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At least 19 recordsLinked to original sources

PubMed1990

Comparison of the effects of fibrinogen and fibrin products and isolated peptide chains on the fibrin-mediated stimulation of plasminogen activation by tissue-type plasminogen activator, and on the fibrin-dependent enhancement of the amidolytic activity of one-chain tissue-type plasminogen activator.

Intact fibrin monomer, the early fibrin degradation product (X-fragment), late fibrinogen degradation products (fragments D and E), fibrinogen cyanogen bromide fragment FCB-2, and isolated peptide chains of fibrinogen and fibrin were investigated for their ability to replace fibrin in the stimulation of one-chain tissue-type plasminogen activator. They were also investigated for their ability to stimulate plasminogen activation by one-chain tissue-type plasminogen activator, which occurs via ternary complex formation. The stimulatory effect of the different fibrin/ogen products decreased in the order: fibrin X-fragment greater than fibrin monomer greater than CNBr-fragment FCB-2 greater than fibrin alpha-chain. Fibrin beta/gamma-chains and fibrinogen peptide chains were found to be weak stimulators. Fibrinogen fragments D and E have almost no effect. The amidolytic activity of one-chain tissue-type plasminogen activator was stimulated by intact fibrin monomer and somewhat more strongly by fibrin X-fragment. This stimulation by fibrin monomer, which occurred via an increase in the kcat value, was competitively inhibited by isolated fibrin alpha-chain (Ki = 0.12 mumol/l). The results show that the fibrin-mediated stimulation of plasminogen activation occurs when both one-chain tissue-type plasminogen activator and plasminogen are bound to fibrin, and that this process is essentially independent of the conformation of the fibrin molecule. In comparison, the fibrin-dependent stimulation of the amidolytic activity of one-chain tissue-type plasminogen activator is a more complex process, which depends on the correct conformation of the fibrin molecule.

Amino Acid Sequence↗

PubMed2000

[Production and clinical application of fibrin sealant with high concentrated fibrin (fibrin patch)].

OBJECT: The sealing properties of fibrin sealant with high concentrated fibrin (fibrin patch) were examined. MATERIAL AND METHODS: A commercial fibrin sealant (Bolheal) produced from pooled human plasma was utilized for this study. The fibrin sealant made of fibrinogen and thrombin solutions mixed in a volume ratio 5:1 was applied as the fibrin sealant with high concentrated fibrin (fibrin patch). The burst pressure of the fibrin clots of either 1:1 or 5:1 mixing ratio, which sealed the small holes, was measured by a water-leak preventing model. The tensile strength of the fibrin patch was measured by the breaking pressure of the fibrin clot. The burst pressure of the fibrin patch, which sealed the dural defect with a diameter of 15 mm, was compared with that of expanded polytetrafluoroethylene (ePTFE). RESULTS: The burst pressure was elevated from 287 +/- 23.1 to 445 +/- 30.5 mmHg by changing the mixing ratio from 1:1 to 5:1. The breaking pressure of the fibrin patch showed 131 +/- 25.4 mmHg and that of the patch mixed at the ratio of 1:1 showed 46.6 +/- 9.9 mmHg. The result of dural repair with the fibrin patch revealed higher sealing effectiveness than that of ePTFE. The burst pressure averaged 70.5 +/- 21.4 mmHg in the fibrin patch samples and 51.4 +/- 13 mmHg in the ePTFE samples. CONCLUSION: The fibrin patch revealed higher performance as a sealant and has the potential to be a candidate for acceptance as the new dural repair material.

Brain↗

PubMed1985

Quantitative assessment of soluble fibrin in plasma by affinity chromatography--a comparative study with desAA-fibrin, desAABB-fibrin and fibrinogen.

