Fibrinogen Solution Research Articles

Effective Use of Fibrin Glue for Acute Aortic Dissection

The use of gelatin-resorcine-formalin (GRF) glue for reconstruction of the vascular wall in the context of acute aortic dissection has become more common. However, anecdotal evidence suggests that use of the GRF glue results in higher rates of postoperative redissection. We describe an alternative method of reinforcing the dissected aorta with fibrin glue that may avoid this complication. A fabric sheet is presoaked in fibrinogen solution and then placed within the false lumen. Thrombin solution is then applied to the fabric sheet. That results in obliteration of the false lumen and effective reinforcement of the dissected wall.

Fibrin Proliferation at Model Surfaces: Influence of Surface Properties

Fibrin proliferation from both human fibrinogen solutions and platelet-poor plasma was studied quantitatively as a function of substrate surface properties. A quartz crystal microbalance was used to monitor both protein adsorption and fibrin proliferation in real time at hydrophobic, hydrophilic, positively charged, and negatively charged surfaces. Scanning electron microscopy was used to characterize the morphology of the polymerized fibrin layers. The observed changes in mass indicate that fibrinogen adsorption occurs rapidly and mediates subsequent fibrin proliferation. Notably, substrate surface properties significantly affect the ability of adsorbed fibrinogen to promote fibrin proliferation.

Angiogenic activity of swine granulosa cells: effects of hypoxia and vascular endothelial growth factor Trap R1R2, a VEGF blocker

The possible role played by hypoxia and vascular endothelial growth factor (VEGF) in the regulation of follicular angiogenesis was studied in a three-dimensional fibrin gel model. Granulosa cells from follicles > 5 mm were subjected to normoxia (19% O 2), partial (5% O 2) or total (1% O 2) hypoxia and their culture media were collected and used to stimulate porcine Aortic Endothelial Cells (AOC) included in the fibrin matrix. A suspension of AOC on microcarrier beads was pipetted in a fibrinogen solution (1 mg/ml PBS) before the addition of 1250 IU thrombine (250 μl) to catalize the gel formation. Granulosa cell conditioned media were tested in the presence or absence of VEGF Trap R1R2 (150 ng/ml), a potent VEGF inhibitor, that had its efficacy tested by adding VEGF (100 ng/ml) to AOC culture. Endothelial cell proliferation was measured at 48, 96, 144, 192 h by means of Scion Image Beta. A significant ( p < 0.01) increase of AOC proliferation at each time of measurement was induced by culture media from granulosa cells subjected to partial (except at the end of the first 48 h) and total hypoxia compared to control and normoxia conditions, and by VEGF. VEGF Trap significantly ( p < 0.01) inhibited the stimulatory effect of media conditioned by granulosa cells cultured in hypoxic conditions. These data suggest that hypoxia stimulates angiogenic activity of granulosa cells possibly by means of VEGF which could represent the main effector in promoting endothelial cell proliferation.

Muscle Cells in Fibrin Matrix Actively Collapse the Network Leading to Rapid Allignment and Formation of Muscle Bundles.

The goal of in vitro organogenesis has been hampered by cell culture techniques that promote two dimensional cell proliferation. Since cell growth stops once confluence is achieved, three-dimensional organization does not occur. Possibly due to a lack of normal spatial orientation and cell-to-cell interactions, cells grown in such systems often assume morphologies significantly different from those assumed in normal biological tissues. We have recently developed a simple technique for growing cells in fibrin gels. This is accomplished by suspending cells in fibrinogen solutions and subsequently mixing the fibrinogen-cell suspension with buffered thrombin. The fibrinogen solution can be prepared from purified protein or from plasma diluted with growth media. The mixture rapidly forms a fibrin gel to which the cells are attached. When muscle cells are cultured in fibrin matrices, they initially form a latticework of proliferating cells. Over the next forty-eight hours, the cells begin to collapse the edge of the structure in a process similar to platelet induced clot retraction. As the fibrin network collapses, muscle cells are brought into linear alignment to form structures closely resembling muscle bundles. This process is relatively rapid. Within forty-eight to seventy-two hours, three-dimensional structures morphologically similar to muscle tissue are formed. By manipulating the gel structure through the imposition of outside forces, the alignment of muscle cells can be caused to occur even more rapidly. This system is simple to use and rapidly results in a muscle culture structure that resembles muscle tissue. This technique holds promise to significantly advance the goal of true in vitro “tissue” formation.

