Role of 'B-b' knob-hole interactions in fibrin binding to adsorbed fibrinogen.

BACKGROUND: The formation of a fibrin clot is supported by multiple interactions, including those between polymerization knobs 'A' and 'B' exposed by thrombin cleavage and polymerization holes 'a' and 'b' present in fibrinogen and fibrin. Although structural studies have defined the 'A-a' and 'B-b' interactions in part, it has not been possible to measure the affinities of individual knob-hole interactions in the absence of the other interactions occurring in fibrin. OBJECTIVES: We designed experiments to determine the affinities of knob-hole interactions, either 'A-a' alone or 'A-a' and 'B-b' together. METHODS: We used surface plasmon resonance to measure binding between adsorbed fibrinogen and soluble fibrin fragments containing 'A' knobs, desA-NDSK, or both 'A' and 'B' knobs, desAB-NDSK. RESULTS: The desA- and desAB-NDSK fragments bound to fibrinogen with statistically similar K(d)'s of 5.8 +/- 1.1 microm and 3.7 +/- 0.7 microm (P = 0.14), respectively. This binding was specific, as we saw no significant binding of NDSK, which has no exposed knobs. Moreover, the synthetic 'A' knob peptide GPRP and synthetic 'B' knob peptides GHRP and AHRPY, inhibited the binding of desA- and/or desAB-NDSK. CONCLUSIONS: The peptide inhibition findings show both 'A-a' and 'B-b' interactions participate in desAB-NDSK binding to fibrinogen, indicating 'B-b' interactions can occur simultaneously with 'A-a'. Furthermore, 'A-a' interactions are much stronger than 'B-b' because the affinity of desA-NDSK was not markedly different from desAB-NDSK.

Insight from studies with recombinant fibrinogens.

Using a two-step cloning strategy, we have synthesized more than 20 variant human fibrinogens for biochemical studies. In preliminary experiments we showed that normal fibrinogen produced in CHO cells serves as an accurate model for plasma fibrinogen. We focus here on those variants whose characterization has provided insight into the mechanism of thrombin-catalyzed polymerization. Analysis of N-terminal variants showed that thrombin specificity dictates the ordered release of fibrinopeptides. Nevertheless, analysis of C-terminal variants indicated that fibrinopeptide B (FpB) release is dependent on polymerization. Changes in the a polymerization site and the high-affinity calcium-binding site were associated with a complete loss of polymerization. These experiments showed that alterations in the calcium-binding site influenced function of the a site; in contrast, alterations in the a site did not alter calcium binding. Analysis of variants in the N-terminus of the B beta chain provided the first direct evidence that this region impacts predominantly on lateral aggregation, as has long been presumed. These experiments also suggested that lateral aggregation facilitated by this region proceeds without the release of FpB. From these studies we learned that individual sites within fibrinogen do not function in isolation. We conclude that thrombin-catalyzed polymerization is mediated by a continuum of concerted interactions.

Decreased lateral aggregation of a variant recombinant fibrinogen provides insight into the polymerization mechanism.

We analyzed the polymerization of BbetaA68T fibrinogen, the recombinant counterpart of fibrinogen Naples, a variant known to have decreased thrombin binding. When polymerized with equal thrombin concentrations, BbetaA68T fibrinogen had a longer lag time and lower rate of lateral aggregation, V(max), than normal recombinant fibrinogen, but a similar final turbidity. At thrombin concentrations that equalized the rates of fibrinopeptide A release, BbetaA68T fibrinogen polymerized with a lag time and V(max) similar to normal, but reached a significantly lower final turbidity. Similar results were produced when BbetaA68T was polymerized with Ancrod, which cleaves fibrinopeptide A at the same rate from either fibrinogen, and when BbetaA68T desA monomers were polymerized. The polymerization of desAB fibrin monomers, which circumvents fibrinopeptide release, was the same for both fibrinogens. We confirmed that turbidity was indicative of fiber thickness by scanning electron microscopy of fibrin clots. Here, we present the first experimental evidence of fibrin polymerization with a normal period of protofibril formation and rate of lateral aggregation, but with a significantly decreased extent of lateral aggregation. We conclude that the decreased lateral aggregation seen in BbetaA68T fibrinogen is due to an altered step in the enzymatic phase of its polymerization process. We propose that during normal polymerization a subtle conformational change in the E domain occurs, between the release of FpA and FpB, and that this change modulates the mechanism of lateral aggregation. Without this change, the lateral aggregation of BbetaA68T fibrinogen is impaired such that variant clots have thinner fibers than normal clots.

