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PubMed2004

Soluble fibrin augments spreading of fibroblasts by providing RGD sequences of fibrinogen in soluble fibrin.

We previously reported that fibroblasts were found to spread far more avidly on NaBr-solubilized fibrin monomer (FM) monolayers than on immobilized fibrinogen (Fbg), indicating that removal of fibrinopeptides by thrombin is a prerequisite for the fibrin-mediated augmentation of cell spreading [J. Biol. Chem. 272 (1997) 8824-8829]. Soluble fibrin (SF), a 1:2 complex of fibrin-monomer and fibrinogen, is known to be present in the circulating blood under the pathological condition in which blood coagulation is activated. However, its physiological roles are still incompletely known. Fibroblasts spread on immobilized purified soluble fibrin. Cells spreading on immobilized soluble fibrin were blocked by the exogenous addition of soluble fibrin and glycine-arginine-glycine-aspartic acid-serine-phenylalanine (GRGDSP)-synthetic peptide but not by the addition of fibrinogen or fibrin monomer. However, cell spreading activity was decreased in the surfaces coated with fragment X, whose Aalpha-chains lack carboxyl-terminal segments including arginine-glycine-aspartic acid (RGD)-2 domain, fibrin monomer complexes. It suggests that the RGD-2 domain of fibrinogen after being complexed with fibrin monomer plays a pivotal role for soluble fibrin-dependent cell spreading. Soluble fibrin in plasma derived from the patients of disseminated intravascular coagulation (DIC) was immuno-purified using the monoclonal antibody (mAb) which specifically recognizes the Ca(++)-dependent conformer of fibrinogen. The purified soluble fibrin consisted of desAA-fibrin monomer and two fibrinogen molecules and did show the cell spreading activity. Thus, soluble fibrin in plasma plays a role as the modulator of thrombogenic process in vivo.

Cell Adhesion↗
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↗
PubMed1986

Binding properties on Sepharose insolubilized fibrinogen and fibrin, of various species of fibrinogen and fibrin solubilized in plasma.

Radiolabelled tracers of fibrinogen, fibrin des-AA and fibrin des-AABB were solubilized in recalcified, prothrombin depleted plasma, adding either 125I-fibrin des-AA or 125I-fibrin des-AABB together with 131I-fibrinogen, and subsequently subjected to affinity chromatography, utilizing short columns of Sepharose insolubilized preparations of fibrinogen, fibrin des-AA and fibrin des-AABB, respectively. Two naturally occurring fibrinogen species, of high molecular weight (HMW; m.w. 340.000) and of low molecular weight (LMW; m.w. 305.000) exhibited similar binding characteristics, as judged by adsorption and desorption experiments. In subsequent studies all tracer preparations were derived from HMW-fibrinogen. Sepharose insolubilized fibrinogen favoured the adsorption of soluble fibrins as compared to fibrinogen in solution; the adsorption of soluble des-AA fibrin was similar to that of soluble des-AABB fibrin. To insolubilized fibrin, adsorption of soluble tracers of fibrinogen and fibrins increased considerably, and soluble fibrins were no longer preferentially adsorbed. The latter observation was supported by similar desorption characteristics of these tracers. These findings may indicate that the E-domains of soluble fibrin become largely inaccessible to the D-domains of Sepharose insolubilized fibrinogen, probably due to complexing fibrinogen in plasma. Furthermore, adsorption was largely related to the a-epitope of insolubilized fibrin.

Animals↗
PubMed1997

Fibrin detected in plasma of patients with disseminated intravascular coagulation by fibrin-specific antibodies consists primarily of high molecular weight factor XIIIa-crosslinked and plasmin-modified complexes partially containing fibrinopeptide A.