Characterization of the fibrinogen-binding surface protein Fbl of Staphylococcus lugdunensis

The fbl gene of Staphylococcus lugdunensis encodes a protein Fbl that is 58 % identical to the clumping factor A (ClfA) of Staphylococcus aureus. The fbl gene was present in eight clinical isolates of S. lugdunensis. When Fbl was expressed on the surface of Lactococcus lactis it promoted adherence to immobilized fibrinogen and cell clumping in a fibrinogen solution. Purified recombinant Fbl region A bound to immobilized fibrinogen in a dose-dependent manner and inhibited the adherence of both Fbl-expressing and ClfA-expressing strains of L. lactis to fibrinogen. Adherence of S. lugdunensis and L. lactis Fbl(+) to immobilized fibrinogen was also inhibited by rabbit anti-Fbl region A antibodies and rabbit anti-ClfA region A antibodies, as well as by human immunoglobulin with a high level of anti-ClfA antibodies. Alignment of the A domains of CflA and Fbl revealed that all of the ClfA residues implicated in binding to the gamma-chain of fibrinogen are conserved in Fbl. Nevertheless Fbl had a tenfold lower affinity for fibrinogen, suggesting that sequence differences that occur elsewhere in the protein, possibly in beta-strand E of domain N2, affect ligand binding.

Quantitative analysis of aggregation in dilute solutions of effectively rigid biomacromolecules via the combination of oscillatory flow birefringence and viscoelasticity measurements: example study of aggregation of bovine fibrinogen in aqueous glycerol, and detection of a large aggregate formed on addition of guanidine hydrochloride

Oscillatory flow birefringence (OFB) properties have been measured for dilute solutions of bovine fibrinogen in 65–68% aqueous glycerol with the Miller–Schrag Thin Fluid Layer (TFL) apparatus employing either titanium or stainless steel surfaces in contact with the solutions. The shearing frequency range was 1 to 2500 Hz, the concentrations ranged from 4 to 8 mg/ml, and measurement temperatures were 9.9, 10.0, and 15.8 °C. The data showed evidence of significant amounts of aggregation that apparently is caused by the presence of glycerol; contributions from the various aggregates were readily detected since the staggered half-overlap aggregation in this system results in substantial differences in the rotational relaxation times of the various effectively rigid aggregates. The combination of oscillatory flow birefringence and viscoelasticity (VE) data provided sensitive and precise characterization of aggregation in these example systems; all aggregates exhibited the expected positive optical anisotropy. The length of unaggregated fibrinogen in solution was found to be that obtained via electron microscopy. Addition of guanidine hydrochloride to hopefully reduce aggregation did so but also resulted in formation of a very large (2800 to 3500 Å), apparently nearly monodisperse, negatively birefringent aggregate, suggesting that this new species might be formed by lateral aggregation.

Modulation of proteins adsorption onto the surface of chitosan complexed with anionic copolymers. Real time analysis by surface plasmon resonance.

