Physiologically clotted fibrin--preparation and characterization for tissue engineering and drug delivery applications.

Fibrin used for biomedical applications is prepared by mixing concentrated solutions of fibrinogen and thrombin in presence of cross-linking agents such as Factor XIII or glutaraldehyde. The main drawbacks associated with this procedure include cost, complexity and time required for fibrin preparation. Hence, present study deals with the characterization of physiologically clotted fibrin (PF) for bone tissue engineering and drug delivery applications. For this the physico-chemical properties of PF were compared with those of the conventionally prepared fibrin (CF). Further MTT and haemolytic assays were performed for both PF and CF to compare their biocompatibility. The amount of alkaline phosphatase produced and calcium secreted by MG-63 cells in the presence of PF and CF were used to relate the osteogenic potency of PF with that of CF. Gallic acid, an anti-cancer drug was loaded within PF and CF and their role in drug delivery was compared.

Biological effects of static electric field: Plasma/serum proteome analysis of rats.

The external static electric field (SEF) of man-made origin brings to the substantially increased SEF background in a human environment the biological activity of which is a moot question. The paper reports on rats blood plasma/serum proteome modifications by means of 1D polyacrilamide gel electrophoresis and clotting process alterations after the short- and long-term SEF exposures of 200 kV/m. The results indicate decrease of fast α1 and α2 globular proteins in plasma coinciding with clotting acceleration after the short-term SEF, and attenuation of clotting-dependent proteome modifications reflected with incomplete coagulation after the long-term SEF exposure. Increased lysozyme activity in serum unlike plasma was observed after both SEF exposures. Applied model of the high-voltage SEF environment indicates dependence of biological systems functioning on the external SEF.

Rat hepatocytes in serum-free primary culture elaborate an extensive extracellular matrix containing fibrin and fibronectin.

Adult rat hepatocytes cultured on type IV collagen, fibronectin, or laminin and maintained in serum-free medium were examined by indirect immunofluorescence using polyclonal antibodies against extracellular matrix proteins. An extensive fibrillar matrix containing fibronectin and fibrin was detected in all hepatocyte cultures irrespective of the exogenous matrix substratum used to support cell adhesion. Fibrils radiated from the cell periphery and covered the entire culture substratum. In addition, thicker fibers or bundles of fibers were localized on top of hepatocytes. This matrix did not contain laminin or the major types of collagen found in the liver biomatrix (types I, III, and IV). Isolation of the fibrillar matrix and analysis on polyacrylamide gels under reducing conditions demonstrated a major 58-kD polypeptide, derived from beta-fibrinogen as indicated by immunoblotting and two-dimensional peptide mapping. Plasmin rapidly dissolved the matrix. Deposition of the fibrin matrix in hepatocyte cultures was arrested by hirudin, by specific heparin oligosaccharides that potentiate thrombin inhibition by antithrombin III, and by dermatan sulfate, an activator of heparin cofactor II-mediated inhibition of thrombin. The results indicate that hepatocytes in culture synthesize and activate coagulation zymogens. In the absence of inhibitory and fibrinolytic mechanisms, a fibrin clot is formed by the action of thrombin on fibrinogen. Fibronectin attaches to this fibrin clot but fails to elaborate a fibrillar matrix on its own in the presence of coagulation inhibitors.

Synthesis and Preclinical Evaluation of the Fibrin-Binding Cyclic Peptide 18F-iCREKA: Comparison with Its Contrasted Linear Peptide.

Purpose: Cys-Arg-Glu-Lys-Ala (CREKA) is a pentapeptide which can target fibrin-fibronectin complexes. Our previous study has built a probe called iCREKA which was based on CREKA and has proved the feasibility and specificity of iCREKA by the fluorescence experiment. The purpose of this study is to achieve the 18F-labeled iCREKA and make preclinical evaluation of the 18F-iCREKA with comparison of its contrasted linear peptide (LP). Methods: CREKA, LP, and iCREKA were labeled by the Al18F labeling method, respectively. These 18F-labeled peptides were evaluated by the radiochemistry, binding affinity, in vitro stability, in vivo stability, micro-PET imaging, and biodistribution tests. Results: 18F-NOTA-iCREKA was stable both in vitro and in vivo. However, 18F-NOTA-CREKA and 18F-NOTA-LP were both unstable. The FITC or 18F-labeled iCREKA could be abundantly discovered only in matrix metalloproteinases- (MMPs-) 2/9 highly expressed U87MG cells, while the FITC or 18F-labeled LP could also be abundantly discovered in MMP-2/9 lowly expressed Caov3 cells. Biodistribution and micropositron emission tomography (PET) imaging revealed that the U87MG xenografts showed a higher uptake of 18F-NOTA-iCREKA than 18F-NOTA-LP while the Caov3 xenografts showed very low uptake of both 18F-NOTA-iCREKA and 18F-NOTA-LP. The tumor-to-muscle (T/M) ratio of 18F-NOTA-iCREKA (9.93 ± 0.42) was obviously higher than 18F-NOTA-LP (2.69 ± 0.35) in U87MG xenografts. Conclusions: The novel CREKA-based probe 18F-NOTA-iCREKA could get a high uptake in U87MG cells and high T/M ratio in U87MG mice. It was more stable and specific than the 18F-NOTA-LP.