Pooled plasma from 40 patients with severe disseminated intravascular coagulation (DIC) secondary to septic conditions was subjected to gel permeation chromatography on Sephacryl S-500 HR after sample pretreatment with KSCN for dissociation of non-covalent fibrin complexes. Fibrin antigen in eluates was detected by an array of ELISA tests, using two monoclonal antibodies against fibrin degradation product D-dimer, a monoclonal antibody against an epitope generated by plasmin cleavage of the D-domain, and an antibody against the neo-N-terminus of the alpha-chain of fibrin exposed by cleavage of fibrinopeptide A. Tag antibodies were a polyclonal antibody against the fibrinogen/ fibrin D-domain, a POD-conjugated version of the monoclonal antibody against fibrin alpha-chain neo-N-terminus, and a polyclonal antibody against fibrinopeptide A. Most fibrin-related material present in the pooled DIC plasma was of higher molecular mass than fibrinogen. Fibrin polymers were reactive with antibodies against D-dimer, plasmin cleaved D-domain, and fibrin alpha-chain neo-N-terminus. Part of the polymers reacted with antibodies against fibrinopeptide A, indicating presence of fibrinogen or desA-fibrin monomer within the covalently linked complex. In conclusion, the primary analytes detected by monoclonal antibodies for D-dimer, plasmin-specific epitopes of fibrin degradation products, as well as sites exposed by fibrinopeptide cleavage in plasma from patients with disseminated intravascular coagulation are high molecular weight factor XIIIa-crosslinked fibrin complexes, containing plasmin-cleaved D-domains, intact fibrin monomer units, and fibrinogen or desA-fibrin monomer.

Antibodies, Monoclonal↗
PubMed1994

Effects of lipoprotein(a) on the binding of plasminogen to fibrin and its activation by fibrin-bound tissue-type plasminogen activator.

Molecular assembly of plasminogen and tissue-type plasminogen activator (t-PA) at the surface of fibrin results in the generation of fibrin-bound plasmin and thereby in the dissolution of a clot. This mechanism is triggered by specific interactions of intra-chain surface lysine residues in fibrin with the kringle domains of plasminogen, and is further amplified via the interaction of plasminogen kringles with the carboxy-terminal lysine residues of fibrin that are exposed by plasmin cleavage. By virtue of its marked homology with plasminogen, apo(a), the specific apolipoprotein component of Lp(a), may bind to the lysine sites available for plasminogen on the surface of fibrin and thereby interfere with the fibrinolytic process. A sensitive solid-phase fibrin system, which allows the study of plasminogen activation at the plasma fibrin interface and makes feasible the analysis of products bound to fibrin, has been used to investigate the effects of Lp(a) on the binding of plasminogen and its activation by fibrin-bound t-PA. Plasma samples from human subjects with high levels of Lp(a) were studied. We have established that Lp(a) binds to the fibrin surface and thereby competes with plasminogen (Ki = 44 nM) so as to inhibit its activation. We have further shown that Lp(a) blocks specifically carboxy-terminal lysine residues on the surface of fibrin. To further explore the role of apo(a) on the Lp(a) fibrin interactions, we have performed ligand-binding studies using a recombinant form of apo(a) that contains 17 kringle 4-like units. We have shown that recombinant apo(a) binds specifically to fibrin (Kd = 26 +/- 8 nM, Bmax = 26 +/- 2 fmol/well) and that this binding increases upon treatment of the fibrin surface with plasmin (Kd = 8 +/- 4 nM, Bmax = 115 +/- 14 fmol/well). Altogether, our results indicate clearly that binding of native Lp(a) through this mechanism may impair clot lysis and may favor the accumulation of cholesterol in thrombi at sites of vascular injury.

Apolipoproteins↗
PubMed1991

Effects of monoclonal antibodies on tissue-type plasminogen activator (t-PA) binding to lysine, fibrin and heparin and on fibrin-mediated enhancement of one-chain t-PA amidolytic activity.

The effects of 4 monoclonal antibodies against human tissue-type plasminogen activator (t-PA) on binding of t-PA to lysine, fibrin, and heparin, and on fibrin-mediated activation of one-chain t-PA-amidolytic activity were investigated. The association constants of the antibodies were determined in a direct assay to be equal to 0.125 l/nmol, 0.225 l/nmol, 0.4 l/nmol, and 0.5 l/nmol for mAB 5, mAB 16, mAB 25, and mAB 31, respectively. All 4 monoclonal antibodies inhibited binding of intact t-PA to lysine-Sepharose and fibrin, and they suppressed fibrin-mediated activation of one-chain t-PA-amidolytic activity. Binding analysis demonstrated that mAB 25 inhibited t-PA binding to lysine-Sepharose and to fibrin as well as fibrin-mediated enhancement of one-chain t-PA-amidolytic activity in a competitive manner with inhibitor constants of 5 nmol/l, 3 nmol/l and 10 nmol/l, respectively. It was also shown that free lysine counteracts the association of t-PA with the antibodies. Binding of t-PA to heparin is only moderately affected by the 4 antibodies. Since t-PA possesses two homologous kringle domains which contain fibrin (lysine) binding sites, the results underline the importance of a lysine binding site for fibrin binding by intact t-PA and show that the binding of the enzyme to fibrin and lysine is mediated by the same binding site of a kringle domain. The parallel effects of antibodies on fibrin binding and on fibrin-mediated enhancement of one-chain t-PA amidolytic activity proves that the site of fibrin binding is identical with the site of fibrin activation. The binding site of heparin apparently differs from lysine and fibrin binding sites.

