIII-A AP variants, S19P, E23K, and D24V, degraded during\npurification, indicating their vital role in FXIII-A<sub>2</sub> stability.\nMutation of P36 to L36/F36 abolished the proteolytic cleavage of AP\nand thus prevented activation. FXIII-A N20S and P27L exhibited slower\nthrombin activation, likely due to the loss of key interdomain H-bonding\ninteractions. Except N20S and P15L/P16L, all activatable FXIII-A*\nvariants (P15L, P16L, S19A, and P27L) showed similar cross-linking\nactivity to WT. By contrast, FXIII-A° P15L, P16L, and P15L/P16L\nhad significantly lower cross-linking activity than FXIII-A°\nWT, suggesting that loss of these prolines had a greater structural\nimpact. In conclusion, FXIII-A AP residues that play crucial roles\nin FXIII-A stability, activation, and activity were identified. The\ninteractions between these AP amino acid residues and other domains\ncontrol the stability and activity of FXIII.