Domain structure and interactions of the type I and type II modules in the gelatin-binding region of fibronectin. All six modules are independently folded.

The gelatin-binding region of fibronectin is isolated easily as a stable and functional 42 kDa fragment containing four type I "finger" modules and two type II "kringle-like" modules arranged in the order I6-II1-II2-I7-I8-I9. This fragment exhibits a single reversible melting transition near 64 degrees C in TBS buffer (0.02 M-Tris buffer containing 0.15 M-NaCl, pH 7.4). The transition is characterized by a calorimetric to van't Hoff enthalpy ratio of 1.6, suggesting a complex domain structure. A 30 kDa fragment with the same NH2 terminus (I6-II1-II2-I7) melts reversibly near 65 degrees C with delta Hcal/delta HvH = 1.3, also consistent with the presence of more than one domain. To elucidate further the domain structure, three non-overlapping subfragments were prepared and characterized with respect to their unfolding induced by heat and guanidinium chloride. The three subfragments, each containing two modules, are designated from amino or carboxyl-terminal location as 13 kDa (I6-II1) 16 kDa (II2-I7) and 21 kDa (I8-I9) according to their apparent Mr in SDS/polyacrylamide gel electrophoresis. All three subfragments exhibited reversible transitions in TBS buffer, behaving in the calorimeter as single co-operative units with delta Hcal/delta HvH close to unity. However, the specific enthalpies and changes in heat capacity associated with the melting of all fragments and subfragments in TBS buffer were low compared to those of most compact globular proteins, suggesting that not all modules are represented. When titrated with guanidinium chloride at 25 degrees C, all fragments exhibited monophasic reversible unfolding transitions detected by changes in fluorescence. Heating in the presence of 6 M-guanidinium chloride revealed three additional transitions not seen in the absence of denaturants. These transitions have been assigned to three of the four type I finger modules (I6, I7 and I9), one of which (I6) was isolated and shown to retain a compact structure as stable as that observed for this module within the parent fragments. Two other modules (II2 and I7) are destabilized when separated from their neighbors. Thus, despite their small size (50 to 60 amino acid residues), all six of the modules in the gelatin-binding region of fibronectin form independently folded domains, three of which (I6, I7 and I9) are unusually stable. Evidence is provided that four of the six modules interact with each other in the parent fragment. This interaction may explain previously noted disruptions in the otherwise uniform strand-like images seen in electron micrographs of fibronectin.

Co-operative domains in fibronectin.

The melting of human plasma fibronectin and its proteolytic fragments has been studied by scanning microcalorimetry to reveal co-operative structural domains in the molecule. It has been established that each of the two similar polypeptide chains of fibronectin has at least 12 structural domains, which differ in stability, size and function. Many of the domains in the N-terminal half of the polypeptide chains appear to be composed of two homologous repeat modules that co-operate to form a single co-operative unit. In the intact fibronectin molecule, the C-terminal regions of both chains seem to interact forming a stable co-operative block.

Domain structure, stability and domain-domain interactions in recombinant factor XIII.

The process of heat denaturation of recombinant factor XIII (rFXIII), as well as its C-terminal 24 kDA and 12 kDa elastase-produced fragments starting at Ser514 and Thr628, respectively, was investigated in a wide range of conditions by fluorescence, CD and differential scanning calorimetry (DSC). It was found that the intact protein melts in two distinct temperature regions reflecting unfolding of different parts of the molecule with different stability. The less stable structures unfold in a low temperature transition with a tm of 69 degrees C or lower depending on conditions. Unfolding of the more stable structures was observed at extremely high temperatures, tm > 110 degrees C at acidic pH < 3.5 and tm = 90 degrees C at pH 8.6 with 2 M GdmCL. Thermodynamic analysis of the low and high temperature DSC-obtained heat absorption peaks indicated unambiguously that the first represents melting of three thermolabile independently folded domains while two thermostable domains melt in the second one giving a total of five domains in each a subunit of rFXIII. Both 24 kDa and 12 kDa fragments exhibited a sigmoidal spectral transition at comparatively high temperature where the thermolabile structures are already denatured, indicating that two thermostable domains are formed by the C-terminal portion of rFXIII and correspond to the two beta-barrels revealed by crystallography. The remaining 56 kDa portion forms three thermolabile domains, one of which corresponds to the N-terminal beta-sandwich and the other two to the catalytic core. Fast accessible surface calculations of the X-ray model of rFXIII confirmed the presence of two structural subdomains in the core region with the boundary at residue 332. The thermolabile domains appear to interact with each other intra- and/or intermolecularly resulting in dimerization the a subunits. At acidic pH, where all domains became destabilized but still remained folded, interdomainial interactions seemed to be abolished, resulting in the reversible dissociation of the dimer as revealed by ultracentrifugation analysis.

