Stimulation of fibrinogen synthesis in cultured rat hepatocytes by fibrinogen fragment E

Because of the inherent difficulties of experimentation in intact animals, we used primary monolayer cultures of non-proliferating adult rat hepatocytes to study the effects of fibrinogen degradation products on fibrinogen biosynthesis. The freshly isolated hepatocytes obtained by collagenase perfusion of the liver in situ were cultured in a chemically defined serum-free medium. The rate of fibrinogen synthesis in control cultures was $\text{40–50}\text{pmol}\text{2.5·10}{}^6$ cells per 24 h. Additions of 20, 60 or 100 μg of homologous stage I fibrinogen degradation products had no effect on fibrinogen synthesis. In contrast, addition of the same amounts of homologous or heterologous (human) stage III fibrinogen degradation products resulted in a concentration-dependent increase in fibrinogen biosynthesis without affecting the rate of synthesis of albumin. When purified stage III fibrinogen degradation products D and E (human) were tested in 10, 30 or 50 μg/3 ml medium only fragment E showed a significant increase in fibrinogen biosynthesis (1.9-, 2.8- and 5.6-fold, respectively, over the control cultures). The presence of excess fibrinogen had no effect. These results suggest that fibrinogen fragment E may be a specific stimulator of fibrinogen biosynthesis which may play an important role in maintaining normal levels of plasma fibrinogen.     

Molecular Characterization of the Interaction of Staphylococcal Microbial Surface Components Recognizing Adhesive Matrix Molecules (MSCRAMM) ClfA and Fbl with Fibrinogen

The ligand-binding domain of Fbl (the fibrinogen binding protein from Staphylococcus lugdunensis) shares 60% sequence identity with ClfA (clumping factor A) of Staphylococcus aureus. Recombinant Fbl corresponding to the minimum fibrinogen-binding region (subdomains N2N3) was compared with ClfA for binding to fibrinogen. Fbl and ClfA had very similar affinities for fibrinogen by surface plasmon resonance. The binding site for Fbl in fibrinogen was localized to the extreme C terminus of the fibrinogen γ-chain at the same site recognized by ClfA. Isothermal titration calorimetry showed that Fbl and ClfA had very similar affinities for a peptide mimicking the C-terminal segment of the fibrinogen γ-chain. The peptide also inhibited binding of Fbl and ClfA to fibrinogen. A series of substituted γ-chain variant peptides behaved very similarly when used to inhibit ClfA and Fbl binding to immobilized fibrinogen. Both ClfA and Fbl bound to bovine fibrinogen with a lower affinity compared with human fibrinogen and did not bind detectably to ovine fibrinogen. The structure of the N2N3 subdomains of Fbl in complex with the fibrinogen γ-chain peptide was modeled based on the crystal structure of the N2N3 subdomains of the ClfA-γ-chain peptide complex. Residues in the putative binding trench likely to be involved in fibrinogen binding were identified. Fbl variant proteins with alanine substitutions in key residues had reduced affinities for fibrinogen. Thus Fbl and ClfA bind the same site in fibrinogen by similar mechanisms.     

