Synthesis and Preclinical Evaluation of the Fibrin-Binding Cyclic Peptide 18F-iCREKA: Comparison with Its Contrasted Linear Peptide.

Purpose: Cys-Arg-Glu-Lys-Ala (CREKA) is a pentapeptide which can target fibrin-fibronectin complexes. Our previous study has built a probe called iCREKA which was based on CREKA and has proved the feasibility and specificity of iCREKA by the fluorescence experiment. The purpose of this study is to achieve the 18F-labeled iCREKA and make preclinical evaluation of the 18F-iCREKA with comparison of its contrasted linear peptide (LP). Methods: CREKA, LP, and iCREKA were labeled by the Al18F labeling method, respectively. These 18F-labeled peptides were evaluated by the radiochemistry, binding affinity, in vitro stability, in vivo stability, micro-PET imaging, and biodistribution tests. Results: 18F-NOTA-iCREKA was stable both in vitro and in vivo. However, 18F-NOTA-CREKA and 18F-NOTA-LP were both unstable. The FITC or 18F-labeled iCREKA could be abundantly discovered only in matrix metalloproteinases- (MMPs-) 2/9 highly expressed U87MG cells, while the FITC or 18F-labeled LP could also be abundantly discovered in MMP-2/9 lowly expressed Caov3 cells. Biodistribution and micropositron emission tomography (PET) imaging revealed that the U87MG xenografts showed a higher uptake of 18F-NOTA-iCREKA than 18F-NOTA-LP while the Caov3 xenografts showed very low uptake of both 18F-NOTA-iCREKA and 18F-NOTA-LP. The tumor-to-muscle (T/M) ratio of 18F-NOTA-iCREKA (9.93 ± 0.42) was obviously higher than 18F-NOTA-LP (2.69 ± 0.35) in U87MG xenografts. Conclusions: The novel CREKA-based probe 18F-NOTA-iCREKA could get a high uptake in U87MG cells and high T/M ratio in U87MG mice. It was more stable and specific than the 18F-NOTA-LP.

The economic impact of poor sample quality in clinical chemistry laboratories: results from a global survey.

Background Despite advances in clinical chemistry testing, poor blood sample quality continues to impact laboratory operations and the quality of results. While previous studies have identified the preanalytical causes of lower sample quality, few studies have examined the economic impact of poor sample quality on the laboratory. Specifically, the costs associated with workarounds related to fibrin and gel contaminants remain largely unexplored. Methods A quantitative survey of clinical chemistry laboratory stakeholders across 10 international regions, including countries in North America, Europe and Oceania, was conducted to examine current blood sample testing practices, sample quality issues and practices to remediate poor sample quality. Survey data were used to estimate costs incurred by laboratories to mitigate sample quality issues. Results Responses from 164 participants were included in the analysis, which was focused on three specific issues: fibrin strands, fibrin masses and gel globules. Fibrin strands were the most commonly reported issue, with an overall incidence rate of ∼3%. Further, 65% of respondents indicated that these issues contribute to analyzer probe clogging, and the majority of laboratories had visual inspection and manual remediation practices in place to address fibrin- and gel-related quality problems (55% and 70%, respectively). Probe maintenance/replacement, visual inspection and manual remediation were estimated to carry significant costs for the laboratories surveyed. Annual cost associated with lower sample quality and remediation related to fibrin and/or gel globules for an average US laboratory was estimated to be $100,247. Conclusions Measures to improve blood sample quality present an important step towards improved laboratory operations.

Physiologically clotted fibrin--preparation and characterization for tissue engineering and drug delivery applications.

Fibrin used for biomedical applications is prepared by mixing concentrated solutions of fibrinogen and thrombin in presence of cross-linking agents such as Factor XIII or glutaraldehyde. The main drawbacks associated with this procedure include cost, complexity and time required for fibrin preparation. Hence, present study deals with the characterization of physiologically clotted fibrin (PF) for bone tissue engineering and drug delivery applications. For this the physico-chemical properties of PF were compared with those of the conventionally prepared fibrin (CF). Further MTT and haemolytic assays were performed for both PF and CF to compare their biocompatibility. The amount of alkaline phosphatase produced and calcium secreted by MG-63 cells in the presence of PF and CF were used to relate the osteogenic potency of PF with that of CF. Gallic acid, an anti-cancer drug was loaded within PF and CF and their role in drug delivery was compared.

[Effects of platelet-rich fibrin extract on MC3T3-E1 cell].

OBJECTIVE: To evaluate the effect of platelet-rich fibrin extract (PRFe) on proliferation and differentiation and F-actin cytoskeleton of osteoblasts. METHODS: The experimental group used the α-minimum essential medium (α-MEM) containing PRFe (10% fetal bovine serum), and the control group used the α-MEM (10% fetal bovine serum). The number of the osteoblasts at 1st, 3rd, 5th d was detected by methyl thiazolyl tetrazolium (MTT) assay, and the differentiation of osteoblast at 1st, 3rd, 5th,7 th d detected by the activity of alkaline phosphatase (ALP).The alizarin red dye was used to observe the number of calcium nodus at 14th, 21st d. The F-actin cytoskeleton was evaluated by confocal laser scanning microscope(CLSM) at 3rd,6th,9th,12th h. The level of osteogenetic biomarkers osteocalcin (OCN) and core-binding factor α1(Cbfα1) at 3rd,7th d were quantified by real-time PCR. RESULTS: A significant increase of absorbance at 1st, 3rd, 5th d was showed in experimental group (0.336 ± 0.011, 0.571 ± 0.039, 0.787 ± 0.050) compared to control group (0.300 ± 0.021, 0.387 ± 0.040, 0.527 ± 0.034) (P < 0.05). The absorbance of experimental group at 1st, 3rd, 5th, 7th d (0.146 ± 0.014, 0.199 ± 0.017, 0.