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PubMed1984

The activated partial thromboplastin time of diluted plasma: variability due to the method of fibrin detection.

The purpose of this study was to determine the effects of the dilution of plasma (1/3 in saline) on the kinetics of fibrin generation in the activated partial thromboplastin time (APTT) assay. The diluted APTT is considered to increase the sensitivity of the APTT test however, studies in our laboratory using an electro-optical fibrin detection system failed to show significant differences in APTT values obtained with diluted and undiluted canine plasma. Seventeen plasmas, including plasmas moderately and markedly deficient in intrinsic factor activity were assayed in the undiluted and diluted APTT assay using two methods for fibrin endpoint detection; a visual "tilt-tube" technique and an electro-optical detection system. In the former technique the endpoint was the formation of a visible fibrin web or clot; in the latter procedure the end point was the first detection of a change in optical density of the plasma. Optical density changes during fibrin formation were also recorded ( thrombokinetograms ). The results indicated that the electro-optical fibrin detection system failed to identify a prolongation of the APTT as a result of 1/3 plasma dilution; a prolongation that was consistently observed with the visual fibrin detection technique. Plasma dilution however, did significantly reduce the rate of fibrin production as indicated by the thrombokinetogram profile. It was concluded that the dilution of plasma with saline, as has been used to increase the sensitivity of the APTT assay procedure, has little effect on the time of onset of fibrin formation in a given plasma. The major effect appears to be on the way in which fibrin forms in that the polymerization/crosslinkage events associated with macroscopic fibrin production are delayed.

Animals↗
PubMed1983

Studies on the mechanism of thrombin. Interaction with fibrin.

Fibrin monomer Sepharose was used to investigate the interactions of thrombin with fibrin. Thrombin binding was found to be reversible and saturable and to depend on the thrombin: fibrin ratio. Scatchard analysis indicated a single class of binding sites with K alpha = 4.9 X 10(5) M-1. Ca2+ ions caused rapid desorption and elution of thrombin from fibrin monomer, and the Ca2+ concentration needed for maximal desorption depended on the fibrin:thrombin ratio. Mg2+, Mn2+, and Sr2+ also released thrombin from fibrin monomer but not as efficiently as Ca2+. These results indicate that divalent metal ions induce a physical change in fibrin monomer which results in desorption of thrombin. Thrombin binding to fibrin in a gel was compared to binding to fibrin monomer. These studies showed that as fibrin monomers polymerize to form the gel network, thrombin is released. Under static conditions the released thrombin remains associated with the gel because diffusion is limited by the gel. However, the thrombin can be readily removed when buffer is allowed to flow through the gel. These results lead to the possibility that thrombin binding to fibrin monomer and its subsequent release, either by Ca2+ or by polymerization, may have important consequences for regulating the effective thrombin concentration in vivo.

Calcium↗
PubMed1993

Regulation by alpha 2-antiplasmin and fibrin of the activation of plasminogen with recombinant staphylokinase in plasma.

