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PubMed1985

Influence of fibrinogen on fibrin polymerization. Ultracentrifugation studies.

During the transformation of fibrinogen to fibrin, excess fibrinogen suppresses further polymerization of fibrin, thereby enabling the nascent fibrin to be transported in a soluble form in blood. The question of possible complex formation between fibrin and fibrinogen was addressed by analyzing fibrin/fibrinogen (1:20, mol/mol) mixtures in the presence of calcium ions in stable linear sucrose density gradients by ultracentrifugation at 37 degrees C. During the period of ultracentrifugation in independent experiments, 40% of desAA-fibrin and 30% of desAABB-fibrin, respectively, precipitated without the participation of fibrinogen. The desAABB-fibrin, recovered in the gradient fractions, appeared as high-molecular-weight polymers (22 S), whereas the recovered desAA-fibrin exhibited only a slight increase in molecular weight (9 S) compared to fibrinogen (8 S). In contrast to this finding, both types of fibrin were totally recovered in gradient fractions provided that fibrinogen was present in the gradient at a uniform concentration of 2 mg/ml. In addition, the presence of fibrinogen but not human serum albumin reduced the size of desAABB-fibrin polymers (17 S). However, stable fibrin-fibrinogen complexes could not be demonstrated, since cosedimentation of differently labelled desAABB-fibrin and fibrinogen was not detectable. These studies suggest a specific but weak interaction of the solubilizing fibrinogen with the soluble fibrin polymers as demonstrated by a rapid exchange of both macromolecules.

Centrifugation, Density Gradient↗
PubMed1962

Significance of cryoprofibrin in fibrinogen-fibrin conversion.

Fibrinogen altered by thrombin-catalyzed liberation of fibrinopeptide A was found to combine with native fibrinogen to form a cold-precipitable complex we have called "cryoprofibrin." The altered fibrinogen lacking fibrino-peptide A polymerized into fibrin, but not until conditions for equilibrium between its incorporation into both cryoprofibrin and fibrin were satisfied. At equilibrium, the concentration of cryoprofibrin was maintained at a threshold proportional to the concentration of fibrinogen. When the concentration of cryoprofibrin was below threshold, fibrin could be depolymerized and solubilized by fibrinogen with resultant formation of cryoprofibrin. Since threshold concentrations of cryoprofibrin appear necessary for precipitation of fibrin, the concentration of cryoprofibrin in plasma provides a basis for determining intravascular deposition of fibrin. Intravascular deposition of fibrin does not appear to occur normally in rabbits, because the concentration of cryoprofibrin in plasma from normal rabbits is far below the threshold for precipitation of fibrin. The applicability of cryoprofibrin as an indicator of fibrin deposition is demonstrated by the occurrence of levels of cryoprofibrin approaching the threshold for precipitation of fibrin in plasma from endotoxin-treated rabbits. The current concept that the fibrinogen molecule can dissociate into subunits can be used to explain the conversion of fibrinogen to cryoprofibrin. As one possibility, the two residues of fibrinopeptide A contained in fibrinogen may be located on two separate subunits of the molecule; cryoprofibrin is produced when one of these subunits is replaced by a subunit altered by loss of fibrinopeptide A. Recombination of native subunits with subunits altered by loss of A would counter dissociation of cryoprofibrin and inhibit polymerization of subunits lacking fibrinopeptide A. As an alternate mechanism, two residues of A may be liberated concurrently from a single subunit. Cryoprofibrin would then correspond to a fibrinogen molecule, containing a subunit with two residues of A, in combination with an altered molecule containing a subunit lacking two residues of A. Liberation of fibrinopeptide B did not contribute measurably to production of fibrin resulting from limited action of thrombin on rabbit fibrinogen. Both fibrin containing B but not A, and fibrin containing neither B nor A, as is produced by extensive action of thrombin, could be solubilized by fibrinogen. Thrombin, or another enzyme utilizing tosyl-L-arginine methyl ester as substrate, appeared reversibly to inhibit polymerization of fibrin containing fibrinopeptide B. This enzyme and fibrinogen were the only proteins appearing to inhibit polymerization in plasma from normal rabbits.