A quantitative determination of soluble fibrin in plasma was carried out by affinity chromatography. For this purpose, desAA-fibrin and fibrinogen immobilized on Sepharose 4B were used at the stationary side whereas batroxobin-induced 125I-desAA-fibrin or thrombin-induced 125I-desAABB-fibrin mixed with plasma containing 131I-fibrinogen represented the fluid phase. The binding characteristics of these mixtures to the immobilized proteins were compared at 20 degrees C and 37 degrees C. Complete binding of both types of fibrin to the immobilized desAA-fibrin was always seen at 20 degrees C as well as at 37 degrees C. However, binding of soluble fibrin was accompanied by substantial binding of fibrinogen that was more pronounced at 20 degrees C. Striking differences depending on the temperature at which the affinity chromatography was carried out, were documented for the fibrinogen-fibrin interaction. At 20 degrees C more than 90% of the applied desAA-fibrin was bound to the immobilized fibrinogen whereas at 37 degrees C only a mean of 17% were retained at the fibrinogen-Sepharose column. An opposite finding with regard to the tested temperature was made with the desAABB-fibrin. Nearly complete binding to insolubilized fibrinogen was found at 37 degrees C (95%) but only 58% of the desAABB-fibrin were bound at 20 degrees C. The binding patterns did not change when the experiments were performed in the presence of calcium ions. The opposite behaviour of the two types of soluble fibrin to immobilized fibrinogen at the different temperatures, together with the substantial binding of fibrinogen in the presence of soluble fibrin to insolubilized fibrin in every setting tested, devaluates affinity chromatography as a tool in the quantitative assessment of soluble fibrin in patient's plasma.

Body Temperature↗

PubMedDate not supplied

Quantification of fibrin deposition in flowing blood with peroxidase-labeled fibrinogen. High shear rates induce decreased fibrin deposition and appearance of fibrin monomers.

To study fibrin incorporation into thrombi at different wall shear rates, a new method to study fibrin deposition on extracellular matrixes underlying stimulated endothelial cells under flow conditions was developed. For this method, we used fibrinogen labeled with peroxidase (Fg-PO). Fg-PO was fully exchangeable for Fg in the clotting assays tested, and PO activity was bound to fibrin-specific fragments. Fg-PO containing fibrin could be stained for microscopic studies with 3,3'-diaminobenzidine and could be quantified by oxidation of phenylenediamine. The absorbance values at 492 nm were converted to fibrin quantities via a standard curve. To study fibrin deposition, Fg-PO was added in trace amounts to whole blood anticoagulated with low-molecular-weight heparin, and perfusion studies were performed over endothelial cell matrixes containing tissue factor. In parallel perfusion studies, 125I-labeled Fg was added in trace amounts to whole blood instead of Fg-PO. Both quantitative methods demonstrated decreased fibrin deposition after perfusions at 1,300 sec-1 compared with fibrin deposition after perfusions at 300 sec-1, while fibrinopeptide A generation was independent of the wall shear rate. The decrease in fibrin deposition at 1,300 sec-1 was accompanied by the appearance of fibrin monomers in the perfusate. This suggested that the decrease in fibrin incorporation at 1,300 sec-1 was due to the impaired polymerization of fibrin monomers. This impairment was probably due to a decrease in local fibrin monomer concentration as a result of the increased removal of monomers from the surface at 1,300 sec-1.

Antibodies, Monoclonal↗

PubMed1997

Fibrinogen and fibrin polymerization: appraisal of the binding events that accompany fibrin generation and fibrin clot assembly.

