Direct Evidence for Specific Interactions of the Fibrinogen αC-Domains with the Central E Region and with Each Other

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Abstract
The carboxyl-terminal regions of the fibrinogen Aα chains (αC regions) form compact αC-domains tethered to the bulk of the molecule with flexible αC-connectors. It was hypothesized that in fibrinogen two αC-domains interact intramolecularly with each other and with the central E region preferentially through its N-termini of Bβ chains and that removal of fibrinopeptides A and B upon fibrin assembly results in dissociation of the αC regions and their switch to intermolecular interactions. To test this hypothesis, we studied the interactions of the recombinant αC region (Aα221−610 fragment) and its subfragments, αC-connector (Aα221−391) and αC-domain (Aα392−610), between each other and with the recombinant (Bβ1−66)2 and (β15−66)2 fragments and NDSK corresponding to the fibrin(ogen) central E region, using laser tweezers-based force spectroscopy. The αC-domain, but not the αC-connector, bound to NDSK, which contains fibrinopeptides A and B, and less frequently to desA-NDSK and (Bβ1−66)2 containing only fibrinopeptides B; it was poorly reactive with desAB-NDSK and (β15−66)2 both lacking fibrinopeptide B. The interactions of the αC-domains with each other and with the αC-connector were also observed, although they were weaker and heterogeneous in strength. These results provide the first direct evidence for the interaction between the αC-domains and the central E region through fibrinopeptide B, in agreement with the hypothesis given above, and indicate that fibrinopeptide A is also involved. They also confirm the hypothesized homomeric interactions between the αC-domains and display their interaction with the αC-connectors, which may contribute to covalent cross-linking of α polymers in fibrin.