The interpolyelectrolyte complex formation between chitosan and anionic polyacrylic derivatives, bearing sulfonic moieties, as well as the protein adsorption onto the chitosan/polyacrylic complexes were studied by surface plasmon resonance (SPR) optical biosensor. This unique technique allows a real time monitoring of different surface molecular interactions with very high sensitivity. The acrylic macromolecules are two families of copolymers of 2-acrylamido-2-methylpropane sulfonic acid (AMPS) and, respectively, 2-hydroxyethylmethacrylate (HEMA) and N,N'-dimethylacrylamide (DMAA). The complexation process was evaluated through the SPR measurements resulting from the flowing of polyacrylic aqueous solution over the sensor previously coated with chitosan. The SPR was able to differentiate strong ionic bonds from other weak and reversible interactions. By means of the coated sensors (uncomplexed and the whole series of complexed chitosan), SPR cold be used for a simple "in vitro" protein adsorption analysis, by flowing aqueous solutions of albumin and fibrinogen. While both proteins were adsorbed on the uncomplexed chitosan, the complexed coatings exhibited different and very promising behaviors. In particular, they showed no adsorption or only selective adsorption of albumin.

Cover Picture: Self‐Assembled Monolayers of Dendritic Polyglycerol Derivatives on Gold That Resist the Adsorption of Proteins (Chem. Eur. J. 11/2004)

The cover picture shows the formation of a self‐assembled monolayer of dendritic polyglycerol on a semitransparent gold‐film and its high protein resistant properties. The polyglycerol derivative is readily obtained by ester coupling with thioctic acid, which provides a stable covalent linker to the gold surface. Several dendritic polyglycerol derivatives as well as the monomer equivalent (thioglycerol) were screened for their protein resistant properties with a fibrinogen solution by surface plasmon resonance spectroscopy (SPR). The excellent protein resistant properties and the high thermal stability of this new material, which classifies for the coating of biomedical devices, is reported in detail by R. Haag et al. on p. 2831 ff.

磁場配向したフィブリン線維を取り巻く水分子のＴ２緩和時間

We investigated the effect of structural differences in fibrin fibers on the T2 relaxation time. Fibrin fibers have the unique characteristic of orienting themselves parallel to high magnetic fields during polymerization. Two fibrin gels were polymerized from a fibrinogen solution with and without a 7.05-T magnetic field. Water molecules in the fibrin gel that was polymerized in the high magnetic field exhibited only one relaxation time, T2 = 0.35 s, whereas water molecules in the fibrin gel that was not exposed to a magnetic field during polymerization had at least two exponential components in the T2 relaxation. The long component, T2 = 0.35 s, was the same order as the T2 of the fibrinogen solution (= 0.41 s) and the fibrin gel polymerized in the high magnetic field. The short component was T2 = 0.07 s. This difference is attributed to a change in the magnetic dipole-dipole interactions between water molecules and fibrin fibers.

The viscosity of fibrinogen subfractions and of EDTA denatured fibrinogen do not differ from that of native fibrinogen

Introduction: Fibrinogen is a major determinant of plasma viscosity. The increased risk of atherothrombotic disease associated with a high fibrinogen concentration may partly be attributed to its effect on viscosity. Since the ratio between the three main fibrinogen subfractions high molecular weight (HMW)-, low molecular weight (LMW)-, and very low molecular weight (LMW′)-fibrinogen is altered during acute phase conditions, and an increased HMW/LMW-fibrinogen ratio is associated with increased thromboembolic risk, we have examined how these subfractions affect viscosity. The viscosity of plasma is usually determined in ethylenediaminetetra-acetic acid (EDTA) plasma at 37 °C. Under such conditions the clotting properties of fibrinogen is affected due to denaturation. Denaturation of plasma proteins may affect their viscosity. Therefore, we have also investigated the effects of EDTA on the viscosity of fibrinogen. Materials and methods: Purified fibrinogen was obtained by beta-alanine precipitation of plasma from healthy donors. Separation of the fibrinogen fractions was performed by gradual precipitation of purified fibrinogen by ammonium sulphate. The viscosity was determined using a Haake Microvisco 2 viscometer. Results: There was no statistically significant difference between the viscosity of native fibrinogen and the three fibrinogen subfractions. A substantial prolongation of the thrombin clotting time was observed in the fibrinogen solution containing EDTA at 37 °C compared to 20 °C. However, the viscosity of EDTA anticoagulated purified fibrinogen and plasma samples did not differ from that of heparin anticoagulated samples. Conclusion: The viscosity of the main fibrinogen subfractions HMW-, LMW- and LMW-fibrinogen did not differ from that of native fibrinogen, and the use of EDTA as anticoagulant did not significantly affect the viscosity of fibrinogen at 37 °C.

