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PubMed2004

Healing of colon anastomoses covered with fibrin glue after immediate postoperative intraperitoneal administration of 5-fluorouracil.

PURPOSE: The aim of this experimental study was to investigate whether covering the colonic anastomoses with fibrin glue can protect the colonic healing from the adverse effects of 5-fluorouracil (5-FU), when it is injected intraperitoneally immediately after colon resection. METHODS: Sixty-four rats were randomized to one of four groups. After resection of a 1-cm segment of the transverse colon, an end-to-end sutured anastomosis was performed. Rats of the control group and the fibrin glue group were injected with 6 ml of solution 0.9 percent NaCl intraperitoneally. Rats in the 5-FU and the 5-FU + fibrin glue groups received 5-FU intraperitoneally. The colonic anastomoses of the rats in the fibrin glue group and in the 5-FU + fibrin glue group were covered with fibrin glue. All rats were killed on the 8th postoperative day and the anastomoses were examined macroscopically. The bursting pressure measurements were recorded and the anastomoses were graded histologically. RESULTS: The leakage rate of the anastomoses was significantly higher in the rats of the 5-FU group than in those of the fibrin glue group and those of the 5-FU + fibrin glue group (37.5 percent vs. 0 percent, P = 0.020). The adhesion formation score was significantly higher in rats of the 5-FU group than in the other groups. Bursting pressures were also significantly lower in the 5-FUgroup than in the other groups ( P < 0.001). Rats in the 5-FU + fibrin glue group developed significantly more marked neoagiogenesis than rats in the other groups. Rats in the 5-FU + fibrin glue group also presented significantly more fibroblast activity than those in the 5-FU group. ( P = 0.004) CONCLUSIONS: The immediate postoperative, intraperitoneal administration of 5-FU inhibited wound healing. However, when the colonic anastomoses were covered with fibrin glue, the injection of 5-FU had no adverse effects on the healing of the anastomoses.

Anastomosis, Surgical↗
PubMed2003

Fibrin glue sealing in the treatment of perineal fistulas.

PURPOSE: The surgical management of complex perineal fistulas, such as high transsphincteric and suprasphincteric fistulas, or those associated with Crohn's disease, radiotherapy, surgical trauma, or cavity or a secondary tract, is associated with the risk of sphincter injury and significant discomfort. Fibrin glue may close fistula tracts without muscle division. Therefore, the aim of this study was to evaluate the use of fibrin glue sealing in treatment of perineal fistulas. METHODS: A retrospective chart review of all patients in whom fibrin glue was used for the treatment of perineal fistula was performed. Patients were contacted by telephone to establish follow-up. RESULTS: Thirty-seven patients underwent injection of fibrin glue for complex perineal fistulas. Twenty-four patients had fibrin glue injection as the principal treatment for the perineal fistula, and 13 had fibrin glue in conjunction with an endorectal advancement flap. The fistula was of cryptoglandular origin in 16 (42 percent) cases and associated with Crohn's disease and trauma in 7 (19 percent) and 14 (38 percent) patients, respectively. At a mean follow-up of 12.1 months, healing occurred in only 15 (41 percent) patients. The healing rate was 33 percent when fibrin glue was the principal treatment, and 54 percent when used with an endorectal advancement flap. Fistulas of noncryptoglandular origin had a higher success rate, although this difference did not reach statistical significance. There was no morbidity associated with the injection of fibrin glue. CONCLUSION: In this study, fibrin glue had moderate success in the definitive treatment of perineal fistulas. However, 33 percent of the patients in whom fibrin glue was the only treatment used were able to avoid more extensive surgery. Fibrin glue is associated with minimal risk, therefore its application should be considered in patients with complex anal fistulas.

Combined Modality Therapy↗
PubMed2005

An indium:calcium phosphate colloid that specifically targets fibrin.