The effects of alpha 2-antiplasmin and fibrin on the activation of plasminogen by recombinant staphylokinase (STAR) were studied in an effort to elucidate further the molecular basis of the fibrin-specificity of this fibrinolytic agent. In purified systems consisting of 1.5 mumol/L intact or low-M(r) plasminogen and 3 mumol/L alpha 2-antiplasmin, at 37 degrees C and in the absence of fibrin, STAR did not induce plasminogen activation and plasmin-alpha 2-antiplasmin complex (PAP) formation. Addition of a purified fibrin clot (30% vol at a concentration of 3 mg/mL) to mixtures containing intact plasminogen caused approximately 40% plasminogen activation within 2 hours, whereas in mixtures containing low-M(r) plasminogen, no activation was observed. In contrast, 10 nmol/L streptokinase (SK) induced 74% to 100% plasminogen activation within 2 hours in mixtures containing either intact or low-M(r) plasminogen, in both the absence and the presence of fibrin. In citrated human plasma in the absence of fibrin, 30 nmol/L STAR did not induce measurable plasminogen activation and PAP formation (< 1.5% within 2 hours), whereas addition of a plasma clot (12% vol) resulted in complete clot lysis and conversion of 19% +/- 8% of the plasminogen to PAP within 2 hours. Addition of a second plasma clot produced 23% +/- 2% additional plasminogen activation. Equipotent concentrations for plasma clot lysis of SK (100 nmol/L) induced 54% +/- 11% plasminogen activation in the absence and 49% +/- 16% in the presence of fibrin. Addition of 50 mmol/L 6-aminohexanoic acid (6-AHA) abolished the effect of fibrin on plasminogen activation with STAR, but not on activation with SK. In alpha 2-antiplasmin-depleted human plasma in the absence of fibrin, 30 nmol/L STAR did not induce fibrinogen breakdown (> 90% residual fibrinogen after 6 hours), whereas 30 nmol/L preformed plasmin-STAR complex induced extensive fibrinogen degradation (70% within 20 minutes). Thus, in the absence of fibrin, alpha 2-antiplasmin inhibits the activation of plasminogen by STAR, by preventing generation of active plasmin-STAR complex. Fibrin stimulates plasminogen activation by STAR via mechanisms involving the lysine-binding sites of plasminogen, probably by facilitating the generation of plasmin-STAR complex and by delaying its inhibition at the clot surface.

Antifibrinolytic Agents↗
PubMed1994

Fibrin induction of interleukin-8 expression in corneal endothelial cells in vitro.

PURPOSE: Classically, acute and chronic inflammations are characterized by fibrin deposition and a dynamic influx of leukocytes. This leukocyte recruitment, and associated activation, is thought to be dependent on the generation of leukocyte chemotactic factors (LCF). Although the functional existence of LCF in ocular tissue has been demonstrated, the identity, source(s), and mechanisms of induction of these LCF are unclear. The authors investigate the hypothesis that in vitro corneal endothelial cells produce LCF in response to fibrin-induced activation. They further hypothesize that in vivo this fibrin-induced expression of LCF contributes to the leukocyte accumulation associated with ocular injury. METHODS: Bovine corneal endothelial cells (BCEC) were co-cultured for 3 to 72 hours with physiologic concentrations of highly purified fibrin (0.125 to 2.0 mg/ml) polymerized in situ. At harvest, the conditioned medium was separated from the fibrin matrix by centrifugation and characterized for the presence and nature of polymorphonuclear leukocyte chemotactic activity. This fibrin-induced LCF was compared to known LCF, such as interleukin-8 (IL-8), using standard physical, chemical, and immunologic parameters. RESULTS: Conditioned medium from fibrin-treated BCEC exhibited a dose- and time-dependent induction of LCF activity, as verified by checkerboard analysis. This LCF activity was not immunoprecipitated by a polyclonal antibody to bovine fibrinogen, and it was heat stable (60 degrees C, 90 minutes) and protease labile. Isoelectric focusing and gel filtration analysis revealed a major peak of chemotactic activity at pH 8.5 to 9.0 and a molecular weight of 10 kd, respectively. Radioimmunoassay of conditioned medium from fibrin-treated BCEC for IL-8 demonstrated an 11-fold increase in IL-8 antigen for fibrin-treated BCEC compared to control BCEC. CONCLUSION: In vitro, fibrin induces BCEC expression of LCF activity that includes IL-8. In vivo, this fibrin induction of LCF from corneal endothelial cells probably serves to control both leukocyte recruitment and activation within the anterior chamber in general and to corneal endothelium in particular. These studies provide a foundation for understanding the nature, sources, and mechanisms of the LCF generation that contributes to endocular inflammation.

Animals↗
PubMed1993

Endothelial cell responses to fibrin mediated by FPB cleavage and the amino terminus of the beta chain.