Recombinant fibrinogen studies reveal that thrombin specificity dictates order of fibrinopeptide release.

During cleavage of fibrinogen by thrombin, fibrinopeptide A (FpA) release precedes fibrinopeptide B (FpB) release. To examine the basis for this ordered release, we synthesized A'beta fibrinogen, replacing FpB with a fibrinopeptide A-like peptide, FpA' (G14V). Analyses of fibrinopeptide release from A'beta fibrinogen showed that FpA release and FpA' release were similar; the release of either peptide followed simple first-order kinetics. Specificity constants for FpA and FpA' were similar, demonstrating that these peptides are equally competitive substrates for thrombin. In the presence of Gly-Pro-Arg-Pro, an inhibitor of fibrin polymerization, the rate of FpB release from normal fibrinogen was reduced 3-fold, consistent with previous data; in contrast, the rate of FpA' release from A'beta fibrinogen was unaffected. Thus, with A'beta fibrinogen, fibrinopeptide release from the beta chain is similar to fibrinopeptide release from the alpha chain. We conclude that the ordered release of fibrinopeptides is dictated by the specificity of thrombin for its substrates. We analyzed polymerization, following changes in turbidity, and found that polymerization of A'beta fibrinogen was similar to that of normal fibrinogen. We analyzed clot structure by scanning electron microscopy and found that clots from A'beta fibrinogen were similar to clots from normal fibrinogen. We conclude that premature release of the fibrinopeptide from the N terminus of the beta chain does not affect polymerization of fibrinogen.

A functional assay suggests that heterodimers exist in two C-terminal gamma-chain dysfibrinogens: Matsumoto I and Vlissingen/Frankfurt IV.

Because it contains three pairs of polypeptides, fibrinogen isolated from heterozygous individuals is expected to be a mixture of homodimers and heterodimers. Nevertheless, heterozygous individuals with only homodimers have been identified. We synthesized two recombinant fibrinogens with the mutations from fibrinogen Vlissingen/ Frankfurt IV (gamma(delta)319, 320) and Matsumoto I (gammaD364H), both identified in heterozygous individuals. We found that polymerization of these fibrinogens was undetectable in 30 min; polymerization of a 1:1 mixture of variant and normal fibrinogen was the same as polymerization of a 1:1 mixture of buffer and normal fibrinogen; polymerization of either plasma fibrinogen was markedly impaired when compared to the 1:1 mixture of the respective variant and normal fibrinogens. We conclude that each plasma fibrinogen is a mix of homodimers and heterodimers, such that the incorporation of heterodimers into the fibrin clot impairs polymerization. We suggest that incorporation of heterodimers can induce clinical symptoms.

Recombinant fibrinogen Vlissingen/Frankfurt IV. The deletion of residues 319 and 320 from the gamma chain of firbinogen alters calcium binding, fibrin polymerization, cross-linking, and platelet aggregation.