Antibodies, Monoclonal↗
PubMed1996

Monoclonal antibodies to human fibrin: interaction with other animal fibrins.

Four monoclonal antibodies have previously been raised in our laboratory for possible use in thrombus imaging and the targeting of thrombolytic agents. These antibodies were raised to various human fibrin-related immunogens and each antibody had been selected for its specificity towards fibrin and not fibrinogen. To study further these antibodies in animal circulation models both in vivo and in vitro, their selectivity towards human fibrin as opposed to other animal fibrins was examined. In this study dissociation constants for each antibody with each of six species fibrins (human, baboon, pig, dog, sheep and rabbit) were estimated using both fibrin clots and monolayers. Some limited data were also obtained with Sepharose-fibrin. Of the antibodies two (denoted 12B3B10 and 12B3A11) are seen to bind almost exclusively to human fibrin with dissociation constants of about 8 x 10(-10) M using fibrin clots and monolayers. These same two mabs bound to baboon fibrin with a dissociation constant of 2 x 10(-9) M, while neither displayed significant levels of binding to the fibrins from dog, pig, sheep and rabbit. The other two antibodies investigated (1H10 and 5F3) were found to bind well to fibrins of human, baboon, pig and dog, with dissociation constants in the range of 1.4-4.2 x 10(-9) M. However neither 1H10 nor 5F3 displayed significant recognition of sheep and rabbit fibrins. Both 1H10 and 5F3 were also found by means of competitive ELISA's to retain their selectivity to baboon, dog and pig fibrins in the presence of their respective fibrinogens.

Animals↗
PubMedDate not supplied

Insoluble "fibrin" in human aortic intima. Quantitative studies on the relationship between insoluble "fibrin", soluble fibrinogen and low density lipoprotein.

A quantitative assay for fibrin or other insoluble fibrin-like antigens ("fibrin") in small samples of intima is described. Tissue samples were subjected to electrophoresis directly from the intima into an antibody-containing gel to remove and measure fibrinogen and other soluble fibrin reactive antigens (FRA). The residual tissue was then exhaustively incubated with plasmin, and the soluble fragments generated from the insoluble "fibrin" were measured by quantitative immunoelectrophoresis. "Fibrin" accounted for about 2% of the tissue dry weight in normal intima and the ratio fibrinogen/"fibrin" was 1-1.5. In the gelatinous lesions, which seem to be the precursors of fibrous plaques, there was a small increase in "fibrin" but a substantial increase in fibrinogen and low density (LD)-lipoprotein, and the ratio fibrinogen/"fibrin" rose to about 3, which suggests that the increase in "fibrin" is secondary to increased permeation of fibrinogen. At the edges of large plaques there was also a threefold increase in fibrinogen, but "fibrin" increased fivefold, and accounted for 10% of the tissue dry weight. The same high concentration was found in the centres of large fibrous plaques with advanced atheroma lipid. Raised levels of "fibrin" were accompanied by raised levels of fibrinogen in most tissue samples. About 80% of the total soluble FRA could be clotted with thrombin; there was no significant difference between normal intima and lesions, and the proportion clotted was not related to "fibrin" content.

Aged↗
PubMed1990

Alpha-thrombin-catalyzed hydrolysis of fibrin I. Alternative binding modes and the accessibility of the active site in fibrin I-bound alpha-thrombin.