Endothelial cells (EC) interact with fibrin at sites of vascular injury, thrombosis, inflammation and tumor growth, whereas they are quiescent when exposed to circulating fibrinogen. To determine the structural basis for specific interaction with fibrin we have characterized the response of EC to fibrin of varying structure. Fibrin was prepared with thrombin, which cleaves both fibrinopeptide A (FPA) and fibrinopeptide B (FPB), with Reptilase, which cleaves only FPA, and with contortrix procoagulant to cleave only FPB. Fibrin with FPB cleavage stimulated release of von Willebrand factor from EC Weibel-Palade bodies and also supported cell spreading. Involvement of the amino terminus of the fibrin beta chain in the response was shown by stimulation of von Willebrand factor release by the peptide beta 15-42. Also, fibrin prepared from a fibrinogen derivative lacking residues 15-42 of the beta chain failed to support EC spreading. EC adhesion was unaffected by the pattern of fibrinopeptide cleavage or by the removal of peptide beta 15-42 from fibrin. The results indicate that separate sites on the fibrin molecule mediate adhesion and spreading, and that the latter requires cleavage of FPB and the new amino terminus of the beta chain. They further suggest that cellular responses to fibrin are regulated by the proteolytic cleavages and conformational changes that convert fibrinogen to fibrin and may also be modulated by plasmic or elastase degradation.

Amino Acid Sequence↗
PubMed1993

A new immunoassay for soluble fibrin enables a more sensitive detection of the activation state of blood coagulation in vivo.

A novel sandwich immunoassay for measurement of soluble fibrin in plasma has been developed. For immunization we used the synthetic heptapeptide Gly-Pro-Arg-Val-Val-Glu-Arg representing the amino terminus of the alpha-chain of human fibrin. A monoclonal IgG1 antibody was obtained by conventional hybridoma technology. To increase convenience, the sandwich immunoassay was developed for the Enzymun-Test systems which are based on streptavidin pre-coated tubes. The new assay was designed with the same fibrin specific antibody both in biotinylated and in peroxidase-labelled form. Fibrin in native plasma samples could only be detected after pre-incubation of the plasma with chaotropic ions. Test results were calculated using a standard curve comprising six fibrin standards (0-50 micrograms/ml). Precision of the method was satisfactory; intra-assay CVs using plasma samples ranged between 5.0% (23.7 micrograms/ml) and 12.4% (0.2 microgram/ml). CVs of interassay precision measurements using standards as samples range between 7.3% (25.0 micrograms/ml) and 11.4% (1.0 micrograms/ml). The lower detection limit was 0.12 microgram/ml. Investigations of normal range in 70 age-matched healthy individuals resulted in a mean of 1.12 micrograms/ml. Linearity was excellent; recovery of high fibrin plasma after dilution with normal plasma was always between 100 and 107%. Fibrin specificity was due to the monoclonal antibody 2B5 used and no cross reactivity with fibrinogen, fibrinogen split products or fibrin D-dimer was observed. Fibrin fragment E1 (studied by Dempfle CE, et al. Blood Coag Fibrinol 1993; 4: 79-86) and fragments X and Y showed moderate cross reactivity in the assay and caused some overestimation of fibrin at high fibrin split product concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1996

Fibrin binding of plasminogen coated liposomes in vitro.

In this study, the fibrin binding properties of liposomes containing a number of plasminogen (Plg) molecules on the outside were compared to those of free (non-liposomal) Plg in an in vitro model system. Fibrin monolayer coated 96-wells plates were used, containing fibrin monomer at a density of around 3.4 to 3.9 x 10(-4) nmol/cm2. These densities are similar to liposomal Plg-densities, thus allowing multivalent interactions to occur. In the panel of experimental conditions that was chosen, binding of free Plg and liposomes with Plg showed three main differences in characteristics. Firstly, in the fibrin binding of Plg-liposomes not all Plg may be involved, but on the average 40% of the total amount of liposomal Plg. This was shown by lysing the liposomes after binding to the fibrin and estimation of truly bound Plg. With Plg-densities on the liposomes below the fibrin binding sites density, the maximal number of bound Plg molecules remains below the amount of available fibrin binding sites. Secondly, a higher binding rate by at least one order of magnitude was observed for liposomes with Plg compared to free Plg. Thirdly, liposomes with Plg exhibit a fibrin binding affinity which increases with Plg-density, because of the multivalent character of interaction. Liposomal Plg can successfully compete for fibrin binding sites with a 100 fold higher concentration of free Plg. These in vitro findings indicate that in view of avid and rapid fibrin binding, liposomes with attached plasminogen may be suitable for in vivo targeting to fibrin based thrombi.