[Stored autologous blood from a pregnant woman showing a considerable amount of agglutinates].

A 30-year-old woman was admitted to our hospital to undergo simultaneous cesarean section and radical hysterectomy when the pregnancy became 30 weeks. An ultrasonic examination had found hypoechoic region at the cervix uteri. Because she wished autologous blood transfusion, 100 ml each of her own blood was obtained 3 times preoperatively. All the stored blood was returned to the patient through a filtering system (40 microm in the pore size) during surgery. However, we found paste-like agglutinates floating in the bags. We transfused the blood carefully while confirming that there were no agglutinates in the reservoir below the filter. The paste-like agglutinates were also found on the filter. By microscopic observation fibrin-like substances attached by blood cells were detected. When the autologous blood from pregnant women is returned, special care should be taken because the blood is likely to form agglutinates even though the blood test data are within normal ranges.

The economic impact of poor sample quality in clinical chemistry laboratories: results from a global survey.

Background Despite advances in clinical chemistry testing, poor blood sample quality continues to impact laboratory operations and the quality of results. While previous studies have identified the preanalytical causes of lower sample quality, few studies have examined the economic impact of poor sample quality on the laboratory. Specifically, the costs associated with workarounds related to fibrin and gel contaminants remain largely unexplored. Methods A quantitative survey of clinical chemistry laboratory stakeholders across 10 international regions, including countries in North America, Europe and Oceania, was conducted to examine current blood sample testing practices, sample quality issues and practices to remediate poor sample quality. Survey data were used to estimate costs incurred by laboratories to mitigate sample quality issues. Results Responses from 164 participants were included in the analysis, which was focused on three specific issues: fibrin strands, fibrin masses and gel globules. Fibrin strands were the most commonly reported issue, with an overall incidence rate of ∼3%. Further, 65% of respondents indicated that these issues contribute to analyzer probe clogging, and the majority of laboratories had visual inspection and manual remediation practices in place to address fibrin- and gel-related quality problems (55% and 70%, respectively). Probe maintenance/replacement, visual inspection and manual remediation were estimated to carry significant costs for the laboratories surveyed. Annual cost associated with lower sample quality and remediation related to fibrin and/or gel globules for an average US laboratory was estimated to be $100,247. Conclusions Measures to improve blood sample quality present an important step towards improved laboratory operations.

Fibrin-fibronectin compounds in human ovarian tumor ascites and their possible relation to the tumor stroma.

Covalently linked heterogeneous fibrin-fibronectin compounds were detected in ascitic fluid of 31 patients with advanced ovarian cystadenocarcinoma by means of enzyme-linked immunosorbent assay techniques, immunoaffinity chromatography, and Western blot analysis. Deposition of fibrin and fibronectin could also be demonstrated immunohistochemically in Carnoy-fixed tissue sections. Fibrin and fibronectin were found in the tumor stroma within tumor nests and more prominently in stroma surrounding the tumor nests. The association of fibrin and fibronectin was especially pronounced in the stroma surrounding the tumor islands. Fibronectin was also found to be associated with stroma cells. Areas within the tumor stroma showed superimposed staining for both fibrin and fibronectin supporting the assumption that the covalently linked fibrin-fibronectin conjugates found in ascitic fluid may stem from the provisional tumor stroma by proteolytic release.

Fibrin induction of tissue plasminogen activator expression in corneal endothelial cells in vitro.