Arginine↗
PubMed2004

Fibrin-filled scaffolds for bone-tissue engineering: An in vivo study.

Recently, fibrin sealants that typically contain supra physiological concentrations of fibrinogen and thrombin have been investigated as matrices to facilitate the delivery of cells within biodegradable scaffolds for tissue engineering applications. It is well known from in vitro experiments that the thrombin concentration present during fibrin polymerization influences the structural properties of fibrin, and these can affect cell invasion. This study was conducted to determine whether the structural properties of fibrin can affect bony wound healing in vivo. Drill hole defects were created in the distal femurs of 20 rats. Four experimental groups were used: nontreated defects, scaffolds alone, and scaffolds filled with fibrin polymerized with either a low thrombin concentration [fibrin(low T)] or a high thrombin concentration [fibrin(high T)]. The area of bone formed at 2, 5, and 11 days after implantation was determined histomorphometrically. After 5 days, scaffolds filled with fibrin(high T) were infiltrated with less bone than empty scaffolds (p < 0.05), but no statistical difference was found between the empty scaffolds and the scaffolds filled with fibrin(low T). After 11 days, both fibrin-filled scaffolds significantly delayed bony wound healing (p < 0.004). Reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of the two fibrin formulations showed no difference in gamma-gamma crosslink formation. This work demonstrates that fibrin sealants in their present state are not ideal for enhancing bone-tissue invasion into scaffolds, and that the structural properties of fibrin matrices may be an important design parameter for maximizing host tissue invasion during wound healing.

Animals↗
PubMed1992

Determination of soluble fibrin: a comparison of four different methods.

The determination of soluble fibrin (SF) in plasma was compared using four different methods. The SF-ELISA immunologically measures the concentration of desAA- and desAABB-fibrin while the SF-tPA-test is based on activation of plasminogen by tissue plasminogen activator (tPA) in the presence of fibrin; the SF-PS-turbidimetry assay relies on the protamine sulphate (PS) -induced aggregation of fibrin in plasma whereas the SF-erythrocyte-agglutination-test (SF-EAT) detects soluble fibrin by its aggregation with fibrin monomers attached to test erythrocytes. Soluble fibrin was generated in vitro by addition of thrombin or ancrod to plasma. In these experiments the soluble fibrin values of the four methods correlated well with each other and with the fibrinopeptide A release, especially in ancrod-induced fibrinogen turnover (r greater than 0.93). This high correlation is remarkable, considering the fact that the methods are based on different principles. Detection of thrombin-induced soluble fibrin was more sensitive; differences between ancrod and thrombin action were observed as well, probably due to different forms of soluble fibrin. A delayed increase of SF-PS-turbidimetry values in particular during the thrombin action can be attributed to a lack of detectable aggregation of soluble fibrin at low concentrations due to its solubility in plasma. Subsequently, soluble fibrin was measured in samples from patients. The SF-ELISA and SF-tPA-test were highly sensitive and correlated better than the other methods with each other, but all correlations were less satisfactory compared with the in vitro studies. These weaker correlations might be explained by the heterogeneity of soluble fibrin determined by inter- and intraindividually varying concentrations of fibrinogen and its different derivatives in plasma samples from patients. All methods provided reliable results with differences in sensitivity, specificity and practicality. The SF-tPA-test, SF-PS-turbidimetry, and SF-EAT are practical methods for routine use whereas the SF-ELISA is a highly reliable and by far the most sensitive and specific method thus offering new insights into pathogenesis of fibrinaemia and related diseases.

Enzyme-Linked Immunosorbent Assay↗
PubMed1993

Thrombin treated plasma employed as a standard for determination of soluble fibrin.

