Leonid V. Medved

The Structure and Function of the αC Domains of Fibrinogen

Abstract: The αC domains have been localized on fibrinogen and fibrin. Several model systems have been developed to study their functions. Analysis of the amino acid sequence of the αC domains suggested that each is made up of a globular and an extended portion. Microcalorimetry confirmed this result and showed that the two αC domains interact intramolecularly. Electron microscopy of fibrinogen with a monoclonal antibody to the αC domains demonstrated that these regions normally interact with the central portion of the molecule. In the conversion from fibrinogen to fibrin there is a large scale conformational change, such that the αC domains dissociate from the central region and are available for intermolecular interaction. Experiments with highly purified and well characterized fragment X monomer, missing either one or both of the αC domains, indicate that intermolecular interactions between αC domains are important for the enhancement of lateral aggregation during fibrin polymerization. Isolated αC fragments polymerized at neutral pH and interacted with the αC domains of fibrin monomer to influence clot formation. Several dysfibrinogenemias in which there are amino acid substitutions in, or truncations of, the αC domains revealed that these changes can have dramatic effects on polymerization and clot structure. The polymerization of Aα251 recombinant fibrinogen, that contains Aα chains truncated at residue 251, was altered, as were the mechanical properties and the rate of fibrinolysis of the clots. Altogether, these results help to define the role of the αC domains in determining the structure and properties of clots.

Localization of Factor IXa and Factor VIIIa Interactive Sites

The contribution of the catalytic and noncatalytic domains of factor IXa to the interaction with its cofactor, factor VIIIa, was evaluated. Two proteolytic fragments of factor IXa, lacking some or all of the serine protease domain, failed to mimic the ability of factor IXa to enhance the reconstitution of factor VIIIa from isolated A1/A3-C1-C2 dimer and A2 subunit. Both fragments, however, inhibited this factor IXa-dependent activity. Selective thermal denaturation of the factor IXa serine protease domain eliminated its effect on factor VIIIa reconstitution. Modification of factor IXa with dansyl-Glu-Gly-Arg chloromethyl ketone (DEGR-IXa) stabilized this domain, and heat-treated DEGR-IXa retained its ability to enhance factor VIIIa reconstitution. These results indicate the importance of the serine protease domain as well as structures residing in the factor IXa light chain (-carboxyglutamic acid and/or epidermal growth factor domains) for cofactor stabilizing activity. In the presence of phospholipid, the A1/A3-C1-C2 dimer produced a saturable increase in the fluorescence anisotropy of fluorescein-Phe-Phe-Arg chloromethyl ketone-modified factor IXa (Fl-FFR-IXa). This effect was inhibited by a factor IXa fragment comprised of the -carboxyglutamic acid and epidermal growth factor domains. The difference in Fl-FFR-IXa anisotropy in the presence of A1/A3-C1-C2 dimer (Δr = 0.043) compared with factor VIIIa (Δr = 0.069) represented the contribution of the A2 subunit. A peptide corresponding to factor VIII A2 domain residues 558-565 decreased the factor VIIIa dependent-anisotropy of Fl-FFR-IXa to a value similar to that observed with the A1/A3-C1-C2 dimer. These results support a model of multiple interactive sites in the association of the enzyme-cofactor complex and localize sites for the A1/A3-C1-C2 dimer and the A2 subunit to the factor IXa light chain and serine protease domain, respectively.

Conformational Changes upon Conversion of Fibrinogen into Fibrin

Abstract: 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 αC‐domains that are directly involved in intermolecular interactions upon fibrin assembly. According to the current view, two αC‐domains that interact intramolecularly in fibrinogen undergo an intra‐ to intermolecular switch to form αC‐polymers in fibrin. The availability of recombinant fragments that correspond to the αC‐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 (Aα148–160 and γ312–324) 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‐Aα148–160 and anti‐γ312–324 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.

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 vant 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.

Fibrinogen αC Domains Contain Cryptic Plasminogen and tPA Binding Sites

Abstract: Surface plasmon resonance and ELISA experiments revealed that recombinant fibrinogen αC fragment (residues Aα221–610) corresponding to the αC domain binds tPA and plasminogen with high affinity. This binding was found to be Lys‐dependent and occurred via independent binding sites. Study with truncated variants of the αC fragment located these sites in its COOH‐terminal half. Binding of tPA and plasminogen to these sites stimulated activation of the latter whereas proteolytic degradation of the αC fragment reduced this effect substantially, suggesting the importance of the αC domains in regulation of fibrinolysis.

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.

Structural and Functional Role of the β-Strand Insert (γ381–390) in the Fibrinogen γ-Module

Abstract: Study of the folding status of the fibrinogen γ‐module (residues γ148–411) revealed that its COOH‐terminal β‐strand (residues γ381–390), 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 γ‐γ crosslinks in fibrin.