Self-assembly of fibrin monomers and fibrinogen aggregation during ozone oxidation

The mechanism of self-assembly of fibrin monomers and fibrinogen aggregation during ozone oxidation has been studied by the methods of elastic and dynamic light-scattering and viscosimetry. Fibrin obtained from oxidized fibrinogen exhibits higher average fiber mass/length ratio compared with native fibrin. Fibrinogen ozonation sharply reduced the latent period preceding aggregation of protein molecules; however, the mechanism of self-assembly of ozonated and non-ozonated fibrinogen cluster was identical. In both cases flexible polymers are formed and reaching a certain critical length they form densely packed structures and aggregate. Using infrared spectroscopy, it has been shown that free radical oxidation of amino acid residues of fibrinogen polypeptide chains catalyzed by ozone results in formation of carbonyl, hydroxyl, and ether groups. It is concluded that fibrinogen peripheral D-domains are the most sensitive to ozonation, which causes local conformational changes in them. On one hand, these changes inhibit the reaction of longitudinal polymerization of monomeric fibrin molecules; on the other hand, they expose reaction centers responsible for self-assembly of fibrinogen clusters.     

Molecular mechanisms of the polymerization of fibrin and the formation of its three-dimensional network

The results of biochemical, immunochemical, and X-ray studies of the structures of fibrinogen and fibrin molecules were analyzed. The mechanisms of the successive formation of the fibrin three-dimensional network were described: the polymerization of monomeric molecules with the formation of bifilar protofibrils, the lateral association of protofibrils, and the embranchment of the forming fibrils. Data on the electron and confocal microscopy of the polymeric fibrin were considered. The role of the known polymerization centers of fibrin which participated in the formation of protofibrils and their lateral association was discussed. Data on the existence of the previously unknown polymerization centers were given. In particular, the experimental results demonstrated that one of such centers which participated in the formation of protofibrils was located in the Bβ12–46 fragment, and did not require the cleavage of fibrinopeptide B for its functioning. The results of the computer modeling of the spatial structure of the fibrin(ogen) molecule and the intermolecular interactions in the course of the fibrin polymerization were presented. The location of the αC domains in the fibrin(ogen) molecule and their role in the polymerization process were discussed. Information on the structure of the calciumbinding sites of fibrin(ogen) and the functional role of Ca²⁺ in fibrin polymerization was published. The structure of factor XIII(a) and the mechanisms of fibrin stabilization by this factor were briefly described.     

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.     

Rheological characterization and dissolution kinetics of fibrin gels crosslinked by a microbial transglutaminase

Various fibrin gels were prepared with a microbial transglutaminase under miscellaneous conditions. The gels were characterized through their rheological properties. The influence of fibronectin addition and that of covalent bonding on the viscoelastic characteristics were evaluated. Gel elasticity is proportional to fibrinogen concentration but shows a nonlinear dependence on transglutaminase concentration. Additional crosslink of fibronectin in fibrin gels has no effect on the rheological character of the matrix. Dissolution kinetics in concentrated urea solutions evidences the role of covalent bonds on gel stability. The rheological properties and gel stability are discussed in relation with the enzyme-catalyzed covalent bonding. The microbial enzyme reactions are compared to those of FXIII and tissue transglutaminases.     

The effect of fibrin on endothelial cell migration in vitro

Endothelial cells are known to migrate and come into contact with fibrin during numerous physiological processes, such as in wound healing and in tumor growth. The present study was initiated to investigate the effect of fibrin on endothelial cell migration in vitro. Endothelial cell migration was assayed by wounding confluent monolayers of bovine aortic endothelial cells with a razor blade and counting the number of cells crossing the wound per unit time. Wound-induced proliferation of endothelial cells was inhibited by mitomycin C-treatment without affecting endothelial cell migration, indicating that in this assay migration could be measured independent of proliferation. Migration of endothelial cells in vitro was inhibited by fibrin in a concentration dependent manner. Endothelial cell migration under fibrin was further reduced by plasminogen depletion of the serum, and fibrin still inhibited the migration of mitomycin C-treated endothelial cells. Kadish et al. (Tissue and Cell, 11, 99, 1979) previously reported that fibrin did not affect EC migration in vitro. The inability to inhibit EC migration with fibrin appears to be due to their assay system which employed agarose, since pre-treating the wounded monolayer with agarose eliminated the inhibition of EC migration by fibrin. The present results indicate that EC migration in vitro can be used as a model system for studying the interaction of fibrin with EC.     