The Coa-set Fibrin Monomer test (CFM-test) is a quantitative method for determination of soluble fibrin in plasma. The fibrin standard of the CFM-test is produced by bathroxobin conversion of purified fibrinogen to fibrin. Plasma treated with minute amounts of thrombin may be considered as a more physiological way of preparing fibrin monomers. Therefore, we have employed such thrombin treated plasma (TTP) as an alternative fibrin standard for the CFM-test. The fibrin concentration of the TTP was determined indirectly by quantitation of released fibrinopeptide A. The TTP was stable during freezing, thawing and storage for 3 months at -70 degrees C. The standard curve obtained using TTP as a standard was linear in the range of 0-275 nmol/l fibrin in plasma, but the slope of the line was less steep than the original standard curve. This difference was probably due to the greater plasminogen activating effect of bathroxobin digested fibrinogen compared to soluble fibrin generated in plasma by thrombin, as observed in a previous study. Because of the less steep slope of the alternative standard curve, fibrin levels in plasma samples from 20 healthy volunteers and 25 patients were found to be higher employing TTP as a standard. Preparation of a fibrin standard by incubation of plasma with minute amounts of thrombin will to some extent mimic the process of fibrin generation in vivo. Since we have found a satisfactory stability of such a standard during freezing, thawing and storage, we think the TTP standard might be useful for quantitation of soluble fibrin in plasma.

Blood Preservation↗
PubMed1988

Fibrin protofibril and fibrinogen binding to ADP-stimulated platelets: evidence for a common mechanism.

The molecular basis of platelet-fibrin binding has been elucidated by studying interactions between platelets and protofibrils, soluble two-stranded polymers of fibrin which are intermediates on the fibrin assembly pathway. The fibrinogen degradation product, fragment D, has been used to block fibrin assembly, thus enabling the preparation of stable solutions of short protofibrils, composed of fewer than twenty fibrin monomer molecules per polymer. Fibrin protofibrils bound to ADP-activated platelets in a time- and concentration-dependent process which was effectively blocked by excess unlabelled fibrinogen, i.e., the binding was specific and appeared to involve a common receptor. ADP-stimulated cells bound approx. 3 micrograms of fibrin protofibrils/10(8) platelets, compared to 4 micrograms of fibrinogen/10(8) cells, following a 30-min incubation period at room temperature. Binding of both ligands was inhibited by high concentrations of fragment D, further indicating a similar mechanism. The kinetic data obtained were well described by an apparent first-order mechanism in which the rate constant for fibrin protofibril binding was found to be 5-fold slower than that measured for fibrinogen. Two monoclonal antibodies, each directed against the platelet glycoprotein IIb-IIIa complex, inhibited the binding of fibrin protofibrils and fibrinogen in a similar, concentration-dependent manner, providing strong evidence for a common receptor. Binding of GPRP-fibrin (soluble fibrin oligomers formed in the presence of 1 mM Gly-Pro-Arg-Pro) to ADP-stimulated platelets was also inhibited by a monoclonal antibody directed against the GPIIb-IIIa complex. Neither fibrin protofibrils nor fibrinogen bound to Glanzmann's thrombasthenic platelets, which lack normal quantities of functional glycoprotein IIb-IIIa complex, further supporting the hypothesis that fibrinogen and fibrin bind to a common platelet receptor present on the glycoprotein IIb-IIIa complex.

Adenosine Diphosphate↗
PubMed2004

Comparative evaluation of absorbable hemostats: advantages of fibrin-based sheets.

Bioactive hemostats and wound dressings consist of either inherently active materials or act as delivery vehicles which contain such materials. Fibrin is a natural hemostat and scaffold, guiding the direction of wound contraction and closure. In order to improve the ease of application of liquid fibrin glue, we have made a freeze-dried form of polymerized fibrin that supports hemostasis and wound healing. The bleeding from the middle ear artery of rabbits was found to be arrested instantaneously on application of fibrin sheets, even when the animal was heparinized systemically. As the fibrin sheet was found to be fragile, gelatin was incorporated to the sheet and thus the mechanical stability was improved without compromising the hemostatic effect. The efficacy of the fabricated fibrin and fibrin-gelatin sheets to seal traumatized rat liver was compared with commercially available hemostats, Abgel (cross-linked gelatin) and Surgicel (cross-linked cellulose). Tissue compatibility of all the hemostats was studied by analyzing the liver tissue 15 days after application. While the hemostatic effect was best with fibrin and fibrin-gelatin sheets, both Surgicel and Abgel were not capable of arresting the bleeding quickly. Gross analysis of tissue on the 15th day of application, visibly, Abgel was not only degraded but resulted in severe adhesions of internal organs and histologically capsule formation around the implant was evident. Though Surgicel was also seen as cream soft material on the site of application that joined two pieces of liver, there was no adhesion of other internal organs and histologically, immune reaction and foreign-body-type giant cells were present in large amounts. Fibrin was not found grossly on application site whereas fibrin-gelatin was seen as a small white spot. Granulation tissue formation and cell migration into the fibrin-based sheets were evident, and therefore, fibrin-based sheets are not only efficient hemostats but showed optimum degradation and wound healing.