We synthesized a variant, recombinant fibrinogen modeled after the heterozygous dysfibrinogen Vlissingen/Frankfurt IV, a deletion of two residues, gammaAsn-319 and gammaAsp-320, located within the high affinity calcium-binding pocket. Turbidity studies showed no evidence of fibrin polymerization, although size exclusion chromatography, transmission electron microscopy, and dynamic light scattering studies showed small aggregates. These aggregates did not resemble normal protofibrils nor did they clot. Fibrinopeptide A release was normal, whereas fibrinopeptide B release was delayed approximately 3-fold. Plasmin cleavage of this fibrinogen was not changed by the presence of calcium or Gly-Pro-Arg-Pro, indicating that both the calcium-binding site and the "a" polymerization site were non-functional. We conclude that the loss of normal polymerization was due to the lack of "A-a" interactions. Moreover, functions associated with the C-terminal end of the gamma chain, such as platelet aggregation and factor XIII cross-linking, were also disrupted, suggesting that this deletion of two residues affected the overall structure of the C-terminal domain of the gamma chain.

Analysis of A alpha 251 fibrinogen: the alpha C domain has a role in polymerization, albeit more subtle than anticipated from the analogous proteolytic fragment X.

Numerous experiments have demonstrated that the C-terminal domain of the fibrinogen Aalpha-chain, the alphaC domain, has a role in polymerization. To further examine the role of this domain, we synthesized a recombinant fibrinogen, Aalpha251 fibrinogen, that lacks the alphaC domain. We examined thrombin-catalyzed fibrinopeptide release and found that the rate of FpB release from Aalpha251 fibrinogen was 2.5-fold slower than FpB release from normal fibrinogen, while the rate of FpA release was the same for both proteins. We examined thrombin-catalyzed polymerization and found that the rates of protofibril formation and lateral aggregation were similar for both proteins, although discernible differences in lateral aggregation were clear. The rate of protofibril formation for Aalpha251 fibrinogen was never less than 85% of normal fibrinogen, while the rate of lateral aggregation for Aalpha251 fibrinogen varied from 64 to 74% of normal. We examined polymerization of fibrin monomers and found that polymerization of Aalpha251 fibrin was similar to normal fibrin at 0.4 M NaCl, but clearly different from normal at 0.05 M NaCl. These results indicate that the alphaC domain has a role in lateral aggregation, but this role is more subtle than anticipated from previous experiments, particularly those with fibrinogen fragment X. We interpret this unanticipated finding as indicative of an important contribution from the N-terminus of the beta-chain, such that protein heterogeneity that includes small amounts of fibrin lacking that N-terminus of the beta-chain leads to markedly altered lateral aggregation.

Severely impaired polymerization of recombinant fibrinogen gamma-364 Asp --> His, the substitution discovered in a heterozygous individual.

During blood coagulation, soluble fibrinogen is converted to fibrin monomers that polymerize to form an insoluble clot. Polymerization has been described as a two-step process: the formation of double-stranded protofibrils and the subsequent lateral aggregation of protofibrils into fibers. Previous studies have shown that gamma chain residues Tyr-363 and Asp-364 have a significant role in polymerization, most likely in protofibril formation. To better define the role of these residues, we synthesized three fibrinogens with single substitutions at these two positions: Tyr-363 --> Ala, Asp-364 --> Ala, and Asp-364 --> His. We found that the release of fibrinopeptides A and B was the same for these variants and normal recombinant fibrinogen, showing that all variants had normal fibrin formation. In contrast, we found that polymerization was significantly delayed for both Ala variants and was almost nonexistent for the His variant. Clottability for the Ala variants was only slightly reduced, and fibrin gels were formed. Surprisingly, clottability of the His variant was substantially reduced, and fibrin gels were not formed. Our data suggest that both protofibril formation and lateral aggregation were altered by these substitutions, indicating that the C-terminal domain of the gamma chain has a role in both polymerization steps.

The conversion of fibrinogen to fibrin: recombinant fibrinogen typifies plasma fibrinogen.

