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Conformational changes upon conversion of fibrinogen to fibrin result in the exposure of multiple binding sites that provide its interaction with various proteins and cells and, thus, its participation in a number of physiological and pathological processes. Here we focus on conformational changes in the fibrinogen D regions (domains) and alpha C-domains that are directly involved in intermolecular interactions upon fibrin assembly. According to the current view, two alpha C-domains that interact intramolecularly in fibrinogen undergo an intra- to intermolecular switch to form alpha C-polymers in fibrin. The availability of recombinant fragments that correspond to the alpha C-domain made it possible to further clarify this mechanism and to reveal novel cryptic sites in this domain for plasminogen and its activator tPA, whose exposure may play an important role in the regulation of fibrinolysis. To elucidate the mechanism of exposure of cryptic sites in the D regions, we tested the accessibility of their fibrin specific epitopes (A alpha 148-160 and gamma 312-324) that are also involved in binding of plasminogen and tPA, in several fragments derived from fibrinogen (fragment D), and crosslinked fibrin (fragment D-D and its non-covalent complex with the E1 fragment, D-D:E1). Neither D nor D-D bound tPA, plasminogen, or anti-A alpha 148-160 and anti-gamma 312-324 monoclonal antibodies. At the same time both epitopes became accessible in the D-D:E1 complex. Melting of D and D-D revealed that their domains have the same stability while in the D-D:E1 complex they became more stable. These results indicate that upon fibrin assembly, driven primarily by the interaction between complementary binding sites of the E and two D regions, the latter undergo conformational changes that cause the exposure of their cryptic sites. They also suggest that the fibrin specific conformation of the D regions is preserved in the D-D:E1 complex.

Conversion of fibrinogen into fibrin results in the exposure of cryptic interaction sites and modulation of various activities. To elucidate the mechanism of this exposure, we tested the accessibility of the Aalpha148-160 and gamma312-324 fibrin-specific epitopes that are involved in binding of plasminogen and its activator tPA, in several fragments derived from fibrinogen (fragment D and its subfragments) and fibrin (cross-linked D-D fragment and its noncovalent complex with the E(1) fragment, D-D. E(1)). Neither D nor D-D bound tPA, plasminogen, or anti-Aalpha148-160 and anti-gamma312-324 monoclonal antibodies, indicating that their fibrin-specific epitopes were inaccessible. The Aalpha148-160 epitope became exposed only upon proteolytic removal of the beta- and gamma-modules from D. At the same time, both epitopes were accessible in the D-D.E(1) complex, indicating that the DD.E interaction resulted in their exposure. This exposure was reversible since the dissociation of the D-D.E(1) complex made the sites unavailable, while reconstitution of the complex made them exposed. The results indicate that upon fibrin assembly, driven primarily by the interaction between complementary sites of the D and E regions, the D regions undergo conformational changes that cause the exposure of their plasminogen- and tPA-binding sites. These changes may be involved in the regulation of fibrin assembly and fibrinolysis.

Study of the folding status of the fibrinogen gamma-module (residues gamma 148-411) revealed that its COOH-terminal beta-strand (residues gamma 381-390), that is normally inserted into its central domain, can be removed without destroying its compact structure. Based on this and other observations we propose a "pull out" hypothesis that suggests a mechanism for the formation of transverse gamma-gamma crosslinks in fibrin.

The high-resolution crystal structure of the N-terminal central region of bovine fibrinogen (a 35-kDa E 5 fragment) reveals a remarkable dimeric design. The two halves of the molecule bond together at the center in an extensive molecular “handshake” by using both disulfide linkages and noncovalent contacts. On one face of the fragment, the Aα and Bβ chains from the two monomers form a funnel-shaped domain with an unusual hydrophobic cavity; here, on each of the two outer sides there appears to be a binding site for thrombin. On the opposite face, the N-terminal γ chains fold into a separate domain. Despite the chemical identity of the two halves of fibrinogen, an unusual pair of adjacent disulfide bonds locally constrain the two γ chains to adopt different conformations. The striking asymmetry of this domain may promote the known supercoiling of the protofibrils in fibrin. This information on the detailed topology of the E 5 fragment permits the construction of a more detailed model than previously possible for the critical trimolecular junction of the protofibril in fibrin.

The crystal structure of the fibrinogen gamma-module (residues gamma143-411) [Yee, V. C., et al. (1997) Structure 5, 125-138] revealed an unusual feature. Namely, residues gamma381-390 in the functionally important COOH-terminal region form a beta-strand that is inserted into an antiparallel beta-sheet of the central domain (gamma192-286), while the rest (gamma393-411) seems to be flexible. To clarify the structural and functional importance of this beta-strand insert, we analyzed the folding status of the plasmin-derived fibrinogen fragment D(3) and several truncated variants of the gamma-module expressed in Escherichia coli. It was found that D(3), in which most of the COOH-terminal domain of the gamma-module (gamma287-379) is removed proteolytically, retains a gamma374-405 peptide that seems to be associated noncovalently with the bulk of the molecule via its beta-strand insert region. A study of the denaturation-renaturation process of D(3) suggested that without this peptide its truncated gamma-module remains folded but is destabilized. This was confirmed directly with the truncated recombinant variants of the gamma-module, including residues gamma148-392, gamma148-373, and gamma148-286. They all were folded, but those devoid of the beta-strand insert were destabilized. The results indicate that although the beta-strand insert contributes to the stabilization of the gamma-module, it can be removed without destroying the compact structure of the latter. On the basis of this finding and some other observations, we propose a mechanism for the function-related conformational changes in the fibrin(ogen) gamma-modules.