Lack of effect of thrombin on fibrin(ogen)-endothelial cell interaction

In the present study we investigate the fibrin(ogen)-endothelial cell binding and the effect of thrombin on the endothelial cells in relation to fibrin(ogen) binding capacity. Endothelial cell fibrinogen binding was concentration and time-dependent, reaching saturation at 1.4 M of added ligand. At equilibrium, the number of fibrinogen molecules bound per endothelial cell in the monolayer was 5.8±0.7×10⁶. When endothelial cells were activated by different concentrations of thrombin (0–0.1 NIH units ml^–1), no increase in fibrinogen binding capacity was observed at all the thrombin concentration tested. Whereas disruption of endothelial cell monolayers was observed at thrombin concentrations higher than 0.05 NIH units ml^–1, no increase in the amount of fibrinogen bound was observed. Therefore, resting and thrombin-activated endothelial cells show the same fibrinogen binding capacity.The adhesion of endothelial cells in suspension on immobilized fibrinogen or fibrin was studied to ascertain whether the behavior of fibrin is similar to that of fibrinogen. The extent of endothelial cell attachment to immobilized fibrinogen and fibrin was similar (4275±130 cells cm^–2 for fibrinogen and 4350±235 cells cm^–2 for fibrin) and represent approximately 40% of the added endothelial cells. However, endothelial cell adhesion to immobilized fibrin was significantly faster than endothelial cell adhesion to immobilized fibrinogen. The maximum binding rate was 66±9 and 46±8 cells cm^–2 min^–1 for fibrin and fibrinogen, respectively. Therefore, the fibrinopeptides released by thrombin from fibrinogen induce qualitative changes which enhance the fibrin interaction with the endothelial cells.     

Babesia argentina: Studies on the nature of an antigen associated with infection

A crude soluble haemagglutination antigen obtained from a mixture of Babesia argentina parasites and infected erythrocyte stromata contained fibrinogen. The fibrinogen was removed by precipitation in an initial attempt to purify the antigen. However, most if not all of the antigenic activity was located in the fibrinogen precipitate. After consideration of the evidence available, it was concluded that the antigen was either a babesial moiety complexed with fibrinogen or a fibrinogen molecule altered by the metabolic activity of the parasite.     

Concentration-dependent effect of fibrinogen on IgG-specific antigen binding and phagocytosis

In this paper, we aim to characterize fibrinogen-IgG interactions, and explore how fibrinogen alters IgG-mediated phagocytosis.Using enzyme-linked binding assays, we found that fibrinogen binding to IgG is optimized for surfaces coated with high levels of IgG. Using a similar method, we have shown that for an antigen unable to specifically bind fibrinogen, fibrinogen enhances binding of antibodies towards that antigen. For binding of IgG antibodies to cells expressing Fc receptors, we found a bimodal binding response, where low levels of fibrinogen enhance binding of antibody to Fc receptors and high levels reduce it. This corresponds to a bimodal effect on phagocytosis of IgG-coated particles, which is inhibited in the presence of excess IgG during coating of the particles with antibodies and fibrinogen.We conclude that fibrinogen can modulate phagocytosis of IgG-coated particles in vitro by changing IgG binding behavior, and that high fibrinogen levels could negatively affect phagocytosis.     

The presence of heat-labile factors interfering with binding analysis of fibrinogen with ferritin in horse plasma

Background

Horse fibrinogen has been identified as a plasma specific ferritin-binding protein. There are two ways in the binding of ferritin-binding protein with ferritin: one is direct binding and the other is indirect binding which is heme-mediated. The aim of this study was to analyze the binding between horse fibrinogen and ferritin.

Findings

Although fibrinogen in horse plasma did not show the binding to ferritin coated on the plate wells, after following heat-treatment (60°C, 30 min) of horse plasma, plasma fibrinogen as well as purified horse fibrinogen bound to plates coated with horse spleen ferritin, but not with its apoferritin which lost heme as well as iron after the treatment of reducing reagent. Binding of purified or plasma fibrinogen to ferritin was inhibited by hemin and Sn-protoporphyrin IX (Sn-PPIX), but not by PPIX or Zn-PPIX.

Conclusions

Heat-treatment of horse plasma enabled plasma fibrinogen to bind to plate well coated with holo-ferritin. From the binding analysis of fibrinogen and ferritin, it is suggested that horse fibrinogen recognized iron or tin in complexed with the heme- or the hemin-ring, and also suggest that some fibrinogens circulate in the form of a complex with ferritin and/or heat-labile factors which inhibit the binding of fibrinogen with ferritin.

