Our recent study established the NMR structure of the recombinant bAα406-483 fragment corresponding to the NH2-terminal half of the bovine fibrinogen αC-domain and revealed that at increasing concentrations this fragment forms oligomers (self-associates). The major goals of the present study were to determine the structure and self-association of the full-length human fibrinogen αC-domains. To accomplish these goals, we prepared a recombinant human fragment, hAα425-503, homologous to bovine bAα406-483, and demonstrated using NMR, CD, and size-exclusion chromatography that its overall fold and ability to form oligomers are similar to those of bAα406-483. We also prepared recombinant hAα392-610 and bAα374-568 fragments corresponding to the full-length human and bovine αC-domain, respectively, and tested their structure, stability, and ability to self-associate. Size-exclusion chromatography revealed that both fragments form reversible oligomers in a concentration-dependent manner. Their oligomerization was confirmed in sedimentation equilibrium experiments, which also established the self-association affinities of these fragments and revealed that the addition of each monomer to assembling αC-oligomers substantially increases the stabilizing free energy. In agreement, unfolding experiments monitored by CD established that self-association of both fragments results in a significant increase of their thermal stability. Analysis of CD spectra of both fragments revealed that αC self-association results in the increase of regular structures implying that the COOH-terminal half of the αC-domain adopts ordered conformation in αC-oligomers and that this domain contains two independently folded sub-domains. Altogether, these data further clarify the structure of the human and bovine αC-domains and the molecular mechanism of their self-association into αC-polymers in fibrin.

Nonsubstrate interactions of thrombin with fibrin play an important role in modulating its procoagulant activity. To establish the structural basis for these interactions, we crystallized D-Phe-ProArg-chloromethyl ketone-inhibited human thrombin in complex with a fragment, E ht, corresponding to the central region of human fibrin, and solved its structure at 3.65-Å resolution. The structure revealed that the complex consists of two thrombin molecules bound to opposite sides of the central part of E ht in a way that seems to provide proper orientation of their catalytic triads for cleavage of fibrinogen fibrinopeptides. As expected, binding occurs through thrombin's anion-binding exosite I. However, only part of it is involved in forming an interface with the complementary negatively charged surface of E ht. Among residues constituting the interface, Phe-34, Ser-36A, Leu-65, Tyr-76, Arg-77A, Ile-82, and Lys-110 of thrombin and the A␣ chain Trp-33, Phe-35, Asp-38, Glu-39, the B␤ chain Ala-68 and Asp-69, and the ␥ chain Asp-27 and Ser-30 of E ht form a net of polar contacts surrounding a well defined hydrophobic interior. Thus, despite the highly charged nature of the interacting surfaces, hydrophobic contacts make a substantial contribution to the interaction.