Amino Acid Sequence↗
PubMed1997

Fibrin induction of ICAM-1 expression in human vascular endothelial cells.

In acute and chronic inflammatory processes, fibrin deposition, and leukocyte accumulation are classic histopathologic hallmarks. Previous studies have shown that fibrin and fibrin degradation products can have biologic effects on vascular endothelial cells and can induce the expression of several endothelial cell-derived factors that may be important in regulating inflammation and tissue repair. We now demonstrate that coculture of human vascular endothelial cells (EC) with fibrin results in the up-regulation of intercellular adhesion molecule-1 (ICAM-1), thus providing a first link between fibrin deposition and adhesion molecule expression, which may lead subsequently to leukocyte accumulation and extravasation. Increased ICAM-1 expression was demonstrated by ELISA, flow cytometry, and functional adhesion assays. EC ICAM-1 expression increased in a time and dose response fashion. Cell surface levels of ICAM-1 induced by fibrin were comparable to, or exceeded, levels induced by IL-1beta. ICAM-1 expression increased beginning at 4 h post-fibrin formation with sustained elevated expression at 48 h. Fibrin-stimulated EC also bound increased numbers of polymorphonuclear neutrophils in cellular adhesion assays. This increase in adhesion could be blocked by Ab to ICAM-1. Inhibition of fibrin polymerization also inhibited the up-regulation of ICAM-1. Culture medium from fibrin-stimulated EC contained elevated levels of soluble ICAM-1. These data suggest that fibrin deposition on vascular EC, in addition to other reported effects on EC metabolism, may also lead to leukocyte accumulation and extravasation through the induction of leukocyte adhesion molecules such as ICAM-1.

Cell Adhesion↗
PubMed1997

Degree of polymer organization decreases the binding of a monoclonal antibody raised against the beta-chain amino terminus of fibrin.

UNLABELLED: Accurate non-invasive diagnosis of deep venous thrombosis and pulmonary embolism remains an elusive goal. Radiolabeled antibodies specific for the epitope exposed on the beta-chain of fibrin after fibrino-peptide B release (anti-beta) enabled in situ imaging of thrombi in experimental subjects with nuclear medicine techniques. When used in patients anticoagulated for thrombo-embolic disease, however, the antibody was unable to reliably image the thrombi. We postulated that the neoepitope on the beta-chain of fibrin is covered up as fibrin organizes into a polymer network and is therefore exposed to the antibody only during active incorporation of fibrin subunits. We determined the equilibrium binding kinetics of an anti-beta monoclonal antibody to fibrin in various stages of organization. The concentration of exposed epitopes on immobilized fibrin monomers was equal to the molar concentration of fibrin beta-chains. The percentage of beta-chains exposed to the antibodies markedly decreased as the fibrin network was allowed to organize, a process catalyzed by calcium. CONCLUSIONS: The beta-chain amino terminus of fibrin is exposed transiently as subunits are added to the enlarging fibrin network. Anti-beta antibodies bind preferentially to actively enlarging fibrin polymers.

Antibodies, Monoclonal↗
PubMed1993

The sequence of cleavage of fibrinopeptides from fibrinogen is important for protofibril formation and enhancement of lateral aggregation in fibrin clots.