The ability of indium to target fibrin in vitro was evaluated. The radionuclide (114m)Indium (114mIn) was prepared as a soluble and colloidal (In:In) form, as well as, a mixed indium:calcium phosphate (In:CaP) colloid. Soluble 114mIn was prepared by maintaining acid pH (50 mM HCl). Colloidal 114mIn (In:In) was prepared under slightly basic conditions (50 mM Tris-Cl, pH 7.6). The mixed In:CaP colloid was prepared by incubation of 114mIn with calcium (10 mM) and phosphate (250 microM) under slightly basic conditions (50 mM Tris-Cl, pH 7.6). To assess fibrin binding, the three 114mIn preparations were mixed with diluted human plasma (source of fibrinogen). Fibrin polymerization was initiated by addition of calcium (5 mM) and thrombin (0.5 U/ml). Following incubation (15 min, 37 degrees C), the fibrin matrix was condensed, removed from the reaction mixture, and washed briefly. Fibrin uptake of 114mIn (soluble, colloidal, or In:CaP) was determined by gamma counting. Results demonstrated that soluble 114mIn exclusively bound a plasma protein electrophoretically and immunologically identified as transferrin. Although both colloidal 114mIn and 114mIn:CaP bound fibrin, the mixed 114mIn:CaP colloid demonstrated substantially higher fibrin binding activity (about 2-fold). The target of indium binding was confirmed as fibrin due to the presence of characteristic cross-linked gamma-gamma dimers (100 kDa) and beta-monomers (58 kDa) by SDS-PAGE. 114mIn colloid and the mixed 114mIn:CaP colloid demonstrated no ability to bind fibrin's precursor, fibrinogen. 114mIn:CaP fibrin binding was associated with formation of CaP, as evidenced by its dependence on phosphate concentration. The biocompatibility of CaP including its ability to bind 114mIn and specifically target fibrin may be of potential value for diagnostic imaging studies to identify regions of occult vascular stenosis (i.e., atherosclerotic plaques, deep vein thrombosis, pulmonary embolus).

Biocompatible Materials↗
PubMed1986

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.

Drug Contamination↗
PubMed1986

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.

Adenine↗
PubMed2005

Control of basic fibroblast growth factor release from fibrin gel with heparin and concentrations of fibrinogen and thrombin.

Basic fibroblast growth factor (bFGF) has been known to stimulate the regeneration of a number of tissues including cartilage, nerve, skin, liver, and blood vessel. Delivery of bFGF for a long period in a controlled manner would enhance stimulative effects. The purpose of the present study is to test the hypothesis that the kinetics of bFGF release from fibrin gels could be controlled with heparin and concentrations of fibrinogen and thrombin. The kinetics of bFGF release from fibrin gels with various concentrations of fibrinogen, thrombin, and heparin was determined. The bioactivity of bFGF released from fibrin gels was assessed using dermal fibroblast cell culture. To examine the therapeutic potential of the bFGF delivery system, bFGF-loaded fibrin gels were injected into mouse ischemic limbs. The addition of heparin to fibrin gels decreased the bFGF release rate. As the thrombin content in fibrin gels increased, the bFGF release rate significantly decreased. Similarly, increased concentration of fibrinogen in fibrin gels decreased the bFGF release rate. Basic FGF released from fibrin gels exhibited significantly higher extents of fibroblast growth than bFGF added in a free form daily into the culture medium, suggesting that the fibrin gels may stabilize the bFGF bioactivity. Immunohistological analysis of mouse ischemic limbs indicated that the microvessel density was much higher in the ischemic limbs treated with injection of bFGF-loaded fibrin gels than in the ischemic limbs with no treatment. This study showed that the rate of bFGF release from fibrin gels can be controlled and that the bFGF delivery system has therapeutic potentials for angiogenesis.

Angiogenesis Inhibitors↗
PubMedDate not supplied

An in vitro and in vivo analysis of fibrin glue use to control bone morphogenetic protein diffusion and bone morphogenetic protein-stimulated bone growth.