Fibrinogen is a complex multifunctional protein comprised of three major domains (two outer D and one central E) which contains constitutive binding sites (e.g. Da, Db, gammaXL, D:D, gamma', thrombin substrate, platelet receptor) as well as binding sites that become exposed or expressed as a result of fibrinogen proteolysis by thrombin and/or that are exposed as a consequence of the polymerization process itself (tPA binding sites). Fibrin-dependent tPA-mediated activation of plasminogen is associated with exposure of polymerization-dependent epitopes (Aalpha148-160, gamma312-324) that are expressed in assembled fibrin and in crosslinked (polymerized) fibrinogen but not in unpolymerized fibrinogen or fibrin. Fibrin polymerization is initiated by thrombin cleavage of fibrinopeptide A from fibrinogen Aalpha chains, exposing two E domain E(A) sites. Cleavage of fibrinopeptide B from fibrinogen Bbeta chains exposes other E domain polymerization sites, termed E(B), that also interact with platelets, fibroblasts and endothelial cells. Fibrin generation is followed by an assembly process of intermolecular end-to-middle D to E associations to form linear and branched double-stranded fibrin fibrils, lateral fibril-fibril associations to form fibers and a branched fiber network. Binding sites in fibrinogen play their roles in fibrin assembly by self-association (gammaXL to gammaXL and D:D to D:D) or by complementary association with exposed sites in fibrin (Da to E(A) and Db to E[B]). Other binding sites in fibrinogen include thrombin substrate recognition sites in each E domain and a non-substrate high affinity thrombin binding site in the carboxy-terminal region of each gamma' chain, which also binds plasma factor XIII. Fibrin possesses low affinity thrombin binding sites in each E domain and retains the gamma' chain nonsubstrate thrombin-binding site.

Animals↗

PubMed2001

Fibrin degradation products, fibrin monomer and soluble fibrin in disseminated intravascular coagulation.

Disseminated intravascular coagulation (DIC) is characterized by activation of hemostasis and fibrinolysis resulting in the formation of thrombin and plasmin, and the characteristic effects of these enzymes on plasma fibrinogen can be useful in diagnosis. Thrombin cleaves fibrinopeptides from fibrinogen, forming fibrin monomer that rapidly polymerizes to form a clot. Small amounts can circulate in plasma as "soluble fibrin," which may have a complex composition and include fibrinogen and a variable amount of cross-linking. Plasmic degradation of cross-linked fibrin forms a heterogeneous group of degradation products reactive in assays for D-dimer, and their levels provide a measure of the amount of fibrin formation and lysis. Caution should be exercised in comparing quantitative results using different assays because of problems with standardization and variable reactivity with different molecular forms. Marked elevations of fibrin(ogen) degradation products are a constant finding in experimental animal models of DIC. In human models of DIC resulting from endotoxin infusion, D-dimer is elevated early and high levels persist, reflecting lysis of microvascular fibrin deposits. Elevated levels of D-dimer and soluble fibrin are very sensitive for the diagnosis of DIC, and a normal level has a high negative predictive value. Serial monitoring of soluble fibrin or D-dimer assays may be of value in evaluating the response to therapy and possibly in identifying at-risk patients.

Animals↗

PubMed1997

Platelet adhesion to fibrinogen, fibrin monomer, and fibrin protofibrils in flowing blood -- the effect of fibrinogen immobilization and fibrin formation.

Platelet fibrin(ogen) adhesive interactions were investigated in whole citrated blood using the rectangular perfusion chamber at wall shear rates of 300 and 1600 s(-1) with regard to the amount and structure of immobilized protein. Only single platelets adhered to adsorbed fibrinogen at both low and high surface fibrinogen concentrations and at 1600 s(-1) almost no adhesion was observed. When using spray-immobilized protein, platelet adhesion was significantly higher than to adsorbed protein. Conversion of adsorbed fibrinogen to fibrin monomer resulted in the formation of pronounced platelets aggregates and with the elevation of wall shear rate 50% decrease of adhesion took place. Degree of platelet adhesion to fibrin monomer was significantly influenced by immobilized protein concentration at both shear rates. However, the morphology (small and dense platelet aggregates) and extent of platelets adhered to fibrin pentamer was nearly the same at both shear rates. Starting with surface-bound fibrinogen and alternating addition of thrombin and fibrinogen fibrin pentamer was prepared using the stepwise synthesis. This methodology is based on the observation that at low concentration immobilized fibrin monomer binds fibrinogen in 1:1 molar ratio. The gradually formed fibrin of a defined size and composition can be a useful tool in the further understanding of the role of fibrin architecture in the pathophysiology of thrombosis.

Adsorption↗

PubMed1994

Important factors influencing the strength of autologous fibrin glue; the fibrin concentration and reaction time--comparison of strength with commercial fibrin glue.

