The activation peptide of coagulation factor XIII is vital for its expression and stability.

BACKGROUND: The human activation peptide of factor XIII (AP-FXIII) comprises the first 37 amino acids of the N-terminus and holds the FXIII in an inactive state. FXIII is activated either proteolytically by cleavage of AP-FXIII by thrombin, or non-proteolytically by high calcium concentrations. OBJECTIVE: To investigate the role of AP-FXIII in the expression and stability of FXIII. METHODS: We cloned 13 FXIII variants with progressive truncations of AP-FXIII from the N-terminus (delN-FXIII-A), expressed them in mammalian cells, and measured their thermostability, activation, and transglutaminase activity. We also used in silico calculations to analyze the stability of hypothetical delN-FXIII dimers and to identify crucial motifs within AP-FXIII. RESULTS: Variants with deletions longer than the first 10 amino acids and an R11Q point mutant were not expressed as proteins. In silico calculations indicated that the sequence (8) FGGR(12) R plays a substantial role in intersubunit interactions in FXIII-A2 homodimers. In agreement with this prediction, the temperature stability of delN-FXIII variants decreased with increasing length of deletion. These results may suggest a role of the N-terminus of AP-FXIII in dimer stability. Substantial sequence homology was found among activation peptides of vertebrate and even invertebrate (crustacean) FXIII-A orthologs, which further supports our conclusion. CONCLUSIONS: We conclude that deletion of 11 or more N-terminal amino acids disrupts intersubunit interactions, which may prevent FXIII-A2 homodimer formation. Therefore, AP-FXIII plays an important role in the stability of the FXIII-A2 dimer.

Congenital factor XIII deficiency in Pakistan: characterization of seven families and identification of four novel mutations.

Deficiency of coagulation factor XIII (FXIII) belongs to the rare bleeding disorders and its incidence is higher in populations with consanguineous marriages. The aims of this study were to characterize patients and relatives from seven families with suspected FXIII deficiency from Pakistan and to identify the underlying mutations. As a first indicator of FXIII deficiency, a 5M urea clot solubility test was used. Plasma FXIII A- and B-subunit antigen levels were determined by ELISA. FXIII activity was measured with an incorporation assay. Sequencing of all exons and intron/exon boundaries of F13A was performed, and a novel splice site defect was confirmed by RT-PCR analysis. Genetic analysis revealed six different mutations in the F13A gene. Two splice site mutations were detected, a novel c.1460+1G>A mutation in the first nucleotide of intron 11 and a previously reported c.2045G>A mutation in the last nucleotide of exon 14. Neither of them was expressed at protein level. A novel nonsense mutation in exon 4, c.567T>A, p.Cys188X, was identified, leading in homozygous form to severe FXIII deficiency. Two novel missense mutations were found in exons 8 and 9, c.1040C>A, p.Ala346Asp and c.1126T>C, p.Trp375Arg, and a previously reported missense mutation in exon 10, c.1241C>T, p.Ser413Leu. All patients homozygous for these missense mutations presented with severe FXIII deficiency. We have analysed a cohort of 27 individuals and reported four novel mutations leading to congenital FXIII deficiency.