Domain organization of the terminal parts in the fibrinogen molecule.

Using limited proteolysis and scanning microcalorimetry it was shown that each terminal part of the fibrinogen molecule is constituted by four co-operative domains. Among these domains two strongly interacting domains are formed by the C-terminal part of the beta-chain, while the two other domains are formed by the C-terminal part of the gamma-chain.

Plasminogen-binding site of the thermostable region of fibrinogen fragment D.

Affinity chromatography of plasminogen and its proteolytic fragments on immobilized fibrinogen TSD fragment has shown that the latter contains a plasminogen-binding site which is complementary to the lysine-binding site(s) of plasminogen molecule 1-3 kringle structures.

The role of fibrinogen alpha C-domains in the fibrin assembly process.

Turbidity development registration and electron microscopic observation of the assembly process of the fibrin monomer and its derivative lacking in intact alpha C-domains (monomeric X1 fragment) have shown that these domains participate in fibrin polymerization, not as structural components, but as a factor promoting the ordered process of fibrin assembly.

Structural organization of C-terminal parts of fibrinogen A alpha-chains.

Calorimetric studies of fibrinogen melting and of its early degradation products have shown that the C-terminal parts of both the A alpha-chains form structural domains which strongly interact with each other in the native fibrinogen molecule.

The cleavage of beta-chain in bovine fibrinogen DH fragment (95 kDa) leads to a significant increase in its anticlotting activity.

It is shown that in the presence of Ca2+ plasmin converts bovine fibrinogen fragment DH (95 kDa) into DLA fragment by the cleavage of its beta-chain Arg372-Thr373 bond. DLA fragment consists of two components (82 and 12 kDa) held together by non-covalent bonds and has 3.5-fold higher anticlotting activity than DH fragment. The DH to DLA fragment conversion leads to the destabilization of thermolabile domains of the latter without the loss of their compact structure. The results obtained show that the activation of DH fragment by the cleavage of its Arg372-Thr373 bond bears some resemblance to the general activation of proenzyme into enzyme.

Localization of a fibrin polymerization site complementary to Gly-His-Arg sequence.

Dansyl-labeled tetrapeptide Gly-His-Arg-Pro which mimics the central fibrin polymerization site was used to investigate its binding to a number of fibrinogen fragments containing different numbers of domains. The tetrapeptide was found to bind to fragments DH(95 kDa), DL(82 kDa) and DY(63 kDa) but not to the TSD(28 kDa) fragment. The DY fragment differs from the TSD by the presence of beta and beta C domains. Therefore these domains, which are formed by the C-terminal part of the beta chain, possess a polymerization site complementary to the Gly-His-Arg containing counterpart.

Fibrinogen-containing membrane-associated structures arising at the surfaces of ADP-stimulated blood platelets.

Ultrastructural studies of ADP-stimulated gel-filtered human platelets incubated with different concentrations of fibrinogen reveal unusual extracellular structures composed at least partly of the aggregated fibrinogen. Development of these structures depends on the exogenous fibrinogen concentration and duration of the incubation. Fibrinogen-containing extracellular material exists in two different structural forms. As was shown earlier, one of them is represented by dense, amorphous intercellular matrix and is localized mainly in platelet microaggregates (Belitser et al., Thromb. Res., in press). Another one consists of huge sheet-like structures bearing individual or clumped platelets bound either to one or to both of their surfaces. After thrombin treatment, these structures could not be found anymore; sometimes they seem to be substituted by the fibrin-like fibers spatially connected to each other and/or to the platelet membranes. It has been suggested that soluble fibrinogen interacting with its specific membrane receptors undergoes conformational changes promoting intermolecular interactions and resulting in the fibrinogen aggregates formation at the surfaces of the activated platelets. A probable physiological significance of the structures described is discussed. It is supposed that under certain conditions in vivo they may play an important role, in particular as a highly concentrated substrate for the thrombin action, providing in such a way a possibility of rapid, effective consolidation of the initial platelet thrombi with the fibrin fibers.

Relationship between exons and domains in the fibrinogen molecule.

