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On the mechanism of ?C polymer formation in fibrin.

ABSTRACT: Our previous studies revealed that the fibrinogen ?C-domains undergo conformational changes and adopt a physiologically active conformation upon their self-association into ?C polymers in fibrin. In the present study, we analyzed the mechanism of ?C polymer formation and tested our hypothesis that self-association of the ?C-domains occurs through the interaction between their N-terminal subdomains and may include ?-hairpin swapping. Our binding experiments performed by size-exclusion chromatography and optical trap-based force spectroscopy revealed that the ?C-domains self-associate exclusively through their N-terminal subdomains, while their C-terminal subdomains were found to interact with the ?C-connectors that tether the ?C-domains to the bulk of the molecule. This interaction should reinforce the structure of ?C polymers and provide the proper orientation of their reactive residues for efficient cross-linking by factor XIIIa. Molecular modeling of self-association of the N-terminal subdomains confirmed that the hypothesized ?-hairpin swapping does not impose any steric hindrance. To "freeze" the conformation of the N-terminal subdomain and prevent the hypothesized ?-hairpin swapping, we introduced by site-directed mutagenesis an extra disulfide bond between two ?-hairpins of the bovine A?406-483 fragment corresponding to this subdomain. The experiments performed by circular dichroism revealed that A?406-483 mutant containing Lys429Cys/Thr463Cys mutations preserved its ?-sheet structure. However, in contrast to wild-type A?406-483, this mutant had lower tendency for oligomerization, and its structure was not stabilized upon oligomerization, in agreement with the above hypothesis. On the basis of the results obtained and our previous findings, we propose a model of fibrin ?C polymer structure and molecular mechanism of assembly.

SUBMITTER: Tsurupa G

PROVIDER: S-EPMC3343699 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

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On the mechanism of αC polymer formation in fibrin.

Tsurupa Galina G Pechik Igor I Litvinov Rustem I RI Hantgan Roy R RR Tjandra Nico N Weisel John W JW Medved Leonid L

Biochemistry 20120315 12

Our previous studies revealed that the fibrinogen αC-domains undergo conformational changes and adopt a physiologically active conformation upon their self-association into αC polymers in fibrin. In the present study, we analyzed the mechanism of αC polymer formation and tested our hypothesis that self-association of the αC-domains occurs through the interaction between their N-terminal subdomains and may include β-hairpin swapping. Our binding experiments performed by size-exclusion chromatogra ...[more]

PMID: 22397628

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Project description:The fibrin polymers formed in solution during the earliest phase of the fibrinogen-fibrin conversion are shown to be stable soluble molecules at pH7.4 and 0.15m- or 0.3m-NaCl. The various sequential soluble fibrin polymers produced from the fibrinogen-thrombin reaction can be observed by gel chromatography and can be isolated for characterization. The mechanism of fibrin polymerization proposed from the present studies suggests that the initial event is the thrombin activation at only one of the Aalpha-chains in fibrinogen. The resulting highly reactive intermediate is the true fibrin monomer and it rapidly, and irreversibly, self-associates to form the stable fibrin dimer (s(20.w)=12S). Fibrin dimer possesses the N-terminal pattern alanine/glycine/tyrosine (1:1:2) per 340000 molecular weight, and possesses the chain structure [(alpha)Aalpha)(Bbeta)(2)(gamma)(2)](2). The fibrin dimer is a soluble inert molecule, but additional thrombin activation of its remaining intact Aalpha-chains leads to new associations into larger inert soluble fibrin polymers. In this manner progressively larger fibrin oligomers are constructed with thrombin continually in control of the process because of the necessity to repeatedly re-activate the various fibrin polymers in solution. The inert character of the soluble fibrin polymers can be explained by the reciprocal alignment of the associating molecules, which mutually consumes their active surfaces and leaves an intact Aalpha-chain at either end of each fibrin oligomer. The soluble fibrin polymers will proceed to further association only if thrombin activates these remaining Aalpha-chains, otherwise the fibrin molecules are stable indefinitely. The intermolecular associations within the soluble fibrin polymers are essentially irreversible under these nearly physiological conditions. However, the bonding is not covalent. This mechanism accounts for the clinical observations of stable fibrinogen-derived polymers in the plasma from patients undergoing thrombotic processes. Since it is shown that the intermediate fibrin polymers, themselves, are stable soluble molecules, it is no longer necessary, nor warranted, to invoke hypothetical ;fibrinogen-fibrin complexes' to explain observations of fibrin solubility.

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On the mechanism of ?C polymer formation in fibrin.

Publications

On the mechanism of αC polymer formation in fibrin.

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