Influence of various structural domains of fibrinogen and fibrin on the potentiation of plasminogen activation by recombinant tissue plasminogen activator

Fibrinogen, fibrin, and related fragments have varying stimulatory effects on the initial rate of the activation of human plasminogen ([Glu¹]Pg) by recombinant tissue plasminogen activator (rt-PA). A detailed analysis of this enhancement was undertaken using various purified and complexed forms of the known domains of fibrin(ogen) with a view to gaining additional knowledge regarding the substructures of fibrinogen and fibrin that are important for their stimulatory capacities. Both arvin-mediated fibrin, as well as fibrinogen fragments generated as a result of its cleavage with CNBr, stimulate the activation in a biphasic manner, most likely as a result of changes in the promoter molecule accompanying the denaturation processes that are normally employed to either solubilize or generate these particular promoters. Using purified fibrinogen and fibrin fragments, it was found that fragment E, which binds to [Glu¹]Pg, does not enhance the activation reaction, while fragment D₁ has a potentiating effect. This suggests that the binding of [Glu¹]Pg to fibrin(ogen) alone is not, in itself, sufficient for stimulation of activation to occur, but that the rt-PA-fibrin(ogen) interaction is fundamental to this same process. All purified and mixtures of fragments containing the fragment D domain (e.g., D₂E, X-oligomer, fragment X) stimulate the reaction to a greater degree than fibrinogen and fragment D₁. It is concluded that the fibrinogen D domain is asine qua non for the enhancement reaction, while structures containing the E domain had a symbiotic effect on enhancement.On study leave from the National Institute for Biological Standards and Control, South Mimms, HERTS EN6 3QG, England.     

Force spectroscopy of the fibrin(ogen)-fibrinogen interaction

Fibrin aggregation is of vital importance in many physiological and pathological processes, such as blood coagulation, wound healing, and thrombosis. In the present study, we investigated the forces involved in the initial steps of the fibrinogen fibrin aggregation by force spectroscopy using the atomic force microscope. Our data confirm the existence of strong specific interactions between fibrin and fibrin(ogen), with unbinding forces ranging from 290 to 375 pN and a logarithmic dependence on the loading rate between 0.8 and 23 nN/s.     

Plasma circulating fibrinogen stability and moderate beer consumption

Moderate beer consumption (MBC) is cardioprotective: it positively influences plasma lipid levels and plasma antioxidant activity in beer-consuming individuals. The connection between MBC and blood coagulation is not clearly defined. Forty-two volunteers were equally divided into experimental (EG) and control (CG) groups following coronary bypass surgery. For 30 consecutive days, only patients of the EG consumed 330 mL of beer per day (about 20 g of alcohol). A comprehensive clinical investigation of 42 patients was done. Blood samples were collected before and after the investigation for a wide range of laboratory tests. The plasma fibrinogen was denatured with 8 M urea and intrinsic fluorescence (IF), hydrophobicity and differential scanning calorimetry (DSC) were used to reveal possible qualitative changes. After 30 days of moderate beer consumption, positive changes in the plasma lipid levels, plasma anticoagulant and plasma antioxidant activities were registered in patients of the EG group. In 17 out of 21 patients of the same group, differences in plasma circulating fibrinogen's (PCF), secondary and tertiary structures were found. The stability of fibrinogen, expressed in thermodynamic parameters, has shown that the loosening of the structure takes place under ethanol and urea denaturation. Also fluorescence stability of PCF was decreased. No changes in the lipid levels, anticoagulant and antioxidant activity or changes in PCF were detected in patients of CG. In conclusion, for the first time after a short term of moderate beer consumption some qualitative changes in the plasma circulating fibrinogen were detected: differences in the emission peak response, fluorescence intensity and all thermodynamic data. Together, with the decrease in the PCF concentration it may lead to an elevation of the blood anticoagulant activity.     

ATP-sensitive potassium channels are altered in ventricular myocytes from diabetic rats

