According to the current view, upon conversion of fibrinogen into fibrin, its αC-domains switch from intra- to intermolecular interaction to form αC-domain polymers (1
). Numerous data suggest that such a switch is connected with conformational changes that result in physiologically active conformations of the αC-domains in fibrin αC-polymers (2
). While the structure of the individual fibrinogen αC-domain has been established (13
), little is known about their structure and function in polymeric fibrin. The major problems in studying of fibrin αC-domain polymers are the complexity of fibrin structure and the presence of multiple domains, some of which duplicate the activity of the αC-domain. In addition, αC-domain polymers cannot be prepared, like some other fibrin(ogen) domains or regions, by limited proteolysis of fibrin, due to their rapid degradation into smaller fragments. In the present study, we overcame these problems by preparing a soluble model of αC-domain polymers using the recombinant Aα221-610 fragment corresponding to the fibrinogen αC region, which includes the αC-domain (Aα392-610). The study revealed that in this model, Aα221-610 fragment forms ordered linear oligomers that are stable in solution and contain compact structure whose thermal stability is similar to that of the αC-domains in fibrin(ogen). Furthermore, in contrast to the monomeric αC-domain, such oligomers exhibit prominent binding to plasminogen and tPA supporting our hypothesis that the αC-domain binding sites become exposed upon formation of αC polymers in fibrin.
Our previous electron microscopy experiments revealed that an Aα223-539 fragment containing the αC-connector and truncated αC-domain, which was prepared by limited proteolysis of bovine fibrinogen, may form ordered oligomers (18
) that could mimic the arrangement of the αC-domains in fibrin. However, preparation of that fragment was complicated by a very low yield due to high susceptibility of the αC-domains to proteolysis and the C-terminal portion of the αC-domain in the resulting fragment was missing (18
). To overcome those problems, we prepared the recombinant bovine Aα224-568 fragment containing the full-length αC-domain and αC-connector, as well as its human analog, the Aα221-610 fragment (25
). While such fragments exhibited some ordered oligomers observed by electron microscopy, those oligomers were unstable and, therefore, not detected in solution where the fragments were preferentially monomeric (28
). Subsequent treatment of these fragments with factor XIIIa resulted in stable, soluble oligomers; however, electron microscopy revealed that they were neither linear nor ordered (28
), suggesting that their structure does not mimic that of αC polymers in fibrin. Thus, although our previous attempts to prepare soluble model of fibrin αC polymers failed, they provided valuable information that prompted us to further searched for a more adequate soluble model of fibrin αC polymers, which would be ordered and stable in solution.
Our search for such a model was facilitated by our recent finding that the recombinant αC-domain, as well as its N-terminal sub-domain, forms ordered oligomers in a concentration-dependent and reversible manner (13
). Another finding that the αC-domain interacts with the αC-connector (19
) suggested that such interaction may further promote formation of oligomers by the full-length αC region and provide a proper alignment of these portions in such oligomers for their efficient cross-linking with factor XIIIa. Therefore, in the present study, we prepared the recombinant Aα221-610 fragment corresponding to this region, confirmed that it forms oligomers, purified such oligomers, and then stabilized them by covalent cross-linking with factor XIIIa to prevent their dissociation in solution.
Several lines of evidence indicate that the prepared cross-linked Aα221-610 oligomers were highly ordered. First, oligomerization of Aα221-610 was concentration-dependent and reversible, indicating highly specific interactions between the αC-domains in the oligomers. Second, the fact that their cross-linking with factor XIIIa was much more rapid than that of the Aα221-610 monomer further confirms the ordered arrangement of the αC-connectors and αC-domains in the oligomers before the cross-linking occurs. Third, electron microscopy revealed that the oligomers appeared as well organized, almost linear arrays with a width of two monomeric molecules. Finally, spectral studies confirmed that the αC-domains in such oligomers were folded into compact cooperative units having a high content of regular structures and their thermal stability was comparable with that of the αC-domains in fibrin (31
). Thus, such highly ordered and reversible oligomerization of isolated Aα221-610 suggests that in fibrin the corresponding αC regions form αC polymers in a similar manner. This implies that cross-linked Aα221-610 oligomers mimic fibrin αC polymers.
