Structure, Stability, and Interaction of Fibrin αC-Domain Polymers

Our previous studies revealed that in fibrinogen the alpha C-domains are not reactive with their ligands, suggesting that their binding sites are cryptic and become exposed upon its conversion to fibrin, in which these domains form alpha C polymers. On the basis of this finding, we hypothesized that polymerization of the aC-domains in fibrin results in the exposure of their binding sites and that these domains adopt the physiologically active conformation only in alpha C-domain polymers. To test this hypothesis, we prepared a recombinant alpha C region (residues A alpha 221-610) including the alpha C-domain (A alpha 392-610), demonstrated that it forms soluble oligomers in a concentration-dependent and reversible manner, and stabilized such oligomers by covalently cross-linking them with factor XIIIa. Cross-linked A alpha 221-610 oligomers were stable in solution and appeared as ordered linear, branching filaments when analyzed by electron microscopy. Spectral studies revealed that the alpha C-domains in such oligomers were folded into compact structures of high thermal stability with a significant amount of beta-sheets. These findings indicate that cross-linked A alpha 221-610 oligomers are highly ordered and mimic the structure of fibrin alpha C polymers. The oligomers also exhibited functional properties of polymeric fibrin because, in contrast to the monomeric alpha C-domain, they bound tPA and plasminogen and stimulated activation of the latter by the former. Altogether, the results obtained with cross-linked Aa221-610 oligomers clarify the structure of the alpha C-domains in fibrin alpha C polymers and confirm our hypothesis that their binding sites are exposed upon polymerization. Such oligomers represent a stable, soluble model of fibrin alpha C polymers that can be used for further structure function studies of fibrin alpha C-domains.