The affinities of Factor XIII (FXIII), Factor XIIIa (FXIIla), and cellular transglutaminase (Tg) for fibrinogen (Fgn), fibrin (Fbn), and fibronectin (Fn) were compared using a solid-phase binding assay. Initial rates of binding were as follows: FXIII bound Fbn 3-fold more than Fgn. FXIII did not bind Fn till 20 min. Increasing the ligands concentrations and binding time, resulted in weak binding of FXIII to Fn. FXIIla bound Fbn 2-fold more than Fgn and 28-fold more than Fn. Tg bound Fn 130-fold more than either Fgn or Fbn. At equilibrium, the extent of binding was determined to be as follows: FXIII bound Fbn 3–15-fold more than Fgn and 8-fold more than Fn. FXIIIa bound Fgn and Fbn equally and 12–25-fold more than Fn. FXIIla bound Fgn or Fbn 2-fold and 25-fold greater than FXIII-Fbn and FXIII-Fgn interactions, respectively. Tg bound about equally to Fgn and Fbn and 10–20-fold less than Fn. The K _d s for FXIIla binding to Fn, Fgn, and Fbn were 100, 23, and 19 nM, respectively. The K _d for Tg binding to Fn was 6.5 nM. The binding hierarchies are: [Tg-Fn]>[FXIIIa-Fgn]=[FXIIIa-Fbn]>[FXIII-Fbn]>[FXIIIFgn]=[FXIIIa-Fn]>[Tg-Fbn]=[Tg-Fgn]>[FXIII-Fn]. Such hierarchies could regulate the cross-linkings by FXIIIa and Tg during hemostasis, wound healing, and cell adhesion.Abbreviations Tg cellular transglutaminase - FXIII coagulation factor XIII - FXIIla factor XIIIa (thrombin-activated FXIII) - Fgn human plasma fibrinogen - Fn human plasma fibronectin - Fbn human plasma fibrin (thrombin-cleaved fibrinogen) - ECM extracellular matrix     

Kinetics of the interaction of desAABB-fibrin monomer with immobilized fibrinogen

The soluble and stable fibrin monomer-fibrinogen complex (SF) is well known to be present in the circulating blood of healthy individuals and of patients with thrombotic diseases. However, its physiological role is not yet fully understood. To deepen our knowledge about this complex, a method for the quantitative analysis of interaction between soluble fibrin monomers and surface-immobilized fibrinogen has been established by means of resonant mirror (IAsys) and surface plasmon resonance (BIAcore) biosensors. The protocols have been optimized and validated by choosing appropriate immobilization procedures with regeneration steps and suitable fibrin concentrations. The highly specific binding of fibrin monomers to immobilized fibrin(ogen), or vice versa, was characterized by an affinity constant of approximately 10(-8)M, which accords better with the direct dissociation of fibrin triads (KD approximately 10(-8) -10(-9) M) (J. R. Shainoff and B. N. Dardik, Annals of the New York Academy of Science, 1983, Vol. 27, pp. 254-268) than with earlier estimations of the KD for the fibrin-fibrinogen complex (KD approximately 10(-6) M) (J. L. Usero, C. Izquierdo, F. J. Burguillo, M. G. Roig, A. del Arco, and M. A. Herraez, International Journal of Biochemistry, 1981, Vol. 13, pp. 1191-1196).     

Surface properties of Vibrio vulnificus

AIMS: Vibrio vulnificus adheres to a diverse range of surfaces, ranging from the chitinous exoskeleton of mollusks to human tissue. To determine whether environmental and human clinical isolates exhibit different adhesion traits, we studied the ability of 10 environmental isolates and 10 clinical isolates to adhere to human epithelial cells and hydrocarbons with log P values ranging from 3.1 to 8.2. METHODS AND RESULTS: All isolates adhered to varying levels to epithelial cells, and were inhibited to various extents from adherence by mannose and fructose. There was a lack of correlation between adherence to either hydrocarbons or cells and colony opacity. Adherence to hydrocarbons was optimal for solvents with a log P < 8.2. CONCLUSIONS: Vibrio vulnificus clinical and environmental isolates exhibit differential adherence to epithelial cells and hydrocarbons. SIGNIFICANCE AND IMPACT OF THE STUDY: The differential adherence of organisms to hydrocarbons based on log P may have utility in drug design and enhancement of food safety.     

Evidence for the Interaction between (−)-Epigallocatechin Gallate and Human Plasma Proteins Fibronectin,Fibrinogen, and Histidine-rich Glycoprotein

Human plasma proteins were subjected to affinity chromatography with (–)-epigallocatechin gallate (EGCg)-agarose, and the bound proteins were examined by sodium dodecylsulfate–polyacrylamide gel electrophoresis. A molecular weight evaluation of the protein bands suggested the presence of three proteins, fibronectin, fibrinogen, and a 75-kDa protein. When human serum was used, the 75-kDa protein dominated the bound fraction. The determination of the partial amino acid sequence of a peptide derived by endopeptidase digestion of this fraction suggested the 75-kDa protein to be histidine-rich glycoprotein (HRG). The presence of these proteins in the bound fraction was confirmed by the immunoblotting method. Affinity chromatography of the individual proteins indicated that fibrinogen and HRG had direct affinity for EGCg. Dot binding assays demonstrated the interaction of EGCg with these proteins. The method also showed that only gallate-containing catechins were bound by these proteins. These data suggest that when EGCg is absorbed in the body through the digestive system, it may interact with these proteins in blood plasma.     