Animals↗
PubMed2004

Evidence that catalytically-inactivated thrombin forms non-covalently linked dimers that bridge between fibrin/fibrinogen fibers and enhance fibrin polymerization.

Phe-pro-arg-chloromethyl ketone-inhibited alpha-thrombin [FPR alpha-thr] retains its fibrinogen recognition site (exosite 1), augments fibrin/fibrinogen [fibrin(ogen)] polymerization, and increases the incorporation of fibrin into clots. There are two 'low-affinity' thrombin-binding sites in each central E domain of fibrin, plus a non-substrate 'high affinity' gamma' chain thrombin-binding site on heterodimeric 'fibrin(ogen) 2' molecules (gamma(A), gamma'). 'Fibrin(ogen) 1' (gamma(A), gamma(A)) containing only low-affinity thrombin-binding sites, showed concentration-dependent FPR alpha-thr enhancement of polymerization, thus indicating that low-affinity sites are sufficient for enhancing polymerization. FPR gamma-thr, whose exosite 1 is non-functional, did not enhance polymerization of either fibrin(ogen)s 1 or 2 and DNA aptamer HD-1, which binds specifically to exosite 1, blocked FPR alpha-thr enhanced polymerization of both types of fibrin(ogen) (1>2). These results showed that exosite 1 is the critical element in thrombin that mediates enhanced fibrin polymerization. Des B beta 1-42 fibrin(ogen) 1, containing defective 'low-affinity' binding sites, was subdued in its FPR alpha-thr-mediated reactivity, whereas des B beta 1-42 fibrin(ogen) 2 (gamma(A), gamma') was more reactive. Thus, the gamma' chain thrombin-binding site contributes to enhanced FPR alpha-thr mediated polymerization and acts through a site on thrombin that is different from exosite 1, possibly exosite 2. Overall, the results suggest that during fibrin clot formation, catalytically-inactivated FPR alpha-thr molecules form non-covalently linked thrombin dimers, which serve to enhance fibrin polymerization by bridging between fibrin(ogen) molecules, mainly through their low affinity sites.

Amino Acid Chloromethyl Ketones↗
PubMed1986

A model for the role of hyaluronic acid and fibrin in the early events during the inflammatory response and wound healing.

A model is presented outlining the molecular and cellular events that occur during the early stages of the wound healing process. The underlying theme is that there is a specific binding interaction between fibrin, the major clot protein, and hyaluronic acid (HA), a constituent of the wound extracellular matrix. This binding interaction, which could also be stabilized by other cross-linking components, provides the driving force to organize a three-dimensional HA matrix attached to and interdigitated with the initial fibrin matrix. The HA-fibrin matrix plays a major role in the subsequent tissue reconstruction processes. We suggest that HA and fibrin have both structural and regulatory functions at different times during the wound healing process. The concentration of HA in blood and in the initial clot is very low. This is consistent with the proposed interaction between HA and fibrin(ogen), which could interfere with either fibrinogen activation or fibrin assembly and cross-linking. We propose that an activator (e.g. derived from a plasma precursor, platelets or surrounding cells) is produced during the clotting reaction and then stimulates one or more blood cell types to synthesize and secrete HA into the fibrin matrix of the clot. We predict that HA controls the stability of the matrix by regulating the degradation of fibrin. The new HA-fibrin matrix increases or stabilizes the volume and porosity of the clot and then serves as a physical support, a scaffold through which cells trapped in the clot or cells infiltrating from the peripheral edge of the wound can migrate. The HA-fibrin matrix also actively stimulates or induces cell motility and activates and regulates many functions of blood cells, which are involved in the inflammatory response, including phagocytosis and chemotaxis. The secondary HA-fibrin matrix itself is then modified as cells continue to migrate into the wound, secreting hyaluronidase and plasminogen activator to degrade the HA and fibrin. At the same time these cells secrete collagen and glycosaminoglycans to make a more differentiated matrix. The degradation products derived from both fibrin and HA are, in turn, important regulatory molecules which control cellular functions involved in the inflammatory response and new blood vessel formation in the healing wound. The proposed model generates a number of testable experimental predictions.