PURPOSE: Fibrin is deposited in the anterior chamber of the eye in response to injury and can damage corneal endothelial cells (CEC). Fibrin degradation is plasmin dependent and is regulated by the balance between plasminogen activators (PA), tissue-PA (t-PA), urokinase-PA (u-PA), and their inhibitors (PAI). Although several factors can modulate PA/PAI expression in cells, the effect of fibrin is inconclusive. We hypothesized that fibrin can regulate fibrinolysis in the anterior segment by modulating PA/PAI expression in CEC. METHODS: Bovine CEC (BCEC) were treated for 3 to 72 hours with in situ polymerized fibrin (2 mg/ml) +/- 35S-methionine, cycloheximide, or actinomycin D. Polymerization was thrombin catalyzed, and control BCEC were incubated with or without thrombin or polymerization by-products. PA and PAI in conditioned medium, fibrin matrix, and cell fractions were analyzed by PA-specific zymographic and enzymatic assays. RESULTS: Fibrin treatment induced a dramatic (> 20-fold) accumulation of extracellular, fibrin-bound PA. This activity was identified as t-PA by its Mw (70 kD) affinity for fibrin and sensitivity to inhibition by Erythrina. Induction of t-PA was not observed in control BCEC under any condition. Fibrin induction of t-PA was selective because the levels of u-PA (45 kD), PAI-1 (50 kD), or protein synthesis in general were unaffected. Fibrin induction of t-PA was not accompanied by changes in cellular t-PA levels and was dependent on both RNA and protein synthesis. CONCLUSIONS: Fibrin selectively induces t-PA expression in CEC. Induced t-PA is released extracellularly and binds exclusively to the fibrin matrix. These findings suggest a role for fibrin and CEC in the regulation of fibrinolysis in the anterior segment of the eye.

Characterization of a monoclonal antibody specific to the amino terminus of the alpha-chain of human fibrin.

A peptide, Gly-Pro-Arg-Val-Val-Glu, corresponding to the first six residues of the amino terminus of the alpha-chain of human fibrin (desAA-fibrin) was prepared by solid-phase peptide synthesis. The peptide was covalently linked to keyhole-limpet hemocyanin (KLH) and used as an immunogen for preparing monoclonal antibodies. A monoclonal antibody specific to the hexapeptide, but not to KLH or fibrinogen, was produced. The antibody did not bind to thrombin-mediated clots prepared from either plasma or purified fibrinogen. However, immunoreactivity was detected when fibrin (prepared from fibrinogen) was solubilized with 8 M urea. In contrast, a monoclonal antibody specific to the amino terminus (Gly-His-Arg-Pro-Leu-Asp-Lys) of the beta-chain of fibrin recognized the epitope in clots. These results indicate that thrombin cleavage of fibrinogen produces a structural change in the amino terminal domain of the alpha-chain that makes it inaccessible to antibody interaction. In addition, our study suggests that the potential clinical application of monoclonal antibodies to localize fibrin-rich thrombi must take into account the final structure of clots.

Defective fibrin crosslinking in acute leukemia.

Fibrin crosslinking was assayed in 22 patients with acute leukemia showing secondary coagulation abnormalities of variable severity. In 9 patients fibrin crosslinking was found to be normal, whereas 10 patients presented impaired polymerization of alpha-chains and 3 of both alpha- and gamma-chains. Only a rough correlation was found between transamidating activity of factor XIII and the fibrin crosslinking pattern in these patients. Moreover, incomplete fibrin crosslinkage occurred at levels of factor XIII far in excess of that required for full polymerization of fibrin in "normal" plasma. This latter finding suggests that, in addition to factor XIII deficiency, other causes are responsible for the high rate of fibrin crosslinking impairment in acute leukemia.

A simple rapid method for isolating soluble fibrin complexes from fibrinogen by treatment with thrombin and t-AMCHA.

Using treatment with thrombin associated with trans-aminomethylcyclohexane carboxylic acid (t-AMCHA), a simple and rapid method for isolating soluble fibrin complexes (SFC) from fibrinogen in the plasma was developed. By this procedure, the recovery rates of SFC and early FDP (mainly X) increased according to the concentration of t-AMCHA, reaching a maximum at 286 mM t-AMCHA. On the other hand, the recovery rate of fibrinogen remained below 1.5% and that of late FDP was almost 100% at all concentrations of t-AMCHA. These results suggested that SFC and FDP could be isolated from fibrinogen by thrombin and t-AMCHA (286 MM) treatment. Moreover, it was possible to isolate SFC from FDP using gel filtration after treatment with thrombin and t-AMCHA. The SFC could be quantified by assay of the eluted fractions containing SFC by the staphylococcal clumping test.

Purification and characterization of the sand crab (Ovalipes bipustulatus) coagulogen (fibrinogen).

From the coagulocytes (amoebocytes) coagulogen (fibrinogen) was isolated, and purified on Sephacryl S-200. The cell homogenate contained one major protein species with a minimum molecular weight of 70,000. This protein clotted in the presence of human thrombin, human factor XIII and Ca++. The coagulogen contained no free thiol groups, however these were detectable in the reduced protein. Using the cyanoethylation procedure, it was estimated that one coagulogen molecule contained two lysine residues which participated in the cross-linking reaction. The total amino acid composition of the crab coagulogen and coagulin (fibrin) was estimated and compared with the amino acid composition of the Limulus polyphemus, lobster, (Panulirus interruptus) and porcine fibrinogen.