Over-expression of Dyrk1A affects bleeding by modulating plasma fibronectin and fibrinogen level in mice

Down syndrome is the most common chromosomal abnormality in humans. Patients with Down syndrome have hematologic disorders, including mild to moderate thrombocytopenia. In case of Down syndrome, thrombocytopenia is not associated with bleeding, and it remains poorly characterized regarding molecular mechanisms. We investigated the effects of overexpression of Dyrk1A, an important factor contributing to some major Down syndrome phenotypes, on platelet number and bleeding in mice. Mice overexpressing Dyrk1A have a decrease in platelet number by 20%. However, bleeding time was found to be reduced by 50%. The thrombocytopenia and the decreased bleeding time observed were not associated to an abnormal platelet receptors expression, to a defect of platelet activation by ADP, thrombin or convulxin, to the presence of activated platelets in the circulation or to an abnormal half-life of the platelets. To propose molecular mechanisms explaining this discrepancy, we performed a network analysis of Dyrk1A interactome and demonstrated that Dyrk1A, fibronectin and fibrinogen interact indirectly through two distinct clusters of proteins. Moreover, in mice overexpressing Dyrk1A, increased plasma fibronectin and fibrinogen levels were found, linked to an increase of the hepatic fibrinogen production. Our results indicate that overexpression of Dyrk1A in mice induces decreased bleeding consistent with increased plasma fibronectin and fibrinogen levels, revealing a new role of Dyrk1A depending on its indirect interaction with these two proteins.     

A fibrin or collagen gel assay for tissue cell chemotaxis: assessment of fibroblast chemotaxis to GRGDSP

Fibroblast chemotaxis is implicated in many physiological processes, including wound healing and morphogenesis. We present a novel assay for chemotaxis of fibroblasts (and other slow-moving tissue cells) in a direct-viewing chamber containing a physiologically relevant three-dimensional fibrin or collagen gel in which long-lasting, spatially continuous gradients have been sustained for at least 24 h, long enough for significant fibroblast migration. This combination of features is not available in any alternative assay of comparable setup simplicity. Using a putative fibroblast chemotactic factor, the fibronectin peptide GRGDSP, we measured human foreskin fibroblast alignment in the direction along the gradient, which followed a biphasic dependence on GRGDSP concentration with an optimal concentration of about 10 nM. Time-lapse video microscopy revealed that cell migration was up the soluble GRGDSP gradient, confirming positive chemotaxis to GRGDSP and rejecting the possibility of dominant haptotaxis down the soluble GRGDSP gradient, that is, up a putative gradient of integrin-mediated adhesion induced by the soluble GRGDSP gradient.     

Hydrodynamic and mass spectrometry analysis of nearly-intact human fibrinogen, chicken fibrinogen, and of a substantially monodisperse human fibrinogen fragment X

The shape and solution properties of fibrinogen are affected by the location of the C-terminal portion of the Aα chains, which is presently still controversial. We have measured the hydrodynamic properties of a human fibrinogen fraction with these appendages mostly intact, of chicken fibrinogen, where they lack 11 characteristic 13-amino acids repeats, and of human fragment X, a plasmin early degradation product in which they have been removed. The human fibrinogen/fragment X samples were extensively characterized by SDS-PAGE/Western blotting and mass spectrometry, allowing their composition to be precisely determined. The solution properties of all samples were then investigated by analytical ultracentrifugation and size-exclusion HPLC coupled with multi-angle light scattering and differential pressure viscometry detectors. The measured parameters suggest that the extra repeats have little influence on the overall fibrinogen conformation, while a significant change is brought about by the removal of the C-terminal portion of the Aα chains beyond residue Aα200.     

Fibrinogen and fibrin in strong magnetic fields. Complementary results and discussion

When fibrin polymerizes in a strong magnetic field, it can be highly oriented. The structural diffraction study of the oriented polymer becomes thus possible. The magnetic birefringence can also be used to study the development of the polymer Fibrinogen in solution is weakly oriented in high magnetic fields. In this work we present complementary results and discussion. The validity of the comparison of the orientation parameters of fibrinogen and fibrin with those of other orientable known biological structures is discussed. The orientation of fibrin formed from fibrin monomer solution is compared to that of fibrin formed by the action of thrombin on fibrinogen. The conditions to obtain highly oriented fibrin gels suitable for three dimensional structure studies are also briefly discussed.