Blood Coagulation↗
PubMed2003

A novel role of fibrin in epidermal healing: plasminogen-mediated migration and selective detachment of differentiated keratinocytes.

Recent studies have shown that fibrin promotes epidermal regeneration in vitro and maintains the stem cell population after transplantation of keratinocytes in vivo. As epidermal keratinocytes do not express integrin alpha(v)beta3, the receptor for fibrin and fibrinogen, the mechanism through which fibrin affects epidermal cells remains elusive. To investigate the role of fibrin in epidermal wound healing, we developed an in vitro model in which fibrin was added to the top of wounded keratinocyte monolayers grown on collagen. With this matrix topology, keratinocytes migrate between the collagen on their basal side and fibrin on their apical side mimicking migration of the epidermis in vivo. Using this model, we found that fibrin promoted keratinocyte migration in low and high calcium concentrations by exposing the cells to plasminogen. The migration rate depended strongly on the concentration of fibrinogen and the rate of plasmin-mediated fibrin degradation. Surprisingly, fibrin and fibrinogen caused significant detachment of keratinocytes which was prevented by the addition of calcium. Further examination using flow cytometry revealed that the detached cells were larger, more granular, and had very low levels of beta1 integrin, which are all signs of differentiated keratinocytes. Our results suggest a novel dual role of fibrin in epidermal healing. First, fibrin promotes keratinocyte migration indirectly by exposing plasminogen to migrating cells, and second, fibrin selectively disrupts adhesion of differentiated keratinocytes. Our data are novel and may have important implications in understanding wound healing and in the use of fibrin as a biomaterial for protein and gene delivery.

Calcium↗
PubMed2006

Autologous fibrin sealant (Vivostat) for mesh fixation in laparoscopic transabdominal preperitoneal hernia repair.

BACKGROUND AND STUDY AIMS: The use of fibrin glue derived from humans or animals has been reported as an alternative method of mesh fixation, instead of staples, in inguinal hernia repair. However, fibrin sealants involve the potential risks of virus transmission or immunological reactions to foreign proteins. This risk could be avoided by using autologous fibrin derived from the patient. A feasibility study on the use of autologous fibrin was therefore carried out in patients undergoing laparoscopic transabdominal inguinal hernia repair. PATIENTS AND METHODS: In a series of 10 patients undergoing laparoscopic transabdominal inguinal hernia repair, autologous fibrin was produced from 120 ml of the patient's blood during the hernia repair. The process took an average of 20 min. The perioperative and postoperative results were compared with those in a control group of 20 patients in whom conventional fibrin was used. RESULTS: Producing and applying the autologous fibrin was uncomplicated. No differences in the outcome were observed between the two groups. One patient in the conventional fibrin group developed a seroma. None of the patients reported persistent pain. No recurrences were observed after a mean follow-up period of 9 months (range 6 - 12 months) in the conventional fibrin group and 7 months (range 6 - 8 months) in the autologous fibrin group. CONCLUSIONS: This feasibility study suggests that autologous fibrin sealant allowed adequate mesh fixation that did not differ from that in a control group in whom conventional fibrin glue was used. Autologous fibrin may be an interesting alternative for a variety of laparoscopic and endoscopic applications.

Female↗
PubMed1999

Antifibrinolytic additives to fibrin glue for laparoscopic wound closure in urinary tract.