Fibrin glue was prepared from citrated plasma of human donors by means of ethanol. The outcome was a fibrinogen concentrate with a mean concentration of 43 mg/ml. The fibrinogen was converted to fibrin by the addition of 0.3 part of thrombin solution, 150 NIH U/ml, containing 100 mM calcium chloride. In a rat model full-thickness skin grafts were sealed with the glue, and the adhesive strength was measured at different fibrin concentrations, and after a variable reaction time, and compared to commercial fibrin glue (Tisseel). The strength of ethanol-prepared glue was directly proportional to the fibrin concentration, and increased rapidly within the first minutes of the reaction time. The strength of the commercial glue could be obtained with autologous fibrin glue at the same fibrin concentration.

Animals↗

PubMed2002

A novel monoclonal antibody to fibrin monomer and soluble fibrin for the detection of soluble fibrin in plasma.

BACKGROUND: Soluble fibrin (SF), composed of fibrin monomer (FM) and fibrinogen, is well known to exist in the circulating blood derived from patients with thrombotic diseases, and its quantification is useful to get some information on the state and degree of intravascular coagulation. However, there was no convenient method for the determination of SF. METHODS: We prepared a novel monoclonal antibody (MoAb) (F405) to FM and SF using desAA-fibrin as the immunogen in the presence of anti-polymerant peptide (Gly-Pro-Arg-Pro, GPRP), and the characterization of the F405 was performed by Western blotting analysis and an enzyme-linked immunosorbent assay (ELISA). We also tried to detect SF in human plasma using an ELISA involving the immobilized F405 and horseradish peroxidase (POD)-labeled anti-fibrinogen polyclonal antibody. RESULTS: The antibody reacted with the fibrin degradation products fragments X, Y and E, but not with fibrinogen or its fragments X, Y, D and E, or the fibrin D-dimer. The epitope recognized by F405 appeared to be the alpha-chain N-terminal region exposed upon removal of the A peptide from the Aalpha-chain because F405 was found to bind to the alpha-chain N-terminal oligo-peptide of fibrin (GPRVVERHQ). Since F405 reacted not only with FM in the presence of GPRP peptide, but also with the SF complex prepared by the addition of thrombin-treated FM to human fibrinogen, we attempted to detect SF in human plasma using ELISA. The analytical range of this method was 1-300 microg/ml. The assay detection limit was < 0.5 microg/ml, and the results of intra- and inter-assay precision studies indicated that this method is accurate and yields reproducible results (< 9.4% and < 10%, respectively). When 56 samples of plasma from patients with disseminated intravascular coagulation (DIC) and 117 control samples from healthy individuals were tested, elevated levels of SF complex were detected in the DIC samples: the mean +/- S.D. of the SF concentration in the DIC and control samples were 63.4 +/- 65.3 microg/ml and 1.9 +/- 1.0 microg/ml, respectively. CONCLUSIONS: The ELISA using F405 is useful for the diagnosis of DIC.

Animals↗

PubMed1985

Clearance characteristics of des-AA fibrin and des-AABB fibrin, and thrombus-related uptake of des-AABB fibrin as compared to fibrinogen.

The following paper presents a short review of previous studies relating to the behaviour in man of radiolabelled fibrins des-AA and des-AABB, as compared to that of radiolabelled fibrinogen. Des-AA fibrin was eliminated with a half-life of 30 to 60 min in eight healthy controls, but its half-life was substantially shorter in eight fibrinaemic patients with no demonstrable fibrinolysis. Clearance of des-AABB fibrin was studied in thirteen patients with established venous thrombosis, all subjected to a concomitant fibrin(ogen) uptake test. There was no essential difference in its half-life in patients with a positive fibrinogen uptake test (n = 7) as compared to those with a negative test (n = 6). The metabolic half-life of des-AABB fibrin was 10 +/- 3.5 hrs. The uptake of labelled des-AABB fibrin by thrombi was similar to that of labelled fibrinogen during the first hours after injection, but only fibrinogen could reflect a continuous build-up of thrombi, due to its longer survival time.