Serum protein adsorption and platelet adhesion on aspartic-acid-immobilized polysulfone membranes

Polysulfone (PSf) membranes that covalently conjugated with aspartic acid (ASP-PSf) were prepared and analyzed for hemocompatability. Compared to PSf or other types of surface-modified PSf membranes, the ASP-PSf membranes had a reduced ability to adsorb protein from either a plasma solution or a mixed solution of albumin, globulin and fibrinogen. This appears to be due to the creation of a hydrophilic surface by the aspartic acid zwitterion immobilized on the ASP-PSf membranes. Furthermore, the analyses of membrane protein adsorption showed that a mixed protein solution recapitulates the cooperative adsorption of proteins that occurs in plasma. We also found that the number of adhering platelets was the lowest on the ASP-PSf membranes and, in general, that platelet adhesion decreased in parallel with fibrinogen adsorption. In summary, aspartic acid immobilized on the ASP-PSf membranes, which have zwitterions with a net zero charge, effectively contributes to the hydrophilic and hemocompatible sites on the surface of the hydrophobic PSf membranes.

Synthesis and characterization of poly(ethylene glycol) methyl ether endcapped poly(ethylene terephthalate)

AbstractLinear and branched poly(ethylene terephthalate) (PET) copolymers with polyethylene glycol) (PEG) methyl ether (700 or 2000 g/mol) end groups were synthesized using conventional melt polymerization. DSC analysis demonstrated that low levels of PEG end groups accelerated PET crystallization. The incorporated PEG end groups also decreased the crystallization temperature of PET dramatically, and copolymers with a high content of PEG (&gt;17.6 wt%) were able to crystallize at room temperature. Rheological analysis demonstrated that the presence of PEG end groups effectively decreased the melt viscosities and facilitated melt processing. XPS and ATR‐FTIR revealed that the PEG end groups tended to aggregate on the surface, and the surface of compression molded films containing 34.0 wt% PEG were PEG rich (85 wt% PEG). PEG end‐capped PET (34.0 wt% PEG) and PET films were immersed into a fibrinogen solution (0.7 mg/mL BSA) for 72 h to investigate the propensity for protein adhesion. XPS demonstrated that the concentration of nitrogen (1.05%) on the surface of PEG endcapped PET film was statistically lower than PET (7.67%). SEM analysis was consistent with XPS results, and revealed the presence of adsorbed protein on the surface of PET films.

Multicomponent proton spin–spin relaxation of fibrin gels with magnetically oriented and randomly oriented fibrin fiber structures

We investigated the effect of structural differences in fibrin fibers on the T2 relaxation time. Fibrin fibers have the characteristic of orienting parallel to high magnetic fields during polymerization. Two fibrin gels were polymerized from a fibrinogen solution with and without a 7.05 T magnetic field. Water molecules in the fibrin gel that were polymerized in the high magnetic field exhibited only one relaxation time T2=0.35 s, whereas, water molecules in the fibrin gel that were not exposed to a magnetic field during polymerization had at least two exponential components in the T2 relaxation. The long component, T2=0.35 s, was the same order as the T2 of the fibrinogen solution (=0.41 s) and the fibrin gel polymerized in the high magnetic field. The short component was T2=0.07 s. This difference is attributed to a change in the magnetic dipole–dipole interactions between water molecules and fibrin fibers.