BACKGROUND AND OBJECTIVES: Fibrinolytic activity of urine may rapidly degrade fibrin glue used in the urinary tract, thereby limiting tissue adhesion. The goals of this study were to verify the ability of antifibrinolytic agents to delay the degradation of fibrin glue in the urinary tract and to assess the results of this delay on subsequent wound healing. MATERIALS AND METHODS: In 25 domestic pigs, a 3.5-cm incision in the urinary bladder was left open (N = 6) or closed laparoscopically with fibrin glue alone (N = 6), fibrin glue containing aprotinin 5000 KIU/mL (N = 6), or fibrin glue containing aprotinin 2500 KIU/mL with (N = 4) or without (N = 3) aminocaproic acid 12.5 mg/mL. At harvest 7 days later, the bladder was tested for leakage. Histologic features were scored by a pathologist blinded to the closure method. RESULTS: There were no significant differences among the groups in the amount of leakage at harvest. Significant fibrin glue material in the wound was noted more often in the pigs treated with fibrin glue plus aprotinin (7 of 13) than in the fibrin glue-only group (0 of 6; P = 0.04). The presence of significant fibrin material in the wound correlated well with absence of granulation tissue (P < 0.001), such that granulation tissue bridging the wound edges was found more often in the fibrin glue-only group (6 of 6) than in the groups treated with fibrin glue plus aprotinin (4 of 13; P = 0.01). CONCLUSIONS: Although aprotinin +/- aminocaproic acid did delay the degradation of fibrin glue used to close a bladder wound, it was associated with inhibition of granulation tissue in the glued wound. These findings suggest that aprotinin alone and aprotinin plus aminocaproic acid are not useful additives to fibrin glue used for wound closure in the urinary tract.

Aminocaproates↗
PubMed2005

Infection-stimulated fibrin deposition controls hemorrhage and limits hepatic bacterial growth during listeriosis.

Bacterial infections are major causes of human mortality. The activation of coagulation pathways leading to the deposition of insoluble fibrin frequently accompanies bacterial infection, and much attention has focused upon the pathological attributes of infection-stimulated fibrin deposition. Nevertheless, here we present conclusive evidence that infection-stimulated fibrin deposition can perform critical protective functions during bacterial infection. Specifically, we demonstrate that coagulation-impaired fibrin(ogen)-deficient mice, in comparison with genetically matched control mice, display increased mortality upon peritoneal infection with the gram-positive facultative intracellular bacterium Listeria monocytogenes. To distinguish effects of fibrinogen from those of fibrin, we treat wild-type mice with warfarin, an anticoagulant that suppresses fibrin formation without impacting fibrinogen levels. Warfarin treatment exacerbates listeriosis, suggesting that fibrin is the key mediator of protection. With regard to the underlying protective mechanisms, we demonstrate that fibrin(ogen) suppresses anemia, reduces hemorrhagic pathology, and limits bacterial growth during listeriosis. Despite confirming a prior report that fibrin(ogen) promotes the peritoneal clearance of the extracellular bacterium Staphylococcal aureus, we demonstrate that fibrin(ogen) plays little role in controlling peritoneal numbers of L. monocytogenes bacteria or the dissemination of L. monocytogenes bacteria from the peritoneal cavity. Rather, fibrin(ogen) primarily limits the growth of these intracellular bacteria within hepatic tissue. While the pathological potential of excessive infection-stimulated fibrin deposition is well appreciated, our findings reveal that fibrin can function protectively, via multiple mechanisms, during bacterial infection.

Animals↗
PubMed2000

Fibrin(ogen)-induced expression of ICAM-1 and chemokines in human synovial fibroblasts.

It has long been recognized that in most inflamed arthritic joints the coagulation system is activated, leading to the local generation of fibrin, and it has long been hypothesized that the local fibrin deposition promotes inflammation and tissue destruction. However, only recently has the direct effect of fibrin on the inflammatory process been seriously investigated, and specific roles assigned to fibrin or its products as mediators of the inflammatory process. Although fibrin and/or fibrinogen (fibrin(ogen)) is abundantly present in inflamed tissues and joints rich in fibroblastic cells, no significant data on fibrin(ogen)-induced gene expression by fibroblasts have been published. We now demonstrate that coculture of human synovial fibroblasts with fibrin(ogen) results in the up-regulation of ICAM-1 as well as increased production of the chemokines IL-8 and growth-related oncogene-alpha. Increased ICAM-1 expression was fibrin(ogen) dose-dependent and was demonstrated by ELISA, flow cytometry, and functional adhesion assays. Levels of ICAM-1 induced by fibrin(ogen) were comparable to those that could be induced by cytokine stimulation. Fibrin(ogen) stimulation of ICAM-1 could be suppressed by pyrrolidinedithiocarbamate, an inhibitor of NF-kappaB activation. Chemokine production was induced by fibrin(ogen) in cell culture supernatants >100-fold as compared with controls. Thus, through its activation of synovial fibroblasts, fibrin(ogen) deposition may promote the recruitment (via chemokines) and retention (via adhesion molecules) of lymphocytes within the arthritic joint.