The correlation between domains structure of the fibrinogen molecule and the exon/intron composition of the genes encoding the three fibrinogen polypeptide chains has been analysed. A coincidence of the borders between structural domains and corresponding exons in the N-terminal parts of the molecule was revealed, while no such correlation was found in the C-terminal parts. The analysis indicates that the domain structure of the fibrinogen molecule and the exon/intron structure of its genes evolved independently during the later stage of evolution. This analysis also allowed delineation of the exact borders between some domains and prediction of the existence of additional domains in the central part of the fibrinogen molecule.

[A model of proteolysis of the fragment DH (95 kD) of the fibrinogen molecule]. / Skhema proteoliza fragmenta DH (95 kDa) molekuly fibrinogena.

A detailed analysis of further proteolytic degradation of fibrinogen fragment DH (Mr 95 kDa) was performed. Two new proteolytic fragments DLA and DL derived from DH-fragment have been purified and analyzed. The results obtained allow to propose a general scheme of DH-fragment proteolysis by stepwise splitting of their individual domains. The borders and molecular masses of these domains were evaluated on the base of proteolysis data.

[Isolation and investigation of structural organization of active and inactive forms of fibrinogen D-fragment molecule]. / Poluchenie i issledovanie strukturnoi organizatsii aktivnoi i neaktivnoi form D-fragmenta molekuly fibrinogena.

High molecular weight fragment D (M.W. 95 000) which is an effective inhibitor of polymerization of fibrin, and its more low molecular weight form (M.W. 82 000) which has no antipolymerization activity was obtained from fibrinogen by proteolysis. It was shown by the method of scanning microcalorimetry that the active D-fragment consists of four cooperative regions: one thermostable and three non-equal thermolabile. The smallest thermolabile cooperative region (M.W. 13 000) in the inactive D-fragment is absent. It is supposed, that it takes part in the formation of the active centre.

[Microcalorimetric studies of temperature transitions in fibrinogen and its proteolytic fragments]. / Mikrokalorimetricheskie issledovaniia temperaturnykh perekhodov v fibrinogene i ego proteoliticheskikh fragmentakh.

Melting of fibrinogen and its proteolytic fragments has been studied by differential scanning microcalorimetry. It has been shown that the fibrinogen molecule contains at least nine cooperative regions. One of them pertains to the central structural block (domain E), two of them are formed by the structures removed at proteolytic fragmentation and the others make part of the terminal structural blocks (domains D). A scheme of localization of melting regions in the fibrinogen molecule is proposed on the basis of the results obtained.

[Domainal organization of the molecules of Lys-plasminogen]. / Domennaia organizatsiia molekuly Lys-plazminogena.

The intramolecular melting of the human Lys-plasminogen and its different fragments were studied by the differential scanning microcalorimetry method. Thermodynamical analysis of melting curves showed that the Lys-plasminogen molecule consists of 7 domains. Five of them are formed by five homologeus regions of the polypeptide chain (kringle), while two domains are formed by the part of the polypeptide chain corresponding to the plasmin light chain. The domains included in the fragments seem to be rather independent, since fragmentation does not lead to noticeable changes of their stability in comparison to that of the intact molecule. It has been shown also that plasminogen-plasmin conversion is accompanied by structural transformation of the molecule which results in the destabilization of one of the light chain domains.

[alphaC domains of fibrinogen molecules as the structures accelerating fibrin assembly]. / alphaC-domeny molekuly fibrinogena kak struktury, uskoriaiushchie samosborku fibrina.

Preparation of monomeric fibrin lacking intact alpha C-domains (monomeric X1-fragment), but fully clottable, is described. The assembly process of both monomeric fibrin and monomeric X1-fragment has been studied by electron microscopy and light scattering methods. It was shown that both proteins form similar fibrils with characteristic cross-banding. Upon dilution a sharp elevation of the differences between the assembly rates of monomeric X1-fragment and monomeric fibrin was revealed. The results obtained show that alpha C-domains take part in fibrin clot formation not as structural components but as the factor accelerating the ordered assembly of complex fibrin structure. The possible mechanism of alpha C-domains participation in fibrin clot formation are regarded.

[Structural organization of the fibrinogen molecule]. / Strukturnaia organizatsiia molekuly fibrinogena.

A brief review of the chemical structure of the fibrinogen molecule, its proteolytic fragmentation, physicochemical and functional properties of proteolytic fragments is presented. The previous notions on the structure of the fibrinogen molecule are considered as well as new data obtained by the methods of scanning microcalorimetry, electron microscopy and by other methods which permit suggesting some models for structural organization of the fibrinogen molecule.