Thrombin cleaves A fibrinopeptides from fibrinogen faster than B; cleavage of the B fibrinopeptides has been associated with enhanced lateral aggregation in the fibrin clot. We show that protofibrils from soluble desA fibrin examined by electron microscopy appear to be more loosely organized than those from desAB fibrin. Protofibril lengths were measured from the micrographs; histograms of the distribution of protofibril sizes at different points in the lag period are different for the two species and indicate that the associations leading to oligomer formation are not random. A kinetic model for oligomer formation was developed to investigate the implications of these histograms for protofibril assembly; the model suggests that oligomer-oligomer reactions are important and that some reactions occur preferentially. The overall appearance of negatively contrasted clots made from fibrin monomer missing both fibrinopeptides was similar to that of clots from molecules missing only the A fibrinopeptide, in contrast to the large differences in clots produced by the action of the enzymes on fibrinogen. However, examination of the band patterns by electron microscopy reveals that fibers from desAB fibrin are less well ordered than those from desA fibrin and often aperiodic. In agreement with the electron microscope results, maximum turbidity values are similar for both types of clots, although the initial turbidity rise is more rapid for desAB soluble fibrin clots. If thrombin is added to desA soluble fibrin during the lag period, there is increased lateral aggregation of fibers observed by electron microscopy and the maximum turbidity is higher. These results indicate that there must be a delay in the cleavage of the B fibrinopeptides for the enhancement of lateral aggregation. Changes that occur upon fibrinopeptide B cleavage from molecules in a fiber, such as an increase in calcium ion binding or conformational changes, cause fibers to aggregate with each other or additional protofibrils to be added to a fiber. On the other hand, if these differences are present initially, as they are on soluble desAB fibrin, these results indicate that all steps occur more rapidly, producing relatively thin fibers that are not well ordered. In conclusion, the usual delay in fibrinopeptide B cleavage appears to be necessary both for normal protofibril and fiber assembly, so that the changes that accompany removal of these peptides preferentially affect lateral aggregation rather than earlier steps of fibrin clot assembly.

Animals↗
PubMed2000

Fibrin D-dimer in thrombogenic disorders.

Measurement of D-dimer (fibrin degradation product) is important for determining not only the activation of fibrinolysis but also the severity of a hypercoagulable state. However, fibrin degradation products are in variable, and the reactivity to cross-linked fibrin degradation products produced during fibrin degradation differs depending on the kind of antibody used against D-dimer. In patients with disseminated intravascular coagulation or earthquake-induced mental and physical stress and in patients after percutaneous transluminal coronary angioplasty, all of which are associated with acute fibrin formation and degradation, some discrepancies between two methods of D-dimer detection, automated latex agglutination assay (LPIA) and enzyme-linked immunosorbent assay (Stago), were found. No discrepancies in persistent fibrin formation and degradation were found among the healthy elderly, patients with lacunar stroke, and patients with coronary artery disease, almost all of whom had levels under 5.0 microg/mL, as determined by both methods. Evidence of persistently increased intravascular coagulation and fibrin turnover in patients with atherosclerotic disease was found. The cleavage of cross-linked fibrin by plasmin results in a production of fibrin degradation products, mostly contained D-dimer domains. Although the clinical utility of D-dimer can be achieved by their detection with specific antibodies, measurement of D-dimer as high-molecular-weight fragments may be useful to determine whether patients will undergo further fibrin degradation. When intermediate products of the degradation process need to be assessed, D-dimer level measurement by LPIA may serve as a suitable marker for ongoing fibrinolysis.

Adult↗
PubMed2006

A new evaluation concept and its measurement: "male sexual anticipating cognitions".