Antioxidants↗
PubMed1986

Macrophage migration in fibrin gel matrices.

Macrophage migration has been extensively studied in vitro on artificial substrates but in vivo macrophages migrate through connective tissue and fibrin gel meshworks that comprise the stroma of many inflammatory reactions and solid tumors. Studies were therefore undertaken to investigate macrophage migration in this more biologically relevant matrix cast either with or without nitrocellulose filter support. Macrophage migration in fibrin gels depended on both fibrin and thrombin concentrations and on the nature of fibrin crosslinking. With gamma-chain crosslinking only, macrophage migration was enhanced as compared with untreated filters at fibrin concentrations of up to 5 mg/ml. However, when fibrin alpha-chains were also crosslinked, as occurs in vivo, macrophage migration was inhibited at approximately 3 mg/ml fibrin and was stopped altogether at 5 mg/ml. Thrombin concentrations of 1 unit/ml favored maximal macrophage migration. Depletion of contaminating fibronectin did not affect these results. The pore size of fibrin gels is well below the minimum that permits macrophage migration through nitrocellulose filters. Thus, to penetrate fibrin gels, macrophages could enlarge effective pore size by local fibrinolysis or by pushing apart fibrin strands. If fibrinolysis is responsible, this process must be highly localized because only small amounts of fibrinolysis accompanied extensive macrophage migration in fibrin gels; moreover, fibrinolysis inhibitors did not affect macrophage migration. We conclude that the fibrin deposited in inflammatory reactions and solid tumors is present in forms and concentrations that either facilitate or inhibit macrophage migration; fibrin may therefore regulate macrophage participation in these and other pathologic reactions.

Animals↗
PubMed1994

Further characterization of the NH2-terminal fibrin-binding site on fibronectin.

The fibronectin (Fn) monomer contains two major sites of fibrin binding affinity present within the NH2-terminal and COOH-terminal domains; they consist of five (1F1-5F1) and three (10F1-12F1) consecutive type 1 modules, respectively. Recently, we have reported that the fourth and fifth type 1 module pair (4F1.5F1) of the NH2-terminal domain of fibronectin demonstrated fibrin binding ability (Williams, M. J., Phan, I., Harvery, T. S., Rostagno, A., Gold, L. I., and Campbell, I. D. (1994) J. Mol. Biol. 235, 1303-1311). In an attempt to further localize fibrin binding activity and to characterize the nature of the interaction between different type 1 modules of Fn and fibrin, we have tested a range of recombinant proteins and subtilisin generated proteolytic fragments of Fn in an enzyme-linked immunosorbent assay (ELISA) and by fibrin affinity chromatography. Of the recombinant proteins, we found that only the 4F1.5F1 exhibited significant fibrin binding activity, while 1F1, 1F1.2F1, 7F1, and 10F1 had little to no affinity for fibrin. On a molar basis, 4-5 times more 4F1.5F1 than a proteolytic fragment, corresponding to 1F1-5F1 (25.9 kDa) was required to cause 50% inhibition (IC50) of intact biotinylated Fn binding to fibrin in a competitive ELISA. This suggests that all five type 1 modules in tandem engender higher fibrin binding activity than the 4F1.5F1 alone. Furthermore, since fibrin binding activity of the intact Fn molecule was inhibited, by 70-80%, by the 4F1.5F1, the 25.9-kDa fragment, and a MoAb mapped to an epitope on the 4F1.5F1, the fibrin-binding site within the 4F1.5F1 contributes greatly to the non-covalent interaction of intact Fn with fibrin. These results provide significant insight into the Fn/fibrin interaction, a major component of the processes of wound repair and fibrin matrix assembly.

Amino Acid Sequence↗
PubMed1998

Fibrin deposition in squamous cell carcinomas of the larynx and hypopharynx.