Detection of fibrin monomer. Comparison of the immune precipitate method with the serial-dilution protamine sulfate test and the ethanol gel test.

In a previous publication, a method for measuring fibrin monomer in plasma with the use of an immune precipitate of fibrinogen was described. The method was found to be more sensitive to unstabilized fibrin monomer than the serial-dilution protamine sulfate test, or the ethanol gel test, and detected fibrin in a mixture of the plasmin digestion products of fibrin. In the present study the sensitivity of the immune precipitate method for detecting specific plasmin digestion products of fibrin X, Y, D, and E, its sensitivity for stabilized fibrin monomer complexes, and its sensitivity for fibrin retaining B-peptide were measured and compared with the corresponding sensitivities of the serial-dilution protamine sulfate test and the ethanol gel test for detecting these same products. The immune precipitate method was found to be highly sensitive to stabilized fibrin monomer complexes and to fibrin retaining B-peptide; to be significantly less sensitive to the plasmin digestion fragments X, Y, and E; and to be insensitive to fragment D. The serial-dilution protamine sulfate test and the ethanol gel test were found to be insensitive to all of the plasmin digestion products of fibrin and less sensitive to the other fibrin complexes.

Flow and antibody binding properties of hydrated fibrins prepared from plasma, platelet rich plasma and whole blood.

Previous studies, using cross-linked fibrin prepared from purified fibrinogen, showed low binding of a fibrin-specific monoclonal antibody designated T2G1 (Procyk et al., Blood 77:1469-75, 1991). In this study we investigated the binding of T2G1 and one other antibody to clots prepared from platelet poor plasma (PPP), platelet rich plasma (PRP) and whole blood. In contrast to our previous study, we used unlabelled antibodies and quantitated the level bound by ELISA, measuring antibody concentration in the non-adsorbed fraction. Antibody T2G1 bound 1.35 +/- 0.10 pmol/pmol fibrin (n = 11) to whole blood columns, 1.64 +/- 0.18 (n = 10) to PRP columns and 1.58 +/- 0.13 (n = 8) to PPP columns. The binding of T2G1 to columns made from purified fibrinogen was 0.78 +/- 0.05 pmol/pmol fibrin (n = 15). An antibody to a conformation-dependent epitope on Fragment D (Fd4-7B3) bound in comparable amounts to the different fibrins. Flow data show that whole blood columns, and also, but to a lesser extent those made with plasma, had a higher flow rate, permeability and fiber mass-length ratio than columns prepared from fibrinogen indicating a more coarse fibrin network. These data show that the presence of other proteins and blood cells, similar to what might occur in vivo, not only lead to an increase in the permeability of gels but also allow for better exposure of some epitopes.

Contaminating fibrin in CPD-blood: solubility in plasma and distribution in blood components following separation.

In order to estimate the solubility of contaminating fibrin in CPD-blood, thrombin induced fibrin polymerzation in CPD-plasma was examined by light scattering and fibrinopeptide A (FPA) determinations. In addition, I125 fibrin monomer enriched CPD-blood was used to investigate fibrin monomer retention in blood bags and transfusion filters (170 microns) and fibrin distribution in blood components derived from CPD-blood. Initial fibrin polymerization in CPD-blood occurred after conversion of 15 per cent of the fibrinogen to fibrin, implying that substantial amounts of fibrin may be kept solubilized in CPD-blood bags. Only minor amounts of I125 fibrin monomers were retained in blood bags (2.4 per cent) and in transfusion filters (2.9 per cent) after sham transfusions. After separating I125-fibrin monomer enriched CPD-blood into its constituent components, the major part of fibrin (75.0 per cent) could be traced in the cryoprecipitate.

Normal and fibrinaemic patient plasma contain high-molecular weight crosslinked fibrin(ogen) derivatives with intact fibrinopeptide A.

Freshly drawn plasma samples from healthy subjects and from fibrinaemic patients were subjected to electrophoresis on SDS-agarose (unreduced material) or on SDS-PAG (1D and 2D, reduced material) and Westernblotted. The blots were immunovisualized using either polyclonal anti-fibrinogen or a monoclonal antibody (Y18) to fibrinopeptide A-containing molecules. The following results were obtained: 1. Normal plasma as well as plasma from patients with fibrinaemia contained FXIII-crosslinked HMW oligomers, stabilized through gamma gamma-dimerization as well as alpha-polymer formation and these oligomers contained molecules with intact A alpha-chain N-termini. 2. Cross-linked material amounted to less than 0.1% of the fibrinogen pool regardless of the sample studied, and apparently less in fibrinaemic patient plasma than in normal plasma. Thus, since the ratio of crosslinked fibrin(ogen) derivatives to that of fibrinogen was lower for fibrinaemic plasma than for normal plasma, it is suggested that the type of soluble fibrin which gives rise to a positive EGT in fibrinaemia patients is not crosslinked.