BACKGROUND CONTEXT: Recombinant human bone morphogenetic protein-2 (rh-BMP2) has become popular for augmenting spine fusion in the lumbar and cervical spine. Concerns exist, however, over bone morphogenetic protein (BMP)-stimulated soft-tissue swelling and bone growth stimulation in areas where bone is not desired, especially as the material "leaks" into such spaces. The most detrimental effects of such leakage might be airway compromise, while heterotopic bone formation into the spinal canal has been reported in animal and human studies. Fibrin glue has been used as a carrier of many osteoinductive materials; however, its efficacy at modulating the clinical effects of BMP are not known. The amorphous nature of fibrin glue makes it a candidate to control diffusion of BMP and possibly limit bone formation by limiting BMP diffusion to areas where such bone is not desired. PURPOSE: To evaluate the use of fibrin glue to limit BMP diffusion and BMP-stimulated bone growth. STUDY DESIGN/SETTING: This is an in vitro basic science study and an in vivo prospective randomized animal study. STUDY SAMPLE: Eighteen Lewis rats. OUTCOME MEASURES: In vitro study: Enzyme-linked immunosorbent assay measurement of rh-BMP2 concentration in saline. In vivo study: At day 60, rats were evaluated for neurologic deficits before sacrifice. Spines were harvested, and the following studies were performed: 1) manual testing for fusion and bone growth; 2) X-ray evaluation; 3) Micro-computed tomography (micro-CT) scans. METHODS: In vitro study: Collagen sponges soaked with BMP at two different concentrations were incubated in saline solution with and without encapsulation by fibrin glue. Saline BMP concentrations were measured at consecutive time points. In vivo study: A rat fusion model using rh-BMP2 for fusion has been developed and tested with resultant100% fusion in over 100 rats. Lewis rats were divided into two groups and treated as follows: I: Exposure of L4-L5 transverse processes, decortication, and placement of BMP sponge in the lateral intertransverse space. II: Exposure and decortication as above and placement of fibrin glue before BMP sponge placement. RESULTS: In vitro study: Peak rh-BMP2 concentrations in saline were 20% and 45% of the maximum possible for fibrin glue encapsulated sponges and controls, respectively, with a more gradual increase to peak concentration in samples encapsulated in fibrin glue. In vivo study: No rats exhibited any neurologic deficits. X-rays revealed at least partial bone formation in all rats. Manual testing of intertransverse fusion spines revealed 100% fusion in rats treated with BMP only, whereas rats treated with fibrin glue before placement of BMP sponges revealed only one possible fusion. Posterior-lateral bone formation was present on X-ray in both groups, and micro-CT imaging revealed bridging bone from transverse processes to the BMP-stimulated bone in the control groups. In spines treated with fibrin glue before rh-BMP2 placement, bone formation could still be seen within the soft tissues; however, bridging bone connecting to the transverse processes was either significantly decreased or not present. CONCLUSIONS: Fibrin glue can limit rh-BMP2 diffusion. Also, because it limited bone formation at the transverse processes, it can be inferred that fibrin glue can limit bone formation when used to separate areas of desired bone formation from areas where bone formation is not desired.

Animals↗
PubMed2005

Migration pattern, morphology and viability of cells suspended in or sealed with fibrin glue: a histomorphologic study.

INTRODUCTION: We studied the migration pattern, morphology and viability of cells suspended in five different fibrin glues. Besides this, the behaviour of chondrocytes seeded on porous matrices comprising different collagen types sealed with fibrin glue was investigated. MATERIAL AND METHODS: In an experiment A, cell suspension (0.5x10(6) cells) was incubated with different fibrin glues. Experiment B was set up to evaluate chondrocytes migration either through a collagen I/III (Chondro-Gide, Geistlich Biomaterials, Switzerland) or collagen II matrix sealed with different fibrin glues in a perfusion chamber system. Analysis were performed by lightmicroscopy (Mayer's hematoxylin-eosin; Masson-Goldner; TUNEL test) and by transmission and scanning electron microscopy. All fibrin glues were measured for TGF-beta 1 and 2 with a specific ELISA. RESULTS: After incubation of cell suspension in autologous fibrin glue, the morphology of cells is chondrocyte-like. Spindly, process-bearing cells were seen in commercial fibrin glue. Cells suspended in commercial fibrin glue revealed a significant higher percentage of TUNEL positive cells compared to fibrin tissue adhesives mixed with autologous serum (p=0.006). The TGF-beta 1 and 2 concentration was significantly higher in partial autologous fibrin sealant (PAF) compared to their commercial counterparts (p=0.001). Cells seeded on the collagen I/III matrix retained their chondrocytic morphology, while in the type II collagen matrix the chondrocytes displayed a fibroblastic phenotype. The ratio of TUNEL positive cells for the collagen I/III matrix was significantly surpassed by the values, when a collagen II matrix was used (p=0.008). No ingrowth of cells was seen in any of the experimental conditions. CONCLUSION: Partial autologous fibrin glue and collagen I/III matrices are favourable in respect to migration pattern, morphology and viability, but definitive conclusions can only be drawn after in vivo studies. This will be addressed in future animal studies.