Plasma fibrinogen is a mixture of multiple molecular forms arising mainly through alternative mRNA processing and subsequent posttranslational modification. Recombinant fibrinogen is synthesized without alternative mRNA processing in a cultured cell system that may generate novel posttranslational modifications. Thus, to show that recombinant fibrinogen can serve as a functional model for plasma fibrinogen, we have examined the conversion of fibrinogen to fibrin, comparing the recombinant with the plasma protein. We examined the kinetics of (1) thrombin-catalyzed fibrinopeptide release, (2) thrombin-catalyzed polymerization of fibrinogen, (3) the polymerization of fibrin monomers, and (4) FXIIIa-catalyzed cross-link formation. We saw small differences in polymerization, suggesting that the ordered assembly of protofibrils and fibers was not identical. In all other analyses, we found that plasma fibrinogen and recombinant fibrinogen were remarkably similar. Using electron microscopy, we examined the structures of individual fibrinogen molecules and fibrin clots. Individual fibrinogen molecules were predominantly three nodule structures for both recombinant and plasma proteins. Both samples also displayed four nodule structures, but fewer four nodule structures were found with recombinant fibrinogen. Fibrin clot structures were essentially indistinguishable. We concluded that recombinant fibrinogen can serve as a accurate model for plasma fibrinogen.

Role of the alpha C domains of fibrin in clot formation.

The role of the carboxyl-terminal portion of the alpha chains of fibrin (alpha C domains) in clot formation was investigated by transmission and scanning electron microscopy and turbidity studies of clots made from preparations of molecules missing one or both of these domains. Highly purified and entirely clottable preparations of bovine fragment X monomer, one containing primarily molecules missing a single alpha C domain (fragment X1) and the other consisting of molecules missing both alpha C domains (fragment X2), were used for these experiments. These preparations were characterized by various methods, including the complete determination of the amino- and carboxyl-termini of all peptides and fragments. These preparations formed clots on dilution to neutral pH. In all cases, clots observed by either scanning or transmission electron microscopy were made up of a branched network of fibers, similar to those formed by thrombin treatment of intact fibrinogen, suggesting that the alpha C domains are not necessary for protofibril and fiber formation or branching. However, both the fiber and clot structure varied with the different fractions, indicating that the alpha C domains do participate in polymerization. The rate of assembly, as indicated by the lag period and maximum rate of turbidity increase, as well as the final turbidity, was decreased with removal of the alpha C domains, suggesting that they accelerate polymerization. preparations of isolated alpha C fragment added to fibrin monomer have striking effects on the turbidity curves, showing a decrease in the rate of polymerization in a dose-dependent manner but not complete inhibition. Electron microscopy of fibrin monomer desA molecules at neutral pH showed that most of the alpha C domains, like those in fibrinogen, remain associated with the central region. Thus, it appears that normally with thrombin cleavage of fibrinogen the effects of the interactions of alpha C domains observed here will be most significant for lateral aggregation.

Carboxyl-terminal portions of the alpha chains of fibrinogen and fibrin. Localization by electron microscopy and the effects of isolated alpha C fragments on polymerization.

The locations of the carboxyl-terminal two thirds of the A alpha chains, or the alpha C domains, were determined for fibrinogen and some of its derivatives by electron microscopy of rotary-shadowed preparations. A monoclonal antibody, G8, to the carboxyl-terminal 150 amino acids of the A alpha chain, binds near the central region of fibrinogen, indicating that the alpha C domains of most molecules are not normally visible because they are on or near the amino-terminal disulfide knot. At pH 3.5, fibrinogen and fibrin monomers appear to be similar, with a projection terminating in a small globular domain from each end of most molecules. In contrast, fragment X monomers, produced by cleavage of the alpha C domains from fibrinogen with plasmin, show no such projections. When fibrin monomer is brought to neutral pH under conditions where polymerization is delayed, individual molecules are still visible showing the alpha C domains as a single additional nodule near the central region. Moreover, analysis of clusters of molecules reveals some intermolecular associations via the alpha C domains. A 40-kDa fragment comprising the alpha C domain has been isolated from a plasmin digest of fibrinogen and characterized by SDS-polyacrylamide gel electrophoresis and determination of amino-terminal amino acid sequences. Electron microscopy of alpha C fragments reveals individual globular structures, as well as oligomeric aggregates. The addition of alpha C fragments to fibrin monomer followed by dilution to neutral pH to initiate polymerization results in lower turbidity, longer lag period, and slower maximum rate of turbidity increase. Also, electron microscopy reveals complexes of alpha C fragments with fibrin monomer at neutral pH. It appears that the free alpha C fragments can bind to the alpha C domains of fibrin, competing with the normal alpha C domain interactions involved in polymerization.