Steady-state kinetic parameters were determined for the action of human alpha-thrombin on human fibrin I polymer, an intermediate in the alpha-thrombin-catalyzed conversion of fibrinogen to the fibrin matrix of blood clots during the terminal phase of the blood clotting cascade. Values of 49 s-1 and 7.5 microM were determined (at 37 degrees C, pH 7.4, gamma/2 0.17) for kcat and Km, respectively. Studies of the effect of fibrin I on alpha-thrombin-catalyzed hydrolysis of the fluorogenic substrate N-p-Tos-Gly-L-Pro-L-Arg-7-amido-4-methylcoumarin (tos-GPR-amc) and the effect of fibrin I on the reaction of alpha-thrombin with antithrombin III (AT) were presented which indicate that the active site of alpha-thrombin is accessible while it is bound to its substrate fibrin I. Fibrin I inhibited alpha-thrombin-catalyzed hydrolysis of tos-GPR-amc in a manner inconsistent with the pure competitive inhibition expected for an alternative substrate, whereas fibrinogen, an alpha-thrombin substrate, behaved as a pure competitive inhibitor of the alpha-thrombin-catalyzed hydrolysis of tos-GPR-amc. The effect of fibrin I on alpha-thrombin-catalyzed hydrolysis of tos-GPR-amc was shown to be consistent with alpha-thrombin binding to fibrin I in alternative orientations. In one orientation both the active site and a site distinct from the active site (an exosite) of alpha-thrombin are occupied by fibrin I. In the other orientation only the exosite of alpha-thrombin is occupied and the active site is freely accessible to other substrates. The values of both kcat (21 s-1) and Km (less than 0.23 microM) determined for fibrin I-bound alpha-thrombin acting on tos-GPR-amc were decreased relative to the values of kcat (180 s-1) and Km (7.3 microM) observed for the action of uncomplexed alpha-thrombin on tos-GPR-amc. This observation suggests that the active site of alpha-thrombin is altered in fibrin I-bound alpha-thrombin. Studies of the effect of fibrin I on the reaction of AT with alpha-thrombin (at 37 degrees C, pH 7.4, gamma/2 0.17) indicated that when alpha-thrombin is bound to fibrin I in an orientation where the active site of alpha-thrombin is accessible, AT reacts with alpha-thrombin with a rate constant (greater than 4.2 x 10(4) M-1 s-1) that is greater than the rate constant (1.5 x 10(4) M-1 s-1) for reaction of AT with the free enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)

Antithrombin III↗
PubMed1994

Inhibition of heparin activity in plasma by soluble fibrin: evidence for ternary thrombin-fibrin-heparin complex formation.

The ability of heparin to dramatically enhance the inactivation of thrombin (IIa) by antithrombin III (ATIII) in buffer is negated through formation of a IIa-fibrin-heparin ternary complex (Hogg and Jackson, Proc Natl Acad Sci USA 86:3619, 1989; Hogg and Jackson, J Biol Chem 265:241, 1990). IIa, in this ternary complex, is protected from inactivation by ATIII. Our aim was to determine whether fibrin also compromises heparin efficacy in plasma. We found that soluble fibrin ablated the heparin-mediated prolongation of the thrombin time with half-maximal effect at 60 nmol/L fibrin. The heparin-mediated prolongation of the activated partial thromboplastin time (APTT) was also reduced by fibrin with half-maximal effects at 140 nmol/L fibrin using 0.12 U/mL heparin and 500 nmol/L fibrin using 0.25 U/mL heparin. The mechanism of inhibition of heparin activity by fibrin in plasma was determined by measuring IIa-ATIII complexes by enzyme-linked immunosorbent assay (ELISA). Fibrin was found to inhibit the heparin-catalyzed inactivation of IIa by ATIII with half-maximal effect at 97 +/- 19 nmol/L fibrin. Fibrin had no effect on the heparin-catalyzed inactivation of factor Xa by ATIII in plasma, using either standard heparin, a heparinoid preparation (Orgaran; Organon, Lane Cove, Sydney, Australia), or low-molecular weight heparin. These findings imply that fibrin is a potent modulator of heparin activity in vivo by inhibiting heparin-catalyzed IIa-ATIII complex formation through formation of ternary IIa-fibrin-heparin complexes.

Amino Acid Sequence↗
PubMedDate not supplied

Precipitation of fibrin monomers and fibrin degradation products by ristocetin.