Aprotinin↗
PubMed1998

Myocardial fibrin deposits in the first month after transplantation predict subsequent coronary artery disease and graft failure in cardiac allograft recipients.

PURPOSE: To determine whether fibrin deposition during the first month following cardiac transplantation predicts development of coronary artery disease and graft failure in cardiac allograft recipients. PATIENTS AND METHODS: We prospectively studied 121 consecutive adult patients who received cardiac transplants between 1988 and 1995. Serial endomyocardial biopsies obtained during the first month posttransplant (2.3 + 0.6 biopsies/patient) were studied immunohistochemically for fibrin deposits. Patients were followed up with annual angiograms (3.2 + 1.7/patient) evaluated with side-by-side comparisons for the presence and progression of coronary artery disease. RESULTS: All pretransplant biopsies were fibrin-negative; 60 allografts (50%) remained without fibrin, and 61 (50%) contained fibrin during the first posttransplant month. Of allografts with fibrin, 72% developed coronary artery disease, while 27% of allografts without fibrin developed the disease (P <0.001). Coronary artery disease was progressive in 61% of allografts with fibrin, and in 25% of allografts without fibrin (P <0.001). Graft failure was more frequent and time-to-graft-failure occurred earlier in patients whose allografts had fibrin during the first month after transplantation (P <0.001). CONCLUSIONS: Fibrin in biopsies during the first month after transplantation identifies patients at high risk for developing coronary artery disease or graft failure, thereby allowing the opportunity to initiate preventive procedures.

Adult↗
PubMed1988

Clots of beta-fibrin. Viscoelastic properties, temperature dependence of elasticity, and interaction with fibrinogen-binding tetrapeptides.

Clots of human beta-fibrin, in which only (or predominantly) the B fibrinopeptide is released, were formed at 14 degrees C by copperhead venom procoagulant enzyme (CVE or venzyme), at pH 8.5, ionic strength 0.45. The shear modulus of elasticity increased slowly and after several days attained a constant value, which was lower than those of alpha-fibrin or alpha beta-fibrin under the same conditions. Before studying the temperature dependence of elasticity, the CVE was then inhibited by introducing phenyl methyl sulfonyl chloride (PMSF) by diffusion. With increasing temperature, the modulus decreased progressively from 5 degrees C to nearly zero at 35 degrees and was essentially reversible with temperature change; recovery of elasticity after change from 34.5 degrees to 14 degrees required approximately 2 d but was considerably faster than the initial buildup of elasticity by CVE at 14 degrees. Creep and creep recovery measurements on unligated clots showed creep rates and irrecoverable deformation that were similar in magnitude to those of alpha-fibrin clots formed with batroxobin and much larger than those of alpha beta-fibrin clots formed with thrombin, under the same conditions. During creep and creep recovery, the differential modulus or compliance remained constant, showing that there was no permanent structural damage, and if network strands are severed in slow flow, they must rejoin in new configurations. Introduction (by diffusion) of the tetrapeptides Gly-His-Arg-Pro (GHRP) and Gly-Pro-Arg-Pro (GPRP), which resemble the B and A binding sites on the E domain of fibrin respectively, reduced the shear modulus and increased the creep rate of beta-fibrin clots to an extent similar to the effect of GPRP on alpha beta-fibrin, much more than that of GHRP on alpha beta-fibrin, but much less than that of GPRP on a-fibrin. A ligated beta-fibrin clot formed with Factor XIIIa (in which the activating thrombin had been neutralized by hirudin) showed essentially perfect elastic behavior, with no creep and with complete recovery after removal of stress, and was inert to GHRP.

Blood Coagulation↗
PubMed2003

Fibrin sealants in supporting surgical techniques: The importance of individual components.