[Role of exogenous fibrinogen in the processes of degranulation of thrombocytes stimulated with thrombin. Ultrastructural study using fibrinogen labeled with colloidal gold]. / Rol' ékzogennogo fibrinogena v protsessakh degranuliatsii trombotsitov, stimulirovannykh trombinom. Ul'trastrukturnoe issledovanie s primeneniem fibrinogena, mechennogo kolloidnym zolotom.

Using colloidal gold-conjugated fibrinogen (F-Au) it is shown that exogenous fibrinogen can participate in the platelet release reaction. In the absence of F-Au, internal secretory vacuoles readily formed in human platelets stimulated with thrombin, but extrusion of their content was delayed. Upon incubation with F-Au, endocytic channels induced by F-Au-receptor interactions, fused with internal vacuoles, thus establishing spatial communications of the latter with the outer medium.

[The role of adhesive proteins and membrane interactions in the processes of thrombocyte aggregation]. / Rol' adgezivnykh belkov i membrannykh vzaimodeistvii v protsessakh agregatsii trombotsitov.

The reported data concerning the role of fibrinogen in platelet aggregation are reviewed and compared to the authors' experimental data obtained by electron microscopy and cytochemical techniques. Using fibrinogen and fibrinogen antibodies bound to colloidal gold, it has been shown that the presence of fibrinogen bridged between the adjoining cells is not necessary for primary platelet microaggregation stimulated by ADP or thrombin. The formation of direct interplatelet contacts resembling "pentalaminar membranes" has been shown to participate in that process. Some mechanisms are proposed to explain the enhanced adhesiveness of the activated platelet surfaces. Redistribution of phospholipids in the membrane bilayer leading to the exposure of negatively charged phospholipids may underlie that phenomenon. The clustering and internalization of "occupied" membrane receptors may also contribute to the formation of close contacts between platelets stimulated by primary agonists in the presence of exogenous fibrinogen and other adhesive proteins.

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.

[Ultrastructural changes of platelets during ADP- stimulated aggregation (in the presence of fibrinogen)]. / Ul'trastrukturnye izmeneniia trombotsitov pri ADF-stimuliruemoi agregatsii (v prisutstvii fibrinogena).

The ultrastructure of resting and stimulated human blood platelets (P) was studied by the transmission electron microscopy. The cells were chemically fixed (using tannic acid and OsFeCN mixture) 1, 3, 5 and 15 min after the addition of ADP and fibrinogen (F). Early changes in P ultrastructure consist in drastic reduction of the electron-dense layer of glycocalyx and in an increase of the plasma membrane permeability. At the early stages of P aggregation the cells contact with each other due to rapidly arising pseudopodia. Later, the extracellular network containing an exogenous F participates in the aggregation process.

[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.

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.

[Analysis of the stability of the intermediate fibrin polymers formed in a fibrinogen-thrombin system]. / Issledovanie stabil'nosti promezhutochnykh polimerov fibrina, obrazuiushchikhsia v sisteme fibrinogen-trombin.

Intermediate fibrin polymers formed from fibrinogen at low thrombin concentrations under physiological conditions are studied for their stability. Smith's statement that intermediate fibrin oligomers are inert molecules whose further self-assembly is possible only under additional thrombin activation was not supported. It is shown that the intermediate fibrin polymer stability at pH 7.4 is not high because of a strong tendency to aggregation and the higher molecular weight of the polymer the less its stability. The polymer stability in solution increases with pH. Stability of soluble fibrin complexes in solution depends on their size, defined by environmental conditions and on the quantity of fibrinogen present.