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

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 sub-fragments, 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. TheαC-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 fibrinopeptides 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 fibrinopeptides B, in agreement with the above hypothesis, and indicate that fibrinopeptides A are 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

Interaction of Fibrin with the Very Low-Density Lipoprotein (VLDL) Receptor: Further Characterization and Localization of the VLDL Receptor-Binding Site in Fibrin βN-Domains

Our recent study revealed that fibrin and the very low-density lipoprotein receptor (VLDLR) interact with each other through a pair of fibrin βN-domains and CR domains of the receptor and this interaction promotes transendothelial migration of leukocytes and thereby inflammation. The major objectives of this study were to further clarify the molecular mechanism of fibrin–VLDLR interaction and to identify amino acid residues in the βN-domains involved in this interaction. Our binding experiments with the (β15–66)₂ fragment, which corresponds to a pair of fibrin βN-domains, and the VLDLR(1–8) fragment, consisting of eight CR domains of VLDLR, revealed that interaction between them strongly depends on ionic strength and chemical modification of all Lys or Arg residues in (β15–66)₂ results in abrogation of this interaction. To identify which of these residues are involved in the interaction, we mutated all Lys or Arg residues in each of the three positively charged Lys/Arg clusters of the (β15–66)₂ fragment, as well as single Arg17 and Arg30, and tested the affinity of the mutants obtained for VLDLR(1–8) by an enzyme-linked immunosorbent assay and surface plasmon resonance. The experiments revealed that the second and third Lys/Arg clusters make the major contribution to this interaction while the contribution of the first cluster is moderate. The results obtained suggest that interaction between fibrin and the VLDL receptor employs the “double-Lys/Arg” recognition mode previously proposed for the interaction of the LDL receptor family members with their ligands. They also provide valuable information for the development of highly specific peptide-based inhibitors of fibrin–VLDLR interaction