Fibrin↗

PubMed2003

Dynamic changes of fibrin architecture during fibrin formation and intrinsic fibrinolysis of fibrin-rich clots.

Clotting and fibrinolysis are initiated simultaneously in vivo, and fibrinolysis usually occurs without any individualized lysis front (intrinsic fibrinolysis). We have developed a novel model to assess whether morphological changes resulting from intrinsic fibrinolysis are similar to those previously reported at the lysis front using externally applied lytic agents. Fibrin assembly and fibrinolysis were followed in real-time by confocal microscopy using gold-labeled fibrinogen molecules. An increase in fiber absorbance (30%, p < 0.01) and a decrease in fiber diameter (60%, p < 0.01) due to the ongoing accumulation and packing of fibrin molecules were the most significant detectable features occurring during fibrin assembly. Similar features with a similar magnitude were observed during fibrin dissolution, but in the reverse order and with a 3-fold increase in duration. Then, lysing fibers were progressively transected laterally, and thinner fibers were cleaved at a 2.5-fold faster rate than thicker fibers (p < 0.001). Frayed lysing fibers were seen to interact progressively with adjoining fibers (agglomeration), leading to a 76 and 88% increase in the network pore diameter (p < 0.05) and fiber diameter (p < 0.01), respectively. At the maximum decrease in fiber absorbance (46%, p < 0.05), the network suddenly collapsed with the release of large fragments that gradually vanished. Morphological changes of fibrin that occur during intrinsic fibrinolysis are similar as those observed next to the lysis front, although they are not restricted spatially to the clot/surrounding milieu interface but are observed through the entire clot.

Algorithms↗

PubMed1986

Soluble fibrin consists of fibrin oligomers of heterogeneous distribution.

Soluble fibrin is observed in patients with intravascular coagulation and represents an intermediary product of conversion of fibrin monomers into a fibrin clot whereby the presence of fibrinogen may suppress fibrin clot formation. The interactions between fibrin and fibrinogen and the occurrence of fibrin oligomers in soluble fibrin were studied by sucrose density ultracentrifugation. Different concentrations of soluble fibrin, prepared by mixing 125I-fibrin (24 nM - 1.5 microM) with a constant concentration of 131I-fibrinogen (6 microM) were analyzed at 37 degrees C in stable linear sucrose density gradients containing a uniform concentration of unlabelled fibrinogen (6 microM) and calcium ions in order to mimic the physiological situation. At any fibrin concentration, 125I-fibrin sedimented faster than 131I-fibrinogen through 5-30% (w/v) sucrose gradients. Sedimentation rates of fibrin increased from 9 S to 23 S depending on the initial fibrin concentration. The relative amount of residual fibrin monomer not incorporated into oligomers was calculated from the sedimentation profiles. At any fibrin concentration, the portion of free monomer was always more than twofold higher for batroxobin-generated (desAA-) fibrin than for thrombin-generated (desAABB-) fibrin. Apparent association constants for desAABB-fibrin were 3-10 times higher than those for desAA-fibrin indicating a stronger interaction between monomers of the former type of fibrin. In the presence of excess fibrinogen the predominant species in soluble desAA-fibrin were monomers and dimers, whereas dimers, trimers and higher-molecular-mass oligomers were present in soluble desAABB-fibrin. Strong interactions between both types of fibrin were demonstrated from their cosedimentation, whereby the size of these copolymers were shown to be governed by the oligomer size of the desAABB-fibrin type. These results provide evidence for the occurrence of differently sized oligomers of fibrin in soluble fibrin and for the concept of a cooperative polymerization process between both types of fibrin devoid of any stable complexes between fibrin and fibrinogen.

Biopolymers↗

PubMed1980

Fibrinogen-fibrin conversion. The mechanism of fibrin-polymer formation in solution.