How high is the true fibrinogen content of fibrinogen standards?

A number of tests are available to measure plasma fibrinogen. Of these, the determination of the thrombin induced rate of plasma clotting is the most widely used in a clinical laboratory. Quantitative fibrinogen assays are calibrated with commercially available standards. There exists no internationally recognized standard against which the manufacturers could calibrate their fibrinogen preparations and lyophilized normal plasmas. In the present study, the amount of clottable material was determined in ten commercially available fibrinogen standards. Following clotting with thrombin, the fibrin clots were extensively washed with citrated saline and subjected to nitrogen analysis. The proportions of intact and partially degraded fibrinogens in each standard were determined by SDS-polyacrylamide gel electrophoresis of the washed clots. Knowing the amino acid composition and the relative proportions of these fractions, the nitrogen contents of the clots were converted to fibrinogen. More than 30% deviation from the declared values was observed in three standards, in one of them even 80%. Our results indicate that fibrinogen standards of markedly differing quality are commercially available and that an accurate, international standard for fibrinogen assay should be established.

Fibrin generation during production of freeze dried antihaemophilic cryoprecipitate.

Thrombin activity during separation and cryoprecipitation of CPD-blood was monitored by fibrinopeptide A (FPA) determinations. After pooling and lyophilization of cryoprecipitate, the total amount of contaminating fibrin was estimated by N-terminal amino acid analyses. In addition, retention of fibrin in standard transfusion filters (170 micron) was examined by gamma counting of 125I des-AA fibrin monomer enriched cryoprecipitate prior and subsequent to filtration. Prior to pooling of cryoprecipitate, thrombin activity, as estimated by FPA levels, was most pronounced during collection of blood from the blood donors and during cryoprecipitation and thawing of the plasma bags. Comparison of these FPA concentrations to the total amount of fibrin in pooled, freeze dried cryoprecipitate, as estimated by N-terminal analyses, revealed a considerable generation of fibrin during the process of lyophilization. In freeze dried cryoprecipitate, 5.3 per cent (range 3.0-7.5 per cent) of the fibrinogen had been converted to fibrin, implying a fibrin content of 20.3-60.3 mg per bottle of 500 U factor VIII. The amount of fibrin in two bottles of a commercially available factor VIII concentrate, also containing 500 U of factor VIII, was 14.1 and 19.8 mg, respectively. Sham transfusions of 125I des-AA fibrin monomer enriched cryoprecipitate revealed that only 1.0 per cent (range 0-2.5 per cent) of the fibrin was retained in the standard transfusion filters. Thus, substantial amounts of fibrin may be transfused to patients upon treatment with freeze dried cryoprecipitate.

Isolation and purification of fibrinogen/fibrin degradation products by chromatography on protamine-agarose.

Protamine-agarose chromatography is introduced as a rapid and efficient method for the isolation of fibrinogen/fibrin degradation products from biological fluids such as human plasma. All fibrinogen/fibrin fragments above a MW of 35000, including fragments D and E are quantitatively adsorbed to insolubilized protamine and can be eluted with a buffer containing 0.2 M sodium citrate/citric acid pH 5.3, following previous elution of non-fibrinogen proteins with a buffer containing 0.8 M NaCl at neutral pH. Fragments D and E are separated by stepwise elution. The efficiency of the method is - evaluated by applying it to plasma samples obtained from healthy donors and from patients with clinical and laboratory evidence of disseminated intravascular coagulation.

Appearance of platelet-clumping activity in heparinized plasma after acidification--it's mainly due to aggregation of fibrinogen.

Platelet-clumping activity was found after acidifying heparinized rabbit plasma to pH 5.5 in the presence of calcium ions. We tried to isolate the activity from plasma by gel filtration under low salt and high salt condition, and we found that the activity was related to the formation of plasma protein aggregates. Immunological examination and the results of fibrin monomer affinity chromatography indicated that fibrinogen was the main component of the plasma aggregates. This phenomenon was not related to the activation of blood coagulation. The binding of fibrinogen in the presence of heparin or calcium ions at low ionic strength may induce the appearance of platelet-clumping activity.