Ristocetin, at relatively low concentrations (1.0 mg/ml-1.5 mg/ml), can selectively precipitate fibrin monomers and fibrin degradation products (fdp) from plasma without effect on fibrinogen or fibrinogen degradation products (FDP). 125I-labeled fibrin monomers and fibrin degradation products were precipitated by ristocetin when their plasma concentrations were greater than 0.25 microgram/ml and 50 microgram/ml, respectively. In order to obtain a visible precipitated, 2 microgram/ml of fibrin monomers of 50 to 100 microgram/ml of fibrin degradation products were necessary. These effects were optimally observed under the following conditions: (1) temperature, 20 C to 37 C; (2) pH, 7.0 to 7.5; and (3) incubation time, 15 to 60 minutes. Late-fibrin degradation products are approximately eight times less sensitive to ristocetin-induced precipitation than early-fibrin degradation products. Plasma medium is essential for the differentiation of fibrin monomers and fibrin degradation products from fibrinogen and fibrinogen degradation products by ristocetin. These results suggest that the specific detection of fibrin monomers and fibrin degradation products in plasma may be easily performed by ristocetin.

Anticoagulants↗
PubMed1999

Evaluation of application techniques of fibrin sealant to prevent cerebrospinal fluid leakage: a new device for the application of aerosolized fibrin glue.

OBJECTIVE: This report evaluates the sealing effects of fibrin sealant applied on the dura mater using different techniques. METHODS: Three application methods were studied: a sequential layer method, a simultaneous method using a cannula, and a spray method using a newly developed spray device. The sealing effects of these methods were compared using in vitro histological analysis and a pressure resistance test. The clinical efficacy of the fibrin sealant to prevent water leakage through the dura mater was retrospectively analyzed in a total of 509 patients. The process of absorption of a clinically applied fibrin clot in vivo was examined using surgical specimens. RESULTS: The fibrin plate made using the spray method withstood a hydrostatic pressure greater than 200 cm H2O. A scanning electron microscopic study of the fibrin clots showed that the sequential and simultaneous methods produced a fibrin fiber network; in contrast, our spray method formed a dense fibrin tissue in which the fibrin molecules fused together forming stratified laminae. Of the 295 supratentorial craniotomies during which spraying was used, postsurgical cerebrospinal fluid leakage occurred in 9 cases (3.1%), whereas of the 214 craniotomies during which spraying was not used, cerebrospinal fluid leakage occurred in 19 cases (8.9%). Histological examinations of 10 surgical specimens obtained during second craniotomies revealed that the spray-made fibrin clots had been gradually replaced by mature granulation composed of collagenous connective tissue. CONCLUSION: The optimal technique for applying fibrin sealant is the spray method that aerosolizes fibrin glue and produces a tough fibrin plate.

Administration, Topical↗
PubMed2004

Comparison of thrombin-catalyzed fibrin polymerization and factor XIIIa-catalyzed cross-linking of fibrin among three recombinant variant fibrinogens, gamma 275C, gamma 275H, and gamma 275A.

BACKGROUND AND OBJECTIVES: We have previously reported that recombinant gamma 275Cys fibrinogen exhibits a marked impairment of functions as well as aberrant fibrin clot and bundle structures, as compared with wild-type, gamma 275Arg, and plasma fibrinogen from a heterozygous proband. Since gamma Arg275His mutations have also been reported in 10 families, we synthesized recombinant gamma 275His fibrinogen and gamma 275Ala fibrinogen (as a control) and analyzed and compared them with gamma 275Cys and gamma 275Arg. METHODS: A variant gamma-chain expression plasmid was transfected into Chinese hamster ovary cells expressing normal human fibrinogen A alpha- and B beta-chains. After purification of the recombinant variant fibrinogens, we performed functional analyzes for thrombin-catalyzed fibrin polymerization and factor XIIIa (FXIIIa)-catalyzed gamma-gamma dimer formation from fibrin or fibrinogen and also ultrastructural analysis of fibrin clots and bundles. RESULTS: By comparison with both gamma 275His and gamma 275Ala fibrinogens, recombinant gamma 275Cys fibrinogen exhibited a more impaired gamma-gamma dimer formation from fibrin or fibrinogen, a more aberrant fibrin clot structure, and thicker fibers in fibrin bundles. In 1 : 1 mixtures of gamma 275Arg and gamma 275Cys fibrinogens or gamma 275Arg and gamma 275His fibrinogens, thrombin-catalyzed fibrin polymerization and both fibrin clot and fiber structures showed some compensation (as compared with gamma 275Cys or gamma 275His alone). CONCLUSION: These results strongly suggest that an amino acid substitution of gamma 275Arg alone disrupts D:D interactions in thrombin-catalyzed fibrin polymerization and the formation of fibrin bundles and fibrin clots. Moreover, the existence of a subsequent disulfide-linked Cys in gamma 275C fibrinogen augments the impairment caused by a His or Ala substitution.