The fibrin polymers formed in solution during the earliest phase of the fibrinogen-fibrin conversion are shown to be stable soluble molecules at pH7.4 and 0.15m- or 0.3m-NaCl. The various sequential soluble fibrin polymers produced from the fibrinogen-thrombin reaction can be observed by gel chromatography and can be isolated for characterization. The mechanism of fibrin polymerization proposed from the present studies suggests that the initial event is the thrombin activation at only one of the Aalpha-chains in fibrinogen. The resulting highly reactive intermediate is the true fibrin monomer and it rapidly, and irreversibly, self-associates to form the stable fibrin dimer (s(20.w)=12S). Fibrin dimer possesses the N-terminal pattern alanine/glycine/tyrosine (1:1:2) per 340000 molecular weight, and possesses the chain structure [(alpha)Aalpha)(Bbeta)(2)(gamma)(2)](2). The fibrin dimer is a soluble inert molecule, but additional thrombin activation of its remaining intact Aalpha-chains leads to new associations into larger inert soluble fibrin polymers. In this manner progressively larger fibrin oligomers are constructed with thrombin continually in control of the process because of the necessity to repeatedly re-activate the various fibrin polymers in solution. The inert character of the soluble fibrin polymers can be explained by the reciprocal alignment of the associating molecules, which mutually consumes their active surfaces and leaves an intact Aalpha-chain at either end of each fibrin oligomer. The soluble fibrin polymers will proceed to further association only if thrombin activates these remaining Aalpha-chains, otherwise the fibrin molecules are stable indefinitely. The intermolecular associations within the soluble fibrin polymers are essentially irreversible under these nearly physiological conditions. However, the bonding is not covalent. This mechanism accounts for the clinical observations of stable fibrinogen-derived polymers in the plasma from patients undergoing thrombotic processes. Since it is shown that the intermediate fibrin polymers, themselves, are stable soluble molecules, it is no longer necessary, nor warranted, to invoke hypothetical ;fibrinogen-fibrin complexes' to explain observations of fibrin solubility.

Amino Acids↗

PubMed1987

Studies on the ultrastructure of fibrin lacking fibrinopeptide B (beta-fibrin).

Release of fibrinopeptide B from fibrinogen by copperhead venom procoagulant enzyme results in a form of fibrin (beta-fibrin) with weaker self-aggregation characteristics than the normal product (alpha beta-fibrin) produced by release of fibrinopeptides A (FPA) and B (FPB) by thrombin. We investigated the ultrastructure of these two types of fibrin as well as that of beta-fibrin prepared from fibrinogen Metz (A alpha 16 Arg----Cys), a homozygous dysfibrinogenemic mutant that does not release FPA. At 14 degrees C and physiologic solvent conditions (0.15 mol/L of NaCl, 0.015 mol/L of Tris buffer pH 7.4), the turbidity (350 nm) of rapidly polymerizing alpha beta-fibrin (thrombin 1 to 2 U/mL) plateaued in less than 6 min and formed a "coarse" matrix consisting of anastomosing fiber bundles (mean diameter 92 nm). More slowly polymerizing alpha beta-fibrin (thrombin 0.01 and 0.001 U/mL) surpassed this turbidity after greater than or equal to 60 minutes and concomitantly developed a network of thicker fiber bundles (mean diameters 118 and 186 nm, respectively). Such matrices also contained networks of highly branched, twisting, "fine" fibrils (fiber diameters 7 to 30 nm) that are usually characteristic of matrices formed at high ionic strength and pH. Slowly polymerizing beta-fibrin, like slowly polymerizing alpha beta-fibrin, displayed considerable quantities of fine matrix in addition to an underlying thick cable network (mean fiber diameter 135 nm), whereas rapidly polymerizing beta-fibrin monomer was comprised almost exclusively of wide, poorly anastomosed, striated cables (mean diameter 212 nm). Metz beta-fibrin clots were more fragile than those of normal beta-fibrin and were comprised almost entirely of a fine network. Metz fibrin could be induced, however, to form thick fiber bundles (mean diameter 76 nm) in the presence of albumin at a concentration (500 mumol/L) in the physiologic range and resembled a Metz plasma fibrin clot in that regard. The diminished capacity of Metz beta-fibrin to form thick fiber bundles may be due to impaired use or occupancy of a polymerization site exposed by FPB release. Our results indicate that twisting fibrils are an inherent structural feature of all forms of assembling fibrin, and suggest that mature beta-fibrin or alpha beta-fibrin clots develop from networks of thin fibrils that have the ability to coalesce to form thicker fiber bundles.