CONTEXT: The development of sex-active drugs justifies the use of scales for evaluating changes induced by such forms of therapy. Among the tools available for evaluating erectile dysfunction (ED), the International Index of Erectile Function is the most widely used. Analysis of the scientific literature shows that these instruments remain primarily focused on functional aspects of ED. The vulnerability factors involved in ED are mainly based upon organic characteristics, e.g., age, cardiovascular diseases, or diabetes, but the psychological factors involved in the occurrence and maintenance of ED are less studied. OBJECTIVE: This conclusion led us to develop a French self-administered instrument, based on patients' subjective sexual experiences, to evaluate "male sexual anticipating cognitions," using Apter's reversal theory as a framework. DESIGN, SETTING, AND PATIENTS: We present here the first stages of the development of this new instrument. We carried out semistructured, one-to-one interviews with patients with psychogenic ED and patients with mixed ED. Eighteen one-to-one interviews were held in several urologic/andrologic and psychiatric sexologist services. Interviews were continued until the required information was obtained. Analyses of the content were performed to generate items for the new instrument. RESULTS: Dimensions isolated were those of (a) sexual beliefs; (b) metamotivational modes; and (c) dysfunctional coping. Pools of items are currently being analyzed by 108 experts (psychologists, sexologists, andrologists, urologists) to determine if additional items are required. The acceptability and the comprehensiveness of this preliminary questionnaire will be tested by 320 patients in a multicentric study, to produce a shorter questionnaire featuring the most relevant items. Once validated, this multidimensional instrument could be used to assess treatment efficacy of the mixed and psychogenic components of ED.

Aged↗
PubMed1984

Effect of crosslinking on the structure of solubilized fibrin degradation products in whole plasma.

The purpose of these studies was to establish the validity of 125I fibrin autoradiography--SDS gel techniques for monitoring degradation products from whole plasma or blood clots. These methods can be used to study fibrin degradation not only in patients with congenital factor XIII deficiency, but also in patients with disseminated intravascular coagulation or deep vein thrombosis during the course of thrombolytic therapy. Such an assay might complement existing immunologic techniques to characterize fibrin degradation in vivo by providing an in vitro analysis of the rate and pattern of fibrin degradation in whole blood or plasma. Fibrin degradation was traced by Coomassie blue staining for protein and by autoradiography on SDS-PAGE of degradation products released from a 125I-labeled fibrin tracer. The degradation of non-crosslinked clots from purified fibrin supplemented with plasmin showed a typical release of X, Y, D, and E fibrin fragments. Subsequently, all X and Y fragments were digested to D and E fragments. The degradation of non-crosslinked washed clots prepared from plasma supplemented with plasmin reflected the same pattern. The degradation of non-crosslinked washed clots prepared from EDTA anticoagulated plasma without added plasmin also showed release of X, Y, D, and E fragments. However, in contrast to the non-crosslinked washed clots supplemented with plasmin, there was no additional degradation of the X and Y fragments. These studies established that the pattern of degradation of the 125I-radiolabeled fibrin tracer was similar to that of the total protein released from the fibrin clot as observed by protein staining.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Coagulation↗
PubMedDate not supplied

Autologous cartilage grafts enhanced by a novel transplant medium using fibrin sealant and fibroblast growth factor.

OBJECTIVE: To introduce and assess a system for the delivery of fibroblast growth factor to autologous cartilage grafts using fibrin sealant and analyze whether this "enhancement" results in reduced rates of cartilage resorption and greater preservation of normal architectural features compared with "unenhanced" cartilage grafts. METHODS: Auricular cartilage segments measuring 1 cm(2) were harvested from 12 New Zealand white rabbits, morselized, and implanted into the subcutaneous dorsum of the upper back for 3 months. The conditions included (1) cartilage alone; (2) cartilage + fibrin sealant; (3/4) cartilage + acidic or basic fibroblast growth factor (aFGF or bFGF); and (5/6) cartilage + fibrin sealant + aFGF or bFGF. Subsequent to graft harvest, gross and microscopic assessments were performed to assess size, structural integrity, and architectural features, with comparisons performed between each of the conditions. RESULTS: The mean areas of the harvested cartilage grafts treated with fibrin sealant + aFGF or bFGF were 1.23 cm(2) and 1.19 cm(2), respectively, while the corresponding value for the untreated (ie, cartilage alone) specimens was 1.03 cm(2). The percentage of decrease in size was 45% for the untreated specimens and 0% for the specimens treated with fibrin sealant + aFGF or fibrin sealant + bFGF. Cartilage treated with fibrin sealant + bFGF had the greatest quantity of elastin fibers of the 6 conditions. Cartilage grafts treated with fibrin sealant alone demonstrated the most intense ground substance staining on a computerized measure of pixel intensity. CONCLUSIONS: Our findings demonstrated significant improvements in graft quality using fibroblast growth factor and fibrin sealant or even fibrin sealant alone. These findings may justify changes in how cartilage grafts are prepared and delivered for facial augmentation procedures to reduce graft resorption and maintain the structural integrity of the cartilage. Further trials will be performed to elucidate the optimal growth factor concentrations for maximum structural and architectural benefits.

