Factor XIIIa-Catalyzed Cross-Linking of Recombinant αC Fragments of Human Fibrinogen

Abstract

Direct measurements of the structure and function of the COOH-terminal portion of the Aα chain (residues 220−610) of human fibrinogen have been hampered by the difficulty of isolating intact fragments derived from this protease-sensitive region. Here, we overcame this problem by expressing two fragments, αC45K (Aα221−610) and a truncated version of it, αC30K (Aα368−610), in Escherichia coli. Both proteins were purified to homogeneous state, and their integrity was confirmed at protein level by sequencing. Upon treatment with factor XIIIa, the αC45K fragment but not the αC30K fragment formed polymers similar to those derived from fibrin clots. Sequence analysis of cross-linked αC45K polymers revealed involvement in the cross-linking reaction of at least three Gln residues (221, 237, 328) in the NH₂-terminal region of the fragment and four Lys residues (539, 556, 580, 601) located in the COOH-terminal part of the molecule. In addition, a fraction of αC45K fragment was found in an intramolecular cross-linked form, suggesting a high level of flexibility of its polypeptide chain and consistent with the location of its donor and acceptor residues in clusters near the ends of the molecule. The αC30K fragment, lacking the NH₂-terminal Gln residues, was not able to form polymers or internally cross-linked monomers. Thus, the C-terminal part of the Aα chain comprises an autonomous, functionally active, and flexible region that plays a key role in α polymer formation and stabilization of fibrin clots by factor XIIIa.