Albumin and fibrinogen adsorption onto phosphatidylcholine monolayers investigated by Fourier transform infrared spectroscopy

Dipalmitoylphosphatidylcholine (DPPC) monolayers were deposited onto a germanium attenuated total reflectance (ATR) crystal using the Langmuir–Blodgett technique. The DPPC-coated crystal was then exposed to human serum albumin or human fibrinogen solutions while measuring the protein adsorption by recording FTIR spectra. The effect of the zwitterionic nature of the DPPC polar headgroup towards protein adsorption has been ascertained by exposing either the phospholipid headgroup or the acyl chains to the protein solution; this was possible by the use of a silanized or a bare germanium crystal. Calibration curves have been made to measure the protein surface concentrations. After 3 h, the albumin surface concentration on DPPC monolayers was about three times higher when the proteins were exposed to the lipid acyl chains instead of the polar headgroups (e.g. 3 vs. 1 μg cm −2). As for fibrinogen (FGN) adsorption, when the lipid polar headgroups were exposed to the protein solution, the FGN adsorption was low reaching a maximum value of 0.5 μg cm −2. When interacting with the lipid acyl chains, the FGN adsorption reached a plateau at a value of 2.1 μg cm −2 after 3 h. Clearly, both albumin and FGN showed a low tendency to adsorb on surfaces where the lipid polar headgroups are exposed toward the protein solution.

A New Method of Purifying Fibrinogen with Both Biological and Immunological Activity from Human Plasma

Using a combination of Cohn ethanol fractionation, virus inactivation, glycine and sodium chloride precipitation, and lysine-Sepharose affinity chromatography, a unique and rapid simplified method was developed to obtain highly purified fibrinogen for diagnostic use with both biological (Clauss method) and immunological (Jacobsson method) activity. Yield was 0.66 g of fibrinogen per liter of starting pooled plasma, and the purified product showed good agreement in activity with the starting material. The purified fibrinogen solution contained over 95% clottable protein and had a clear appearance. No degradation was observed after urokinase treatment and the preparation provided good precision in fibrinogen measurement compared to pooled plasma. The simplified method was, thus, shown to result in a high-purity fibrinogen preparation, suitable for in vitro diagnostic use, as well as for use to prepare a fibrinogen reference material and to perform fibrinogen quality control using an automated coagulation analyzer.

Protofibrils within fibrin fibres are packed together in a regular array

The inner structure of fibrin fibres grown from fibrinogen solution activated by human alpha-thrombin was investigated by means of an Energy Dispersive X-ray Diffraction technique. The experiments show evidence for the well-characterized 22.5 nm repeat distance, which indicates the high order of protofibril arrangement in the longitudinal direction of fibres. The diffraction pattern also manifested a further pronounced peak at 18.1 nm (and its second order reflection at 18.1/ radical 2) demonstrating the existence in fibrin of a high degree of lateral order. The reported results directly confirm, on unperturbed wet samples, that protofibrils closely associate giving rise to a crystalline axial and equatorial packing according to the conclusions of the multibundle model.

Blood response to plasticized poly(vinyl chloride): dependence of fibrinogen adsorption on plasticizer selection and surface plasticizer level.

The high level of plasticizer in plasticized poly(vinyl chloride) (PVC) ensures that plasticizer selection has an important influence on the suitability of PVC to function in blood-contacting applications. In this study, three types of plasticized PVC in sheet form, with di-(2-ethylhexyl)phthalate (DEHP), tri-(2-ethylhexyl)trimellitate (TEHTM) and n-butyryltri-n-hexyl citrate (BTHC) as plasticizer, were selected for assessment and single solute fibrinogen adsorption was utilized as an initial index of interactions with blood components. Fibrinogen adsorption behavior shows a strong dependence on the plasticizer selection, plasticizer level at the surface and the adsorption conditions, such as adsorption time and fibrinogen solution concentration. Results indicate that BTHC plasticized PVC possesses the lowest adsorption capacity in the three types of plasticized PVC, while TEHTM plasticized PVC seems to have the strongest reactivity in certain fibrinogen solution concentrations. The alteration of surface plasticizer level was achieved by a methanol-cleaning treatment with a variety of cleaning times and the fibrinogen adsorption on plasticized PVC decreases with the reduction of surface plasticizer level. The migration behavior of two phthalate esters (DEHP and TEHTM) was evaluated using UV-Spectrophotometer to determine the plasticizer level at the surfaces. In addition, the fibrinogen adsorption mechanism was examined with Freundlich adsorption modeling.

