On the mechanism of αC polymer formation in fibrin.

Fibrinogen αC-domains mediate fibrin(ogen) activity in various physiological and pathological processes. By interacting with each other, they promote lateral aggregation of protofibrils during fibrin assembly1,2 and play an important role in determining the structure and properties of fibrin clots and their susceptibility to fibrinolysis.3 The αC-domains interact with factor XIII,4 which covalently cross-links them upon activation thereby reinforcing fibrin clots, and control activation of this factor.5 In addition, they modulate fibrinolysis by interacting with plasminogen and its activator, tPA, and inhibitor, α2-antiplasmin.6–8 They promote cell adhesion and spreading through the interaction of their Aα572–574 RGD sequence with cell surface integrins9,10 and may be involved in modulation of fibrin-dependent angiogenesis.11 The αC-domains may also contribute to the development of atherothrombosis through their interaction with apo(a) component of lipoprotein(a).12 Each of the two αC-domains of the fibrinogen molecule is formed by the C-terminal portion of the Aα chain (amino acid residues Aα392–610) and is tethered to the bulk of the molecule with a flexible αC-connector (residues Aα221–391); together they comprise a structure called the αC region.12,13 The αC-domain contains a compact structure whose presence was established by differential scanning calorimetry14–16 and electron microscopy.17–19 Recent study revealed that each αC-domain consists of two independently folded units, N- and C-terminal subdomains formed by the Aα392–503 and Aα504–610 residues, respectively.20 The NMR solution structure of the recombinant Aα406–483 fragment corresponding to the N-terminal subdomain of the bovine fibrinogen αC-domain revealed that this fragment contains two β-hairpins which form a mixed four-stranded parallel/antiparallel β-sheet.21,22 The recombinant Aα425–503 fragment corresponding to the N-terminal subdomain of the human fibrinogen αC-domain was shown to have a similar fold.20 Thus, the three-dimensional structure of the N-terminal subdomain of the αC-domain has been established, but that of its C-terminal subdomain remains to be defined. Numerous studies suggest that in fibrinogen two αC-domains interact with each other and with the central E region of the molecule, whereas upon conversion of fibrinogen into fibrin they dissociate and switch from intra- to intermolecular interaction, thus promoting lateral aggregation of protofibrils.1–3,15,19,23,24 Such intermolecular interaction between the αC-domains in fibrin results in formation of αC polymers whose structure is reinforced by cross-linking with activated factor XIII (factor XIIIa). Other studies revealed that the αC-domains are inert in fibrinogen; however, in fibrin they become reactive and interact with a number of proteins including plasminogen, tPA, α2-antiplasmin, and apolipoprotein(a).6,8,12 Altogether, these studies suggest that conversion of fibrinogen into fibrin is accompanied by conformational changes in the αC-domains, resulting in the exposure or formation of their multiple binding sites. A recent study confirmed that the αC-domains adopt a physiologically active conformation upon self-association (polymerization) and partially clarified the structure of these domains in αC polymers.25 However, the mechanism of such self-association, as well as the detailed structure of αC polymers, is still unclear. The ability of the isolated αC-domains to self-associate into soluble oligomers was demonstrated in the studies with the recombinant fragments corresponding to human and bovine αC-domains and their N-terminal subdomains.20,22 This process was shown to be concentration-dependent and reversible, suggesting that it mimics self-association (polymerization) of the tethered αC-domains in fibrin.20,25 It was also found that the thermodynamic stability of the monomeric forms of these fragments is low while that of their oligomeric forms is significantly increased.20,22 On the basis of these and some other findings, we hypothesized that self-association of the αC-domains into αC polymers in fibrin occurs through the interaction between their N-terminal subdomains and may include β-hairpin swapping.20,22 However, this hypothesis does not take into account the recently discovered interaction between the αC-domain and αC-connector,24 which may also contribute to αC polymer formation. It is also unclear whether the C-terminal subdomains contribute to self-association of the αC-domains. The major goal of the present study was to test this hypothesis and to further clarify the mechanism of self-association of the αC-domains and the structure of fibrin αC polymers.