Fibrin sealants have many different uses across a broad range of surgeries, where they have proved successful in controlling bleeding, providing suture support and tissue sealing. The action of all fibrin sealants depends on the thrombin-catalyzed formation of a fibrin clot. However, neither the purity nor the concentration of the main components of fibrin sealants (thrombin and fibrinogen) is uniform across all commercial products and this will affect performance. In addition, the optional inclusion of other components such as factor XIII and antiproteolytic inhibitors may also influence the quality of clot formation. Properties that vary among different fibrin sealants, such as the clotting rate, viscosity, adhesiveness, clot strength and resistance to proteolysis, are all-important considerations for the surgeon. The application of fibrin sealants in a very wide spectrum of surgical procedures means that some fibrin sealants may be more suitable for a particular procedure than others. One of the advantages of commercial fibrin sealants is that the high level of quality control ensures that their composition is extremely consistent between batches. On the other hand, blood bank-derived fibrin sealants may vary in their composition from one preparation to the next and hence be less predictable in their performance. This paper discusses how individual components contribute to the overall performance of fibrin sealants, thereby providing to the surgeon the necessary information to select the optimal fibrin sealant for a specific procedure.

Antifibrinolytic Agents↗
PubMed1986

Structural basis for the species selectivity of a fibrin-specific monoclonal antibody.

The structural determinant underlying the species specificity of a monoclonal anti-fibrin antibody (59D8) is the leucyl residue at position 5 in beta-chains of human fibrin. Anti-fibrin antibody 59D8 which had been elicited by immunization with human beta(1-7) peptide, Gly-His-Arg-Pro-Leu-Asp-Lys, binds to human and canine fibrins but not to bovine, ovine, or porcine fibrins. A comparison of the available amino acid sequence data suggested that the ability of anti-fibrin antibody 59D8 to discriminate among various fibrin beta-chains might be due to the amino acid at position 5. This was confirmed by competitive inhibition studies using synthetic fibrin-like peptides and determination of the amino acid sequences of the N-termini of ovine and porcine fibrin beta-chains. Edman degradation employing o-phthalaldehyde blocking permitted use of fibrin monomer rather than its separated constituent polypeptide chains. The same sequencing strategy was used to obtain partial sequence data for the alpha-chains of bovine, ovine, and porcine fibrin.

Amino Acid Sequence↗
PubMed2002

Interaction of fibrin(ogen) with fibronectin: further characterization and localization of the fibronectin-binding site.

The interaction of fibronectin with fibrin and its incorporation into fibrin clots are thought to be important for the formation of a provisional matrix that promotes cell adhesion and migration during wound healing. However, it is still unclear whether fibronectin interacts with both fibrin and fibrinogen or fibrin only and whether fibronectin binds exclusively to the fibrin(ogen) alphaC domains. To address these questions, we studied the interaction of fibronectin with fibrinogen, fibrin, and their proteolytic and recombinant fragments. In both ELISA and surface plasmon resonance (SPR) experiments, immobilized fibrinogen did not bind fibronectin at all, but after conversion to fibrin, it bound fibronectin with high affinity. To test which regions of fibrin are involved in this binding, we studied the interaction of fibronectin with the fibrin-derived D-D:E(1) complex and a recombinant alphaC fragment (residues Aalpha221-610) corresponding to the alphaC domain that together encompass the whole fibrin(ogen) molecule. In ELISA, when fibronectin was added to the immobilized D-D:E(1) complex or the immobilized alphaC fragment, only the latter exhibited binding. Likewise, when fibronectin was immobilized and the complex or the alphaC fragment was added, only the latter was observed to bind. The selective interaction between fibronectin and the alphaC fragment was confirmed by SPR. The fibronectin-binding site was further localized to the NH(2) terminal connector region of the alphaC domain since in ELISA, the immobilized recombinant Aalpha221-391 sub-fragment bound fibronectin well while the immobilized recombinant Aalpha392-610 sub-fragment exhibited no binding. This finding was confirmed by ligand blotting analysis. Thus, the results provide direct evidence for the existence of a cryptic high-affinity fibronectin-binding site in the Aalpha221-391 region of the fibrinogen alphaC domain that is not accessible in fibrinogen but becomes exposed in fibrin.