Animals↗
PubMed1997

Three-dimensional visualization and quantitation of fibrin in solid tumors by confocal laser scanning microscopy.

Fibrin forms part of the stroma essential for growth of solid tumors. Anticoagulants reduce primary tumor growth and tumor metastasis in murine and some human tumors. These effects may be partly mediated by reduction of intra-tumor fibrin, although there are no quantitative data to support this hypothesis. We therefore evaluated the effect of warfarin on fibrin deposition in a subcutaneously (s.c.) implanted murine tumor using confocal laser scanning microscopy (CLSM). AJ mice received no treatment (n = 6) or sodium warfarin (3.5 mg/L in drinking water, n = 5). All animals received 2 x 10(6) syngeneic Neuro2a neuroblastoma cells s.c. After 14 days, primary tumors were excised and placed in liquid nitrogen. Warfarin treatment resulted in a small, but significant (P < 0.05), decrease in wet tumor weight. Frozen sections (20 microns) were incubated with goat anti-mouse fibrin(ogen) or normal goat serum (isotypic control) and stained with FITC-conjugated rabbit anti-goat antibody. Using a Multiprobe 2001 CLSM (Molecular Dynamics, Sunnyvale, CA), 20 serial optical sections were taken from five, randomly chosen, high power fields (60x objective) for each slide. A threshold excluded all fluorescence except that from structural components within the tumor stroma (fibrin). The volume of fibrin in each section series was determined, and the percentage of tumor volume occupied by fibrin calculated. Intra- and inter-assay variation were assessed on serial frozen tumor sections from an untreated animal. The percentage fibrin volume was not significantly different among or within experiments, indicating that the procedure was reproducible. In controls, the median (range) volume occupied by fibrin was 8.1% (2.4-22.3%), whereas in anticoagulated animals, this was reduced to 3.7% (0.4-14.0%; P < 0.001). This is the first quantitative demonstration that warfarin reduces fibrin deposition in solid tumors. We conclude that three-dimensional CLSM is useful for the quantitation of tissue antigens and that the technique may have clinical value.

Animals↗
PubMed2005

Quantitative evaluation of the fibrin clot extension on different implant surfaces: an in vitro study.

The aim of the present study was a quantitative evaluation of the in vitro fibrin clot extension on different implant surfaces. Forty-five disk-shaped commercially pure Grade 2 titanium samples with three different surface topographies (machined, DPS, and Plus) were used in the present study. For the quantitative evaluation of the fibrin clot, 30 specimens were used (10 per group); human whole blood was employed. Venous blood was drawn from three healthy adult volunteers, and 0.2 mL were immediately dropped onto the surface of each specimen. Contact time was 5 min at room temperature; then the samples were rinsed with saline solution and fixed in a buffered solution of glutaraldehyde and paraformaldehyde. Samples were washed again with buffer and dehydrated in an ascending alcohol series. Specimens belonging to all groups were observed under SEM at a magnification of 1000x. From each sample, 50 random micrographs were collected in .tif format with an N x M 1024 x 768 grid of pixels. Quantitative analysis of fibrin clot extension showed the following results: in machined samples fibrin clot extension was 345987.2 +/- 63747.7 pixels(2) (mean +/- SD), in DPS samples fibrin clot extension was 375930.9 +/- 54726.86 pixels(2) (mean +/- SD), and in Plus samples, fibrin clot extension was 612333.6 +/- 46268.42 pixels(2) (mean +/- SD). With ANOVA it was possible to find that there were significant differences among the groups. The Tukey test revealed that the extension of the fibrin clot of Plus samples was statistically higher compared to both machined and DPS samples. The results of this in vitro study indicate that there is a correlation between implant surface morphology and fibrin clot extension. Improvement in surface microtexture complexity seems to determine the formation of a more extensive and three dimensionally complex fibrin scaffold. Further investigations are necessary to explain in more detail the mechanisms that regulate the fibrin clot formation on different implant surfaces.