Alanine↗
PubMed1980

Cross-linking of alpha 2-plasmin inhibitor to fibrin by fibrin-stabilizing factor.

The concentration of alpha 2-plasmin inhibitor in blood plasma is higher than that in serum obtained from the blood clotted in the presence of calcium ions, but is the same as that in serum obtained in the absence of calcium ions. Radiolabeled alpha2-plasmin inhibitor was covalently bound to fibrin only when calcium ions were present at the time of clotting of plasma or fibrinogen. Whereas, when batroxobin, a snake venom enzyme that lacks the ability to activate fibrin-stabilizing factor, was used for clotting fibrinogen, the binding was not observed. When fibrin-stablizing, factor-deficient plasma was clotted, the specific binding of alpha 2-plasmin inhibitor to fibrin did not occur even in the presence of calcium ions and the concentration of alpha 2-plasmin inhibitor in serum was the same as that in plasma. Monodansyl cadaverine, a fluorescent substrate of the fibrin-stablizing factor, was incorporated into alpha 2-plasmin inhibitor by activated fibrin-stablizing factor. All these findings indicate that alpha 2-plasmin inhibitor is cross-linked to fibrin by activated fibrin-stabilizing factor when blood is clotted. Analysis of alpha 2-plasmin inhibitor-incorporated fibrin by sodium dodecyl sulfate gel electrophoresis showed that the inhibitor was mainly cross-linked to polymerized alpha-chains of cross-linked fibrin. Cross-linking of alpha 2-plasmin inhibitor to fibrin renders fibrin clot less susceptible to fibrinolysis by plasmin.

Calcium↗
PubMed1992

Comparison of fibrin-mediated stimulation of plasminogen activation by tissue-type plasminogen activator (t-PA) and fibrin-dependent enhancement of amidolytic activity of t-PA.

Studies in the past 10 years have shown that there are two different, but related pathways for the acceleration of tissue-type plasminogen activator (t-PA) catalysis: (1) fibrin-dependent enhancement of t-PA amidolytic activity by fibrin binding; (2) fibrin-mediated stimulation of plasminogen activation by t-PA via the formation of a ternary complex of fibrin, t-PA and plasminogen. The common characteristic of both phenomena is the affinity of t-PA for fibrin, which is realized by the same enzyme binding site. However, a comparison of the kinetic data, the participating fibrin structures and the differences between single-chain and two-chain t-PA (sct-PA and tct-PA, respectively) shows that both phenomena have different causes. Fibrin-mediated stimulation of plasminogen activation involves both sct-PA and tct-PA and different fibrinogen derivatives such as fibrin, fibrinogen cyanogen bromide fragment FCB-2, fibrin alpha-chain and poly-lysine. This mechanism is described by a marked apparent decrease in the KM value. In contrast, fibrin-dependent enhancement of t-PA activity against low molecular weight peptides is exclusive to sct-PA and is characterized by an increase in the kcat value and, depending on the nature of the substrate, by an increase in kcat and a decrease in KM. Thus, sct-PA activity modulation depends strictly on the correct three-dimensional folding of fibrin and is not mediated by fibrinogen fragment FCB-2 or isolated fibrin chains.(ABSTRACT TRUNCATED AT 250 WORDS)

Amides↗
PubMed1990

Trophoblast interaction with fibrin matrix. Epithelialization of perivillous fibrin deposits as a mechanism for villous repair in the human placenta.