Electron microscopy images of cross-linked Aα221-610 oligomers revealed linear arrays, or long, thin filaments. The diameter of the filaments making up these polymers was slightly less than twice the diameter of the monomers, and the globular regions of each pair of side-by-side monomers were slightly offset longitudinally. In addition, the uncross-linked monomer preparations also contained dimeric structures. These results suggest that the filaments are possibly made up of monomers interacting end-to-end and side-by-side requiring at least three interacting sites for each monomer (front, back, and side), and common for long biological filaments (33
). Electron micrographs of the cross-linked oligomers demonstrate that besides individual filaments, they also contain branched polymers. Analysis of the width of the branched network revealed that some of the polymers had the same thickness before branching as the individual filaments (two monomers thick) while others were twice as thick. This suggests at least two mechanisms of branching. One mechanism may involve each of the two monomers at the end of a single filament initiating a new filament (see inset in ). These monomers could be cross-linked to each other by factor XIIIa through unoccupied reactive Gln and Lys residues. The other type of branching points would occur through lateral association of two individual filaments (see inset in ). This mechanism may require specific lateral interactions between individual αC filaments. The existence of any such specific interactions to form such a uniform network of filaments in vitro
is evidence that they are intrinsic to the binding and cross-linking sites of the αC region itself. Furthermore, the existence of such regular interactions suggests that they could also occur between αC polymers in fibrin, in which they are located close to each other, although this remains to be demonstrated.
Our CD study indicates that individual αC-domains in cross-linked Aα221-610 oligomers are also highly ordered. While Aα221-610 monomer exhibited a substantial amount of unordered structure (53%), the content of such structures in the oligomers did not exceed 35%. The increase in regular structure content upon oligomerization is, most probably, connected with folding of the C-terminal sub-domains, which was shown earlier to adopt a folded conformation (preferentially consisting of β-sheet structure) in oligomers (14
). However, this sub-domain and the N-terminal sub-domain together represent only about a half of the Aα221-610 fragment (αC region) while another half belongs to the αC-connector, which is considered to be flexible and unordered (1
). These imply that the αC-connector or its portion(s) may also be ordered in cross-linked Aα221-610 oligomers. This is in agreement with the results of the fluorescence study that were obtained by monitoring the fluorescence of Trp residues upon unfolding of such oligomers. Although our previous experiments with the Aα221-610 fragment performed by CD confirmed the presence of a compact cooperative structure in its αC-domain, in the present study we did not observe any sigmoidal transition when heat-induced unfolding of Aα221-610 monomer was monitored by fluorescence (not shown). This is because the αC-domain of the Aα221-610 fragment does not contain any Trp residues; all of them are located in the unordered αC-connector (34
) and, therefore, completely exposed to the solvent. In contrast, Trp residues were responsive to unfolding in the oligomers (), most probably because they, or at least some of them, were in an ordered environment. Thus, the results of the present study suggest that the αC-connector or portion(s) of it adopt a regular conformation upon formation of αC polymers. This is in agreement with the previous finding that conversion of fibrinogen into fibrin is accompanied by a significant increase of the β-sheet structure, which was speculated could occur due to interactions between the C-terminal parts of the α chains in polymeric fibrin (35
Having developed a model mimicking fibrin αC polymers, we used this model to test our hypothesis that polymerization of the αC-domains in fibrin results in the exposure of their multiple binding sites. Since it was established that the αC-domains interact with plasminogen and tPA (2
), these two ligands were used as molecular probes for testing such exposure. Our ELISA and SPR experiments revealed that these ligands did not interact with the monomeric Aα221-610 fragments kept in solution. In SPR, no interaction was also observed with monomeric Aα221-610 immobilized on the surface of a sensor chip. This is in contrast to our previous SPR study that revealed a significant binding of tPA and plasminogen to immobilized Aα221-610 (2
). One of the possible reasons for such a discrepancy could be that in the previous study the aggregation state of the Aα221-610 fragment was not tested and we cannot exclude the possibility that the immobilized fragment was in an oligomeric state while in this study immobilized Aα221-610 was monomeric. Whatever the reason for the discrepancy is, the present study clearly indicates that the monomeric Aα221-610 fragment including the αC-domain does not interact with plasminogen or tPA. In contrast, these ligands exhibited a prominent binding to cross-linked Aα221-610 oligomers, supporting the above hypothesis. Moreover, these oligomers exhibited a prominent stimulating effect on activation of plasminogen by tPA while that of Aα221-610 monomer was very low, further supporting the above hypothesis. Altogether, these results indicate that the αC-domains adopt a physiologically active conformation only upon their polymerization in fibrin.
In summary, in the present study, we prepared soluble Aα221-610 oligomers containing fibrin(ogen) αC-domains and stabilized their structure by covalent cross-linking with factor XIIIa. Physico-chemical and biochemical studies of these oligomers revealed that their oligomerization occurs through highly specific interactions between monomeric units and results in formation of compact, ordered, linear polymers that most probably reflect the structure αC polymers in fibrin. They also confirmed our hypothesis that the αC-domains adopt a physiologically active conformation in such polymers. Thus, cross-linked Aα221-610 oligomers represent a simple model that mimics structural and functional properties of the αC-domain in fibrin αC polymers. This model can be used for further studying the structure and function of fibrin αC-domains.