     

Functional binding analysis of human fibrinogen as an iron- and heme-binding protein

Human fibrinogen is a metal ion-binding protein, but its mechanism of binding with iron and heme has not been elucidated in detail. In this study, human fibrinogen was immobilized on CNBr-activated Sepharose 4B beads. The fibrinogen beads bound hemin (iron–protoporphyrin IX: PPIX) as well as iron ion released from ferrous ammonium sulfate (FAS) more efficiently than Sepharose 4B beads alone. Hemin bound to fibrinogen still exhibited pseudo-peroxidase activity. The affinity of fibrinogen binding to hemin, Sn–PPIX, Zn–PPIX and metal-free PPIX followed the order Sn–PPIX < metal-free PPIX < hemin < Zn–PPIX; PPIX bound more non-specifically to control beads. FAS significantly enhanced the binding of hemin to fibrinogen beads. These results suggest that human fibrinogen directly recognizes iron ion, the PPIX ring and metal ions complexed with the PPIX ring, and that the binding of hemin is augmented by iron ions.     

Mechanism of enzymatic crosslinking of fibrinogen molecules

The mechanism of enzymatic covalent crosslinking of fibrinogen molecules under the effect of plasma transglutaminase (factor XIIIa) has been studied. Using the methods of elastic and dynamic light scattering combined with viscosimetry and electrophoresis of the reduced samples, it has been shown that fibrinogen oxidation strongly accelerates self-assembly of the covalently crosslinked fibrinogen polymers. IR-spectroscopy data indicate that the degree of fibrinogen oxidation positively correlates with the amount of ε/(γ-glu)lys isopeptide covalent bonds whose formation is catalyzed by the factor XIIIa. The results of this and our previous studies cast doubt on the widespread concept that native fibrinogen is the substrate for factor XIIIa. In our opinion, only structurally defective fibrinogen molecules that have undergone oxidative structural conversion in the D-domain region are involved in the enzymatic reaction leading to the formation of covalent ε/(γ-glu)lys isopeptide bonds and self-assembly of fibrinogen polymers.     

Localization of fibrinogen during ADP- or thrombin-induced aggregation of washed rabbit platelets

In contrast to human platelets, which aggregate poorly in response to ADP unless fibrinogen is present in the external medium, washed rabbit platelets form large aggregates in response to ADP without fibrinogen in the suspending medium. Addition of fibrinogen to the suspending medium of rabbit platelets frequently has little or no effect on the extent of ADP-induced platelet aggregation. We examined washed rabbit platelets by immunocytochemistry during ADP-induced aggregation and deaggregation and during thrombin-induced aggregation when the external medium did not contain added fibrinogen to determine if (a) fibrinogen was expressed on the surface of rabbit platelets that could support aggregation when the platelets were stimulated, or (b) fibrinogen secreted from the alpha granules supports platelet aggregation. Glutaraldehyde-fixed samples were prepared at different times after addition of ADP or thrombin, embedded in Lowicryl K4M, sectioned, incubated with sheep anti-rabbit fibrinogen, washed, reacted with gold-labeled anti-sheep IgG, and prepared for electron microscopy. The alpha granules of rabbit platelets were heavily labeled with immunogold; the platelet membrane was not labeled. During platelet aggregation and deaggregation in response to ADP, fibrinogen was not detectable on the platelet surface. In response to thrombin, large aggregates formed before fibrinogen was secreted from the alpha granules; fibrinogen was detectable focally at sites of granule discharge by 30-60 sec and fibrin formed by 3 min. Therefore, stimulated washed rabbit platelets can adhere to each other without large amounts of fibrinogen taking part in the close platelet-to-platelet contact, since aggregation occurs before detectable secretion, and large areas where the platelets are in contact are devoid of fibrinogen between the adherent membranes. Adhesion mechanisms not involving fibrinogen may support the aggregation of washed rabbit platelets.     