Allosteric Effects Potentiating the Release of the Second Fibrinopeptide A from Fibrinogen by Thrombin

Fibrin formation depends on the release of the two N-terminal fibrinopeptides A (FPA) from fibrinogen, and its formation is accompanied by an intermediate, alpha-profibrin, which lacks only one of the FPA. In this study, we confirm that the maximal levels of alpha-profibrin found over the course of thrombin reactions with human fibrinogen are only half of what would be expected if the first and second FPA were being released independently with equal rate constants. The rapidity of release of the fibrinopeptides by thrombin had been shown to depend on an allosteric transformation that is induced when Na(+) binds to a site defined by the 215-227 residues of thrombin, a transformation that results in the exposure of its fibrinogen-binding exosites transforming the thrombin from a slow to a fast acting form toward fibrinogen. When choline was substituted for sodium to transform thrombin to its slow form, the maximal levels of alpha-profibrin rose to those expected for independent release of the two FPA. Thus, it is only the fast thrombin that releases the second FPA fast, and that fast release only occurs when both FPA are present because of a partial coupling of its release with that of the first FPA. The release of the FPA from purified alpha-profibrin with the first FPA already missing is no faster than the release of any FPA. Surprisingly, we also found that slow thrombin became increasingly transformed to a fast form in the absence of sodium when the fibrinogen was elevated to high concentrations. This potentiation by concentrated fibrinogen also occurs with the recombinant mutant thrombin (Y225P), which is otherwise slow in both the presence and absence of Na(+). The potentiation of thrombin by fibrinogen must be short-lived so that the thrombin reverts to its slow acting form in the interim among encounters with other fibrinogen molecules in dilute fibrinogen solutions lacking Na(+), whereas at high fibrinogen concentrations the thrombin encounters other molecules before it reverts back to the slow form.

Domain-Driven Binding of Fibrin(Ogen) onto Silk Fibroin Biomaterials

Studies have demonstrated that serum protein adsorption onto silk fibroin-based biomaterials dramatically changes when the conformation of this natural polymer is rearranged by engineering procedures. In the present study, attention was paid to the binding of fibrin(ogen) to fibroin fibers and regenerated films. The fibroin specimens were incubated either in human plasma or in a fibrinogen solution to which thrombinwas added to activate the polymerization of the precursor into the final product, fibrin. The experiments were carried out in the presence and absence of calcium to investigate the role of calcium-dependent enzymes in the binding process. The two types of samples were analyzed by SEM, the micrographs showed completely different interactions with fibrinogen. Films did not show any visible fibrin polymerization, whereas the fibers were bound to the fibrin bundles by calcium-independent mechanisms.

The impact of rheology of human fibronectin–fibrinogen solutions on fibre extrusion for tissue engineering

Fibronectin–fibrinogen composite materials form a basis for natural constructs for applications in soft tissue engineering including skin repair, blood vessel replacement and nerve regeneration. Scaleable methods for the preparation of such scaffolds are a prerequisite for their widespread use. Here, we report data on the extrusion of fibronectin-based constructs in the form of fibres. The results suggested that events occurring in the extrusion head and coagulation bath were critical in determining the ultimate mechanical strength of the extruded fibres. These events were controlled by interaction between the rheology of the dope, the geometry of the extrusion device and the operating parameters of the extrusion process. The rheology of the dope was successfully controlled by incorporation of sodium alginate, and urea was added to the formulation to produce oriented fibres. We previously reported that oriented fibronectin fibres have the capacity to induce desirable cell responses. Measurements indicated that for a given formulation, the mechanical properties and morphological features depended on the diameter of the extruded fibre, the rate of shear in the extrusion pipe and the time of exposure to it.