Binding Sites↗
PubMed1987

Inhibited thrombins. Interactions with fibrinogen and fibrin.

Fibrin-monomer-Sepharose was used to study thrombin binding to fibrin and the role of the enzyme active centre in this interaction. Binding properties of preformed enzyme-inhibitor complexes, as well as inhibition of thrombin already adsorbed to fibrin monomer, were investigated. No apparent difference was found in binding properties of phenylmethanesulphonyl fluoride-, D-Phe-Pro-Arg-CH2Cl- and dansylarginine NN-(3-ethylpentane-1,5-diyl)amide-inhibited thrombins. Also, the elution profile of phenylmethane-sulphonyl fluoride-inhibited thrombin from fibrinogen-Sepharose was identical with that of active thrombin from fibrin-monomer-Sepharose. Thus far the only low-Mr inhibitor that prevents thrombin from binding to fibrin monomer is pyridoxal 5'-phosphate. Preformed hirudin-thrombin complexes do not interact with fibrin. The extent to which the active centre of thrombin associated with fibrin is still accessible to substrates and inhibitors was also studied. Thrombin bound to fibrin hydrolyses a synthetic substrate at the same rate as the free enzyme. Water-soluble low-Mr inhibitors such as D-Phe-Pro-Arg-CH2Cl and dansylarginine NN-(3-ethylpentane-1,5-diyl)amide can readily modify the active centre of the fibrin-associated enzyme, and the active centre is exposed to the degree that displacement of dansylarginine NN-(3-ethylpentane-1,5-diyl)amide by D-Phe-Pro-Arg-CH2Cl is possible without disturbing the binding. Hirudin disrupts the affinity between thrombin and fibrin. These data indicate that the active centre of thrombin associated with fibrin through extended binding is fully exposed and freely accessible. It is possible that extended binding may play a regulatory role in the activation of Factor XIII by thrombin, as well as inactivation of this enzyme by antithrombin III.

Amino Acid Chloromethyl Ketones↗
PubMed2003

FGF-2 but not FGF-1 binds fibrin and supports prolonged endothelial cell growth.

Endothelial cell viability and growth are dependent on both polypeptide growth factors, and integrin-mediated matrix interactions. We have now examined the ability of fibrin-binding and non-binding growth factors to support long-term endothelial cell growth in the presence or absence of the soluble form. Endothelial cells were cultured on a fibrin surface, with or without FGF-1 or FGF-2, and proliferation was determined by (3)H-thymidine incorporation. Cells cultured on fibrin with no growth factor showed minimal proliferation up to 96 h. In contrast, when FGF-2 was incorporated into fibrin, proliferation was increased 6.5 +/- 0.6-fold, equal to growth on a fibrin surface with FGF-2 continually present in the medium. Thymidine incorporation was similar when cells were cultured on a fibrin surface that had been incubated with FGF-2 and then the growth factor removed (8.6 +/- 0.5-fold). In contrast to results with FGF-2, a surface of fibrin exposed to FGF-1 supported minimal growth, whereas growth was comparable to either FGF-1 or FGF-2 present in the medium. Comparable results were observed when proliferation was quantitated by cell counting at times up to 48 h. Binding studies demonstrated no high-affinity interaction of FGF-1 with fibrinogen or fibrin. We conclude that FGF-2 bound to fibrin supports prolonged endothelial cell growth as well as soluble FGF-2, whereas FGF-1 does not bind to fibrin and can support endothelial cell growth only if continually present in soluble form. Fibrin may serve as a matrix reservoir for FGF-2 to support cell growth at sites of injury or thrombosis.

Cell Division↗
PubMed1995

The role of the lysyl binding site of tissue-type plasminogen activator in the interaction with a forming fibrin clot.

