OC 28.5 - Exploring Interdomain Interactions Mediated by the Factor XIII Activation Peptide

Background: The unique activation peptide (AP) of FXIII protects it from auto-activation and degradation. However, interactions between the AP and other domains of the enzyme which control stability and activity are not well characterized. In plasma, FXIII is activated proteolytically (FXIII-A*) by thrombin and Ca2+, while in the cytoplasm, it is activated non-proteolytically (FXIII-A°) with increased Ca2+ concentrations.

Aims: The objective was to elucidate the role of individual parts of FXIII AP in protein stability, thrombin activation, and transglutaminase activity.

Methods: Recombinant FXIII-A AP variants (P15L, P16L, S19P/A, N20S, E23K, D24V, P27L, and P36L/F) were expressed, and SDS-PAGE was used to monitor thrombin hydrolysis at the AP cleavage site R37-G38. Transglutaminase activities were assessed by crosslinking GEE (Lys-mimic) to Fbg αC (233 – 425, Gln-substrate) and monitored using MALDI-TOF MS. Assays performed in triplicate and reported as mean ± SD.

Results: Among the FXIII-A AP variants expressed, S19A, E23K, and D24V degraded during FXIII-A purification indicating their vital role in FXIII stability. Mutation of P36 to L36/F36 abolished the proteolytic cleavage of AP, and thus prevented proteolytic activation. N20S and P27L exhibited slower thrombin activation by 2-fold compared to WT (P ≤ 0.001), likely due to the loss of key interdomain H-bonding interactions. Except N20S, all activatable FXIII-A* variants (P15L, P16L, S19P, and P27L) showed similar crosslinking activity to WT (P > 0.05). By contrast, FXIII-A° P15L and P16L had significantly lower crosslinking activity than FXIII-A° WT (P ≤ 0.05), suggesting that loss of these prolines had a greater structural impact. Transglutaminase activities of FXIII-A WT and all variants demonstrated the trend FXIII-A* > FXIII-A°.

Conclusion(s): In conclusion, we identified FXIII Activation Peptide residues that play crucial roles in FXIII-A stability and activation. The interactions between these AP amino acid residues and other domains control the stability and activity of FXIII.