Blood Coagulation↗

PubMed1999

Reactivity of soluble fibrin assays with plasmic degradation products of fibrin and in patients receiving fibrinolytic therapy.

The ability to identify the products of thrombin and plasmin action on fibrinogen is important in patients with thrombotic and fibrinolytic disorders. New assays have been developed for "soluble fibrin" which represents soluble derivatives other than fibrinopeptides formed from fibrinogen by thrombin. These assays are either immunological, using antibodies for fibrin-specific neoepitopes, or functional and based on the cofactor activity of soluble fibrin in the tissue plasminogen activator (t-PA)-mediated conversion of plasminogen to plasmin. As plasmic derivations of fibrin share structural features with soluble fibrin, they may be reactive with assays for soluble fibrin. Therefore, we prepared plasmic digests of fibrin and determined the degree of reactivity with four soluble fibrin assays. Three assays used Mabs directed toward the fibrin-specific neoepitopes at alpha17-23 (A), gamma312-324 (B) and alpha17-78 (D). A fourth (C) was based on t-PA co-factor activity. Tests A and C demonstrated marked crossreactivity with fibrin degradation products, and digests containing the largest derivatives showed greatest reactivity. Plasmic derivatives of crosslinked fibrin had greater reactivity than those of non-crosslinked fibrin. Tests B and D demonstrated minimal reactivity with plasmic derivatives of crosslinked or of non-crosslinked fibrin. Samples from patients with lower limb peripheral arterial occlusion were assayed for soluble fibrin, D-dimer and fibrinogen at presentation and eight hours after thrombolytic therapy. Variable results were seen at presentation with elevations in 13, 1, 0 and 4 of 19 patients using Tests A, B, C and D, respectively. After fibrinolytic therapy, marked increases in soluble fibrin levels were observed up to 600-fold above normal. A strong correlation between baseline levels was observed with Test B and Test D, which showed the least cross-reactivity with plasmic derivations. After thrombolytic therapy there were either weak or no correlations among the different assays. The results demonstrate that assays for soluble fibrin may react with plasmic derivatives of fibrin and this must be considered in interpreting clinical results.

Anticoagulants↗

PubMed1993

Binding of a new monoclonal antibody against N-terminal heptapeptide of fibrin alpha-chain to fibrin polymerization site 'A': effects of fibrinogen and fibrinogen derivatives, and pretreatment of samples with NaSCN.

A novel murine monoclonal antibody against the fibrin alpha-chain N-terminus is presented, which reacts with desAA- and desAABB-fibrin. In immunoblot procedures, the antibody reacted with fibrin degradation products X and Y of non-crosslinked fibrin, and fragment E. No binding was observed to the fibrin fragment D-dimer, and fibrinogen fragments D and E. Minor binding to fibrinogen fragments X, and Y, and desBB-fibrin were presumably due to minor contamination with (desAA)-fibrin. A prerequisite for binding was release of fibrinopeptides A (FpA), the binding site being a fibrin-specific neo-epitope. No binding was observed to fibrinogen or to thrombin-treated dysfibrinogen MANNHEIM III (A alpha 16 Arg-->Cys) molecules, which do not release FpA. The antibody bound to abnormal fibrin molecules prepared from dysfibrinogen MANNHEIM I (A alpha 19 Arg-->Gly), albeit to a lesser extent than to normal fibrin. Binding of the antibody to the fibrin epitope was greatly enhanced by denaturation, e.g. by heat, or by treatment with chaotropic ions. Soluble fibrin in clinical samples is generally found as a complex with fibrinogen, since polymerization sites 'A' exposed by release of FpA react with complementary binding sites 'a' on the D-domains of other fibrin and fibrinogen molecules. Treatment of samples with NaSCN caused dissociation of fibrin monomer complexes. Reassociation was prevented by denaturation of both polymerization sites 'A' and 'a'. The antibody in combination with NaSCN-treatment of samples was useful for specific detection of fibrin monomer in plasma samples. Measurement was not influenced by fibrinogen degradation products, whereas fibrin degradation products at very high concentration caused some underestimation of fibrin monomer concentration.