Ozone-induced oxidative modification of fibrinogen: Role of the D regions

Native fibrinogen is a key blood plasma protein whose main function is to maintain hemostasis by virtue of producing cross-linked fibrin clots under the influence of thrombin and fibrin-stabilizing factor (FXIIIa). The aim of this study was to investigate mechanisms of impairment of both the molecular structure and the spatial organization of fibrinogen under ozone-induced oxidation. FTIR analysis showed that ozone treatment of the whole fibrinogen molecule results in the growth of hydroxyl, carbonyl, and carboxyl group content. A similar analysis of fibrinogen D and E fragments isolated from the oxidized protein also revealed transformation of distinct important functional groups. In particular, a remarkable decay of N–H groups within the peptide backbone was observed along with a lowering of the content of C–H groups belonging to either the aromatic moieties or the aliphatic chain CH₂ and CH₃ units. The model experiments performed showed that the rather unexpected decay of the aliphatic CH units might be caused by the action of hydroxyl radicals, these being produced in the water solution from ozone. The observed dissimilarities in the shapes of amide I bands of the fibrinogen D and E fragments before and after ozone treatment are interpreted in terms of feasible local conformational changes affecting the secondary structure of the protein. Taken as a whole, the FTIR data suggests that the terminal D fragments of fibrinogen are markedly more susceptible to the ozone-induced oxidation than the central E fragment. The data on elastic and dynamic light scattering provide evidence that, in the presence of FXIIIa, both the unoxidized and the oxidized fibrinogen molecules bind to one another in an “end-to-end” fashion to form the flexible covalently cross-linked fibrinogen homopolymers. The γ and α polypeptide chains of the oxidized fibrinogen proved to be involved in the enzymatic cross-linking more readily than those of unaffected fibrinogen. The experimental data on fibrinogen oxidation acquired in the present study, combined with our earlier findings, make it reasonable to suppose that the spatial structure of fibrinogen could be evolutionarily adapted to some reactive oxygen species actions detrimental to the protein function.     

The comparison of peroxynitrite action on bovine, porcine and human fibrinogens

Subjects of bovine and porcine flocks are sometimes susceptible to death before time of slaughter, and some of those deaths may be due to cardiovascular problems connected with stress. The role of oxidative stress in farm animals is yet unexplored. Human fibrinogen seems to be highly susceptible to nitration. Peroxynitrite produced from superoxide and nitric oxide initiates noticeable changes in the structure of human fibrinogen molecule. The objective of this work is to compare the in vitro interactions of peroxynitrite with human fibrinogen and with fibrinogen from mammals of great economic importance, namely cows and pigs. Using western blots and ELISA we show that porcine fibrinogen is susceptible to tyrosine nitration induced by peroxynitrite whereas, bovine fibrinogen is more resistant. Moreover, porcine fibrinogen polymerization is susceptible to peroxynitrite action, whereas bovine fibrinogen is the least susceptible to inhibition of polymerization caused by peroxynitrite. These observed differences may result from differences in amino acid sequence of fibrinogen chains, mostly including tyrosine content and location in the Aα chain. Protection against toxic effects of peroxynitrite activity in the circulatory system seems to be important in avoiding cardiovascular diseases and may prevent production loss in pig breeding herds.     

Interaction between fibronectin and purified staphylococcal clumping factor

Clumping of Staphylococcal aureus was observed in the presence of fibrinogen as well as fibronectin. In order to elucidate the mechanism of this clumping, binding of radiolabelled fibrinogen and fibronectin to S. aureus cultures was studied. Cultures of S. aureus reacted with ¹²⁵I-labelled fibrinogen as well as fibronectin. The binding of labelled fibrinogen to S. aureus could be completely inhibited by unlabelled fibronectin, whereas the binding of labelled fibronectin was only partially inhibited by unlabelled fibrinogen. This suggested an interaction of fibronectin with clumping factor which is the binding protein for fibrinogen in staphylococci. The clumping factor was purified from S. aureus strain K 807 by affinity chromatography on fibrinogen-Sepharose followed by HPLC. The purified clumping factor inhibited the binding of fibrinogen and fibronectin to staphylococci. In western blots the purified clumping factor reacted with fibrinogen as well as fibronectin. Thus, the direct interaction of clumping factor with fibronectin might be responsible for the clumping of staphylococci in fibrinogen depleted plasma or serum.     

