On the Mechanism of αC Polymer Formation in Fibrin

Our previous studies revealed that the fibrinogen alpha C-domains undergo conformational changes and adopt a physiologically active conformation upon their self-association into alpha C polymers in fibrin. In the present study, we analyzed the mechanism of alpha C polymer formation and tested our hypothesis that self-association of the alpha C-domains occurs through the interaction between their N-terminal subdomains and may include beta-hairpin swapping. Our binding experiments performed by size-exclusion chromatography and optical trap-based force spectroscopy revealed that the alpha C-domains self-associate exclusively through their N-terminal subdomains, while their C-terminal subdomains were found to interact with the alpha C-connectors that tether the alpha C-domains to the bulk of the molecule. This interaction should reinforce the structure of alpha C polymers and provide the proper orientation of their reactive residues for efficient cross-linking by factor XIIIa. Molecular modeling of self-association of the N-terminal subdomains confirmed that the hypothesized beta-hairpin swapping does not impose any steric hindrance. To freeze the conformation of the N-terminal subdomain and prevent the hypothesized beta-hairpin swapping, we introduced by site-directed mutagenesis an extra disulfide bond between two beta-hairpins of the bovine A alpha 406-483 fragment corresponding to this subdomain. The experiments performed by circular dichroism revealed that A alpha 406-483 mutant containing Lys429Cys/Thr463Cys mutations preserved its beta-sheet structure. However, in contrast to wild-type A alpha 406-483, this mutant had lower tendency for oligomerization, and its structure was not stabilized upon oligomerization, in agreement with the above hypothesis. On the basis of the results obtained and our previous findings, we propose a model of fibrin alpha C polymer structure and molecular mechanism of assembly.