Animals↗

PubMed1995

Orientation of the carboxy-terminal regions of fibrin gamma chain dimers determined from the crosslinked products formed in mixtures of fibrin, fragment D, and factor XIIIa.

There are two schools of thought regarding the orientation of the intermolecular epsilon-amino-(gamma-glutamyl) lysine isopeptide bonds formed between gamma chains in the D domains of assembled fibrin fibers. Some investigators believe that these bonds are oriented parallel to the direction of fiber growth (longitudinally) at the contacting ends of fibrin D domains ('DD-long'), whereas others believe that these bonds are oriented across the two-stranded fibril, between D domains in opposing strands ('DD-transverse'). To distinguish between these two possibilities, the structure of crosslinked products formed in mixtures of fibrin, plasmic fragment D, and factor XIIIa were analyzed, based upon this rationale: Complex formation between D fragments and a fibrin template depends upon the non-covalent 'D:E' interaction between each fibrin E domain and two D fragments ('D:fibrin:D'). If carboxy-terminal gamma chains in the D:fibrin:D complex become aligned in a DD-long configuration, only crosslinked fragment D dimers ('D-D') will result and the fibrin 'template' will not become crosslinked to the associated D fragments. If instead, gamma chain crosslinks form transversely between the D fragments and fibrin, covalently linked D-fibrin complexes will result. SDS-PAGE of factor XIIIa crosslinked mixtures of fibrin and fragment D demonstrated products of a size and subunit composition indicating D-fibrin and D-fibrin-D formation. Small amounts of D dimers were also formed at the same levels as were formed in mixtures of fragment D and factor XIIIa alone. Electron microscopic images of D-fibrin-D complexes prepared under physiological buffer conditions demonstrated that the D fragments were associated with the central E domain of the fibrin molecule, but that they could be dissociated from this non-covalent association in 2% acetic acid. These findings indicate that gamma chain crosslinks occur transversely in D:fibrin:D complexes and permit the extrapolated conclusion that gamma chain crosslinks are also positioned transversely in an assembled fibrin polymer.

Fibrin↗

PubMed2000

Influence of fibrin network conformation and fibrin fiber diameter on fibrinolysis speed: dynamic and structural approaches by confocal microscopy.

Abnormal fibrin architecture is thought to be a determinant factor of hypofibrinolysis. However, because of the lack of structural knowledge of the process of fibrin digestion, relationships between fibrin architecture and hypofibrinolysis remain controversial. To elucidate further structural and dynamic changes occurring during fibrinolysis, cross-linked plasma fibrin was labeled with colloidal gold particles, and fibrinolysis was followed by confocal microscopy. Morphological changes were characterized at fibrin network and fiber levels. The observation of a progressive disaggregation of the fibrin fibers emphasizes that fibrinolysis proceeds by transverse cutting rather than by progressive cleavage uniformly around the fiber. Plasma fibrin clots with a tight fibrin conformation made of thin fibers were dissolved at a slower rate than those with a loose fibrin conformation made of thicker (coarse) fibers, although the overall fibrin content remained constant. Unexpectedly, thin fibers were cleaved at a faster rate than thick ones. A dynamic study of FITC-recombinant tissue plasminogen activator distribution within the fibrin matrix during the course of fibrinolysis showed that the binding front was broader in coarse fibrin clots and moved more rapidly than that of fine plasma fibrin clots. These dynamic and structural approaches to fibrin digestion at the network and the fiber levels reveal aspects of the physical process of clot lysis. Furthermore, these results provide a clear explanation for the hypofibrinolysis related to a defective fibrin architecture as described in venous thromboembolism and in premature coronary artery disease.

Fibrin↗