Synthesis and secretion of fibrinogen and albumin by isolated rat hepatocytes

In order to investigate the regulation of synthesis of some of the plasma proteins, especially fibrinogen, at the cellular level, we have chosen to work with suspensions of hepatocytes isolated by the perfusion of rat liver with crude bacterial collagenase. By adding soybean trypsin inhibitor to the collagenase and by avoiding mechanical damage, we have prepared cell suspensions that synthesize and secrete fibrinogen and albumin and that survive for longer than twenty hours. The fibrinogen secreted is clottable and shows the same pattern in acrylamide gel electrophoresis as fibrinogen purified from rat plasma. After a three hour lag, the rate of synthesis of fibrinogen, as measured by a solid-phase radioimmunoassay, continually accelerates, so that rates several fold greater than the $\text{in vivo}$ rate have been observed after twenty hours incubation. Cycloheximide (0.1 mM) completely abolished the appearance of fibrinogen in the medium; whereas colchicine (0.3 mM) reduced the rate by 85%. Insulin and cortisol succinate enhanced fibrinogen synthesis and secretion. The albumin secretion profile differs in several respects from that of fibrinogen, reflecting differences in intracellular pool levels and probably distinct regulatory mechanisms.     

Antigenicity of human fibrinogen and its derivatives

Serological characterization of fibrinogen and its derivatives was attempted by using antibody-coated latex agglutination and counterelectrophoresis. It was found that: 1) fibrinogen was not only capable of agglutinating uncoated latex particles but also fixed to the latex particles; 2) the sensitivity of some of the latex reagents used for the detection of fibrinogen may reflect the antibody activity to the derivatives with altered antigenicity in which FDP-E antigen is more readily available than in intact fibrinogen molecules, as the one contained in Liquid Human Plasma but absent in its batches prepared in the presence of plasmin inhibitors; 3) increase in FDP-E antigenicity was noticed in the course of fibrinogenolysis so its availability would indicate the occurrence and extent of proteolytic process on fibrinogen; and 4) FDP-E-antigen developed in the course of plasmin digestion may not be shared by intact fibrinogen since the former was reactive in counterelectrophoresis against anti-FDP-E serum even when the latter had been absorbed with fibrinogen.     

Affinity purification of fibrinogen using an Affimer column

BackgroundFibrinogen is an abundant plasma protein with an essential role in blood coagulation and haemostasis thus receiving significant research interest. However, protein purification is time consuming and commercial preparations often have protein contaminants. The aim of this study was to develop a new method to purify high quality and functional fibrinogen.MethodsFibrinogen-specific Affimer protein, isolated using phage display systems, was immobilised to SulfoLink resin column and employed for fibrinogen purification from plasma samples. Fibrinogen was eluted using a high pH solution. Commercial human fibrinogen was also further purified using the Affimer column. Fibrinogen purity was determined by SDS-PAGE and mass spectrometry, while functionality was assessed using turbidimetric analysis.ResultsAffimer-purified fibrinogen from human plasma showed purity at least comparable to commercially available preparations and was able to form physiological fibrin networks. Further purification of commercially available fibrinogen using the Affimercolumn eliminated multiple contaminant proteins, a significant number of which are key elements of the coagulation cascade, including plasminogen and factor XIII.ConclusionsThe Affimercolumn represents a proof of concept novel, rapid method for isolating functional fibrinogen from plasma and for further purification of commercially available fibrinogen preparations.General significanceOur methodology provides an efficient way of purifying functional fibrinogen with superior purity without the need of expensive pieces of equipment or the use of harsh conditions.     