Adsorption↗
PubMed2003

Protective fibrin-sealed plication of the small bowel in recurrent laparotomy.

PURPOSE: Adhesions after recurrent abdominal operations remain extremely common and are sources of severe morbidity. Fibrin-glued plication of the small gut in a meander-like formation is supposed to guarantee a decreased risk of intestinal obstruction postoperatively. This retrospective study analyses the clinical outcome after recurrent laparotomy in children treated with bowel plication by fibrin sealant. METHODS: The surgical technique of performing the fibrin-glued plication is rather simple and quick: after taking off all adhesions two to four loops of the small gut are positioned so that they lie side by side. Beginning proximal fibrin [Tissucol fibrin sealant (Baxter)] is applied between the loops; approximately 20-30 s are needed to keep the loops in position until the fibrin dries. This manoeuvre is continued until all of the small gut is fixed in one block. The gut is brought back into the abdominal cavity without loosening the loops. This fixed formation by sero-serosal adhesions or mesenterial plications is supposed to guarantee postoperative free passage. The charts of 60 children who had undergone a fibrin plication of the small bowel between 1991 and 1999 were evaluated. Additionally, questionnaires were sent to all patients, and they were invited for an examination. RESULTS: Sixty patients (38 boys and 22 girls) received a fibrin sealant plication because of recurrent laparotomies with heavily serosal defects or recurrent ileus because of adhesions. The youngest baby was 10 days. Since 23 patients were premature the oldest patient was 11 years old. There were no intraoperative complications attributed to the method. In the postoperative period 7/60 (12%) patients had a recurrent ileus or subileus, leading in three (5%) patients to an early relaparotomy. CONCLUSION: The fibrin-glued plication of the small bowel decreases the risk of recurrent ileus or subileus considering the high figures in the literature concerning this issue. The time-saving method is very simple and easily feasible. No side effects after the treatment with fibrin glue were observed.

Child↗
PubMed2003

Fibrin glue for anal fistulas: long-term results.

PURPOSE: The aim of this study was to evaluate the long-term success and complication rate of fibrin-glue treatment of anal fistulas. METHODS: Patients with an anal fistula presenting to a single surgeon over a three-year period were enrolled in this study. At their first operation, all 48 patients (26-72 years old) underwent anoscopy, biopsy, destruction of the internal gland, and placement of a draining seton. Approximately two months later after preoperative bowel preparation, the seton was removed, the internal opening closed with a single suture, and fibrin glue instilled by way of the external opening to seal the fistula tract. Patients were followed closely to document the results of treatment and any complications. Long-term follow-up was done by telephone interview. RESULTS: Cause of the anal fistula was cryptoglandular in 36 (75 percent) patients, Crohn's disease in 5 (10 percent), and miscellaneous in 7 (15 percent). Median follow-up was 22 months (range, 6-46 months). After a single treatment with fibrin glue, 29 (60 percent) fistulas closed. Retreatment with fibrin glue brought the successful number of fistula tracts closed to 33 (69 percent). The 15 (29 percent) patients who failed either one or two treatments with fibrin glue were successfully treated with either fistulotomy or advancement flap. Bowel function and fecal incontinence were not altered by the fibrin-glue treatment. In one patient who failed fibrin glue, the fibrin-glue treatment may have created a more complicated fistula tract. Late recurrences (>6 months) occurred in three (6 percent) patients, two of whom were successfully retreated with fibrin glue. CONCLUSIONS: Fibrin-glue treatment of anal fistulas is successful in up to 69 percent of patients if initial failures are retreated. This sphincter-saving technique is associated with minimal complications and no functional detriment. Late recurrences are unusual.

Adult↗