Extravascular fibrin deposition is frequently observed within and around neoplastic tissue and has been implicated in various aspects of tumor growth. The distribution of fibrin deposits was investigated in squamous cell carcinomas representing different stages of tumor progression of the larynx (n = 25) and hypopharynx (n = 9) by immunofluorescent techniques. Double and treble labelings were used to detect fibrinogen and fibrin in combination with marker antigens for tumor cells (cytokeratin), endothelial cells (von Willebrand factor), macrophages (recognized by KiM7), as well as factor XIII subunit A (FXIIIA) and tenascin (an embryonic extracellular matrix protein newly expressed during tumorigenesis). All tissue samples showed specific staining for fibrinogen/fibrin. Fibrin deposition was localized almost exclusively in the connective tissue compartment of tumors with characteristic accumulation at the interface of connective tissue and the tumorous parenchyma. In certain tumor samples showing highly invasive characteristics, fibrin deposits were observed in close association with tumor blood vessels in the tumor cell nodules. The overlapping reactions with polyclonal antibody to fibrinogen/fibrin and monoclonal antibody to fibrin indicate the activation of the coagulation cascade resulting in in situ thrombin activation and fibrin formation. Fibrin was crosslinked and stabilized by FXIIIA as revealed by urea insolubility test. Accumulation of phagocytozing macrophages detected by Ki M7 monoclonal antibody could be seen in areas of fibrin deposition. The blood coagulation factor XIIIA was detected in and around the cells labeled with Ki M7 antibody. Tenascin and fibrin deposits were found in the same localization in the tumor stroma and in association with tumor blood vessels within the tumor cell nodules. Neither fibrin nor tenascin were detected in the histologically normal tissue adjacent to tumors. The close association between fibrin deposits and macrophage accumulation strongly suggests the active participation of tumor-associated macrophages in the formation of stabilized intratumoral fibrin that facilitates tumor matrix generation and tumor angiogenesis.

Adult↗
PubMed1983

Inhibition of fibrin polymerization by fragment d is affected by calcium, Gly-Pro-Arg and Gly-His-Arg.

Fibrinopeptides A and B were removed from purified human fibrinogen by bovine thrombin, whereas the snake venom protease batroxobin only split fibrinopeptide A from fibrinogen. Aggregation of the resulting desAB- and desA-fibrin monomers was evaluated by recording the turbidity of incubation mixtures. Fibrin assembly was strongly accelerated by increasing the calcium concentration from 10(-5) to 10(-3) M. Fragment D was obtained from fibrinogen by proteolytic degradation with plasmin in the presence of Ca2+. At a 4-fold molar concentration relative to fibrinogen, fragment D dramatically inhibited fibrin polymerization at up to 10(-4) M Ca2+. This anticlotting activity was, however, much less pronounced at 10(-3) M Ca2+. The thrombin clotting time, measured on human plasma, was prolonged by fragment D in a dose-dependent manner. In citrate-containing plasma, the fibrinogen clotting was significantly delayed by an equimolar concentration of fragment D. In barium sulfate-adsorbed oxalated plasma, containing 2.5 mM Ca2+, the same amount of fragment D hardly affected fibrin polymerization. We conclude that fragment D has no important anticlotting effect under physiological conditions. The synthetic peptide Gly-Pro-Arg, corresponding to the amino-terminal sequence of the fibrin alpha-chain, inhibited aggregation of both desA-fibrin and desAB-fibrin at 10(-3) M Ca2+. The inhibition of desAB-fibrin polymerization by Gly-Pro-Arg was abolished at 10(-5) M Ca2+. In addition, Gly-Pro-Arg depressed the anticlotting activity of fragment D at low calcium concentration. An analogue of the amino-terminus of fibrin beta-chain, Gly-His-Arg, strongly accelerated aggregation of desA-fibrin monomers, but only moderately enhanced polymerization of desAB-fibrin monomers at 10(-5) M Ca2+, both in the presence and in the absence of fragment D. This activating effect of Gly-His-Arg was abolished at 10(-3) M Ca2+. It is suggested that the binding of calcium, Gly-His-Arg, and possibly also Gly-Pro-Arg, induces a conformational change in fibrin monomers and thus accelerates the polymerization process.

Animals↗