Fibrinogen distribution on surfaces and in organelles of ADP stimulated human blood platelets

The fibrinogen distribution in platelet organelles after ADP-stimulation was investigated with anti-human fibrinogen using protein A-gold applied to serial sections. Fibrinogen was detected in the so-called alpha-granules of platelets and also in granule protrusions which were observed after ADP-stimulation. The ends of these protrusions were formed as coated membranes and the tips were often in apposition to the surface connected membranes or the plasmalemma. At such places fusion events and hence signs of an exocytosis could be demonstrated by means of cryofixation and cryosubstitution. Examination of serial sections revealed fibrinogen on all these granule profiles. Surface connected membranes, free surfaces and the characteristic structure of the contact zones of aggregated platelets were also labelled by gold particles but less than anticipated. On the platelet surfaces and surface connected membranes fibrinogen was rarely demonstrable with ferritin-labelled anti-human fibrinogen on washed or thrombin-stimulated, almost fibrinogen free platelets. After addition of human fibrinogen to the thrombin stimulated and disaggregated platelets a part of the platelets aggregated spontaneously and formed characteristic contact zones. Anti-human fibrinogen was observed on the free surfaces, in filamentous bridges between the contact spaces and in a tubular surface connected membrane system with involvement of coated membranes at the central ends of these structures. The results indicate the following: all alpha-granules contain fibrinogen; after ADP-stimulation secretion takes place with involvement of coated membranes; during aggregation fibrinogen binds to platelet surfaces and forms contact spaces; fibrinogen is taken up by the surface connected system with involvement of coated membranes.     

Mapping the Fibrinogen-Binding Domain of Serum Opacity Factor of Group A Streptococci

Serum opacity factor (SOF) is a large, extracellular, and cell-bound protein of group A streptococci that has two known functions, opacification of serum and binding of fibronectin. Herein, we describe a new function of SOF, the binding of fibrinogen. Utilizing purified, truncated recombinant SOF proteins, the fibrinogen-binding domain was localized to a region in the C-terminus of SOF encompassing amino acid residues 844–1047. Western-blot analysis revealed that SOF bound primarily to the β subunit of fibrinogen. A SOF-negative mutant bound 50% less fibrinogen than did its wild-type parent. Furthermore, fibrinogen blocked the binding of SOF to fibronectin. These data suggest that fibrinogen and fibronectin bind to the same domain within SOF. It remains to be determined whether the binding of fibrinogen to SOF contributes to the virulence of group A streptococci. Received: 13 June 2001 / Accepted: 20 July 2001     

Fibrinogen-binding and platelet-aggregation activities of a Lactobacillus salivarius septicaemia isolate are mediated by a novel fibrinogen-binding protein

The marketplace for probiotic foods is burgeoning, measured in billions of euro per annum. It is imperative, however, that all bacterial strains are fully assessed for human safety. The ability to bind fibrinogen is considered a potential pathogenicity trait that can lead to platelet aggregation, serious medical complications, and in some instances, death. Here we examined strains from species frequently used as probiotics for their ability to bind human fibrinogen. Only one strain (CCUG 47825), a Lactobacillus salivarius isolate from a case of septicaemia, was found to strongly adhere to fibrinogen. Furthermore, this strain was found to aggregate human platelets at a level comparable to the human pathogen Staphylococcus aureus. By sequencing the genome of CCUG 47825, we were able to identify candidate genes responsible for fibrinogen binding. Complementing the genetic analysis with traditional molecular microbiological techniques enabled the identification of the novel fibrinogen receptor, CCUG_2371. Although only strain CCUG 47825 bound fibrinogen under laboratory conditions, homologues of the novel fibrinogen binding gene CCUG_2371 are widespread among L. salivarius strains, maintaining their potential to bind fibrinogen if expressed. We highlight the fact that without a full genetic analysis of strains for human consumption, potential pathogenicity traits may go undetected.