The authors have used morphometric, immunocytochemical, and electron optical techniques to study fibrin deposits associated with villi from 14 normal term placentas, and have examined the response of cultured cellular trophoblast to fibrin matrix in vitro. Morphometric analysis of 3477 villous profiles showed that 5.5% of villi examined had fibrin deposition at sites of syncytial denudation and that fibrin deposition was highly associated with villous epithelial denudation, as evidenced by loss of cytokeratin staining. The perivillous fibrin deposits were strongly immunoreactive for the B beta chain of fibrin II, consistent with local thrombolytic cleavage of fibrinogen to fibrin. Deposits were frequently surfaced by a discontinuous layer of cytokeratin-positive trophoblastic cells that showed type IV basement membrane collagen immunoreactivity at the interface between trophoblast and fibrin. Ultrastructurally, damage to the syncytial trophoblast was apparent at the edge of some deposits, where syncytial denudation was accompanied by a fibrin coating of residual cellular trophoblast and the trophoblastic basal lamina. Other deposits were surfaced by syncytial trophoblast with underlying cellular trophoblast and a new basal lamina external to the basal lamina of the villous core. Cultured cellular trophoblast grown on a fibrin matrix, but not on uncoated plastic, showed morphologic differentiation into a trophoblast layer like that on term villi. The authors suggest that epithelialization of perivillous fibrin deposits is a form of villous repair and that trophoblast-fibrin interactions can modulate trophoblastic differentiation.

Chorionic Villi↗
PubMed1993

Initial interaction between fibrin and tissue plasminogen activator (t-PA). The Gly-Pro-Arg-Pro binding site on fibrin(ogen) is important for t-PA activity.

Gly-Pro-Arg-Pro (GPRP) is a potent inhibitor of fibrin polymerization. It also causes a concentration-dependent inhibition of the activation of plasminogen by t-PA when soluble desAABB-fibrinogen (fibrin II) or fibrinogen are used as promoters of t-PA. Fibrinogen has a much weaker promoter activity than does fibrin II. Experiments were undertaken to explain the mechanism of action of GPRP on plasminogen activation. Kinetic data indicated that when GPRP was present in the assay system, the fibrin(ogen)-GPRP complex was ineffective as a t-PA promoter. Only the free forms of fibrin II or fibrinogen were promoters of t-PA. GPRP specifically eluted t-PA, which was previously bound to a fibrin-Sepharose column, and also inhibited t-PA binding to fibrin-Sepharose in a concentration-dependent manner. These experiments were compared to those with other tetrapeptides: GHRP, GPGG, and GRGD. Only GHRP, which is known to bind more weakly to fibrin(ogen) than GPRP, had any effect. These results suggest that the GPRP binding site on the fibrin(ogen) molecule is important for t-PA activation and is a binding site for t-PA in the initial interaction between t-PA and fibrin. We propose that at or near the GPRP binding site on fibrin there is an initial binding site for t-PA. We hypothesize that kringle 1, which contains the sequence G128RRP, may be involved in the initial binding of t-PA to fibrin.

Amino Acid Sequence↗
PubMed2001

The fibrin assay comparison trial (FACT): correlation of soluble fibrin assays with D-dimer.

Soluble fibrin (SF) is regarded as an indicator of acute fibrin formation and a precursor of fibrin thrombi. Using a set of clinical plasma samples, and fibrin derivatives, five assays for measurement of SF, including two chromogenic assays, two ELISA systems, and one latex-enhanced photometric immunoassay were compared. Correlation between SF assays was moderate (Spearman's rho values between 0.344 and 0.805). Re-calibration with serial dilutions of desAABB-fibrin monomer resulted in adjustment of the numerical scale of the assays without improving correlation. All SF assays reacted with purified crosslinked fibrin derivatives. Using clinical plasma samples, Spearman's rho of SF assays with D-dimer consensus values based upon results of 23 quantitative D-dimer assays were between 0.491 and 0.911. Although all SF assays react with desAABB-fibrin monomer complexes, SF assays are heterogeneous concerning reactivity with fibrin compounds observed in clinical plasma samples. The prospect of a common calibrator for SF assays therefore seems to be remote. Since SF assays react with crosslinked fibrin derivatives, it is not possible to clearly distinguish between acute fibrin formation, and fibrin dissolution on the basis of the results of current SF assays.

Calibration↗