To describe the role of the lysyl binding site in the interaction of tissue-type plasminogen activator (t-PA, FGK1K2P) with a forming fibrin clot, we performed binding experiments with domain deletion mutants GK1K2P, K2P, and the corresponding point mutants lacking the lysyl binding site in the absence and the presence of epsilon-amino caproic acid (EACA). Occupation of the lysyl binding site in the K2 domain with EACA has a pronounced effect on the binding of FGK1K2P to a fibrin clot (C50 = 77 +/- 11 nM versus 376 +/- 45 nM with EACA). Deleting the lysyl binding site in the K2 domain (substitution D236N) also impairs fibrin binding but to a lesser extent (C50 = 169 +/- 20 nM). Although the binding of K2P to a fibrin clot is weak (C50 = 1163 +/- 490 nM), it still is 2 orders of magnitude stronger than the binding of EACA to K2P. Therefore it was surprising to find that deletion of the lysyl binding site in K2P completely abolishes fibrin binding. Even when both the F domain and the lysyl binding site were deleted, considerable fibrin binding is still observed (C50 = 557 +/- 126 nM), suggesting other than F and K2-mediated interactions. The binding of FGK1K2P, FGK1K2P (D236N), GK1K2P, and GK1K2P (D236N) to fibrin could be competitively inhibited by FGK1K2P and K2P, indicating that all molecules recognize the same interaction sites on a fibrin clot. Based on these results, a new model for the interaction of t-PA with a forming fibrin clot is proposed. The fibrin binding sites in t-PA are not confined to the F and K2 domain. The main role of the lysyl binding site in the K2 domain of t-PA appears indirect rather than direct, most likely stabilizing a conformation favorable for fibrin binding.

Amino Acid Sequence↗
PubMed2001

Regulation of plasmin-dependent fibrin clot lysis by annexin II heterotetramer.

In a previous report we showed that plasmin-dependent lysis of a fibrin polymer, produced from purified components, was totally blocked if annexin II heterotetramer (AIIt) was present during fibrin polymer formation. Here, we show that AIIt inhibits fibrin clot lysis by stimulation of plasmin autodegradation, which results in a loss of plasmin activity. Furthermore, the C-terminal lysine residues of its p11 subunit play an essential role in the inhibition of fibrin clot lysis by AIIt. We also found that AIIt binds to fibrin with a K(d) of 436 nm and a stoichiometry of about 0.28 mol of AIIt/mol of fibrin monomer. The binding of AIIt to fibrin was not dependent on the C-terminal lysines of the p11 subunit. Furthermore, in the presence of plasminogen, the binding of AIIt to fibrin was increased to about 1.3 mol of AIIt/mol of fibrin monomer, suggesting that AIIt and plasminogen do not compete for identical sites on fibrin. Immunohistochemical identification of p36 and p11 subunits of AIIt in a pathological clot provides important evidence for its role as a physiological fibrinolytic regulator. These results suggest that AIIt may play a key role in the regulation of plasmin activity on the fibrin clot surface.

Animals↗
PubMed1995

Fibrin II induces endothelial cell capillary tube formation.

We studied the formation of capillary tubes by endothelial cells which were sandwiched between two fibrin gels under serum-free conditions. After formation of the overlying fibrin gel, the endothelial cell monolayer rearranged into an extensive net of capillary tubes. Tube formation was apparent at 5 h and was fully developed by 24 h. The capillary tubes were vacuolated, and both intracellular and intercellular lumina were present. Maximal tube formation was observed with fibrin II (which lacks both fibrinopeptide A and B), minimal tube formation with fibrin I (which lacks only fibrinopeptide A), and complete absence of tube formation with fibrin 325 (which lacks the NH2-terminal beta 15-42 sequence, in addition to fibrinopeptides A and B). The inability of fibrin 325 to stimulate capillary tube formation supports the idea that beta 15-42 plays an important role in this process, and its importance was confirmed by the finding that exogenous soluble beta 15-42 inhibited fibrin II-induced capillary tube formation. This effect was specific for fibrin, since beta 15-42 did not inhibit tube formation by endothelial cells sandwiched between collagen gels. The interaction of the apical surface of the endothelial cell with the overlying fibrin II gel, as opposed to the underlying fibrin gel upon which the cells were seeded, was necessary for capillary tube formation. These studies suggest that the beta 15-42 sequence of fibrin interacts with a component of the apical cell surface and that this interaction plays a fundamental role in the induction of endothelial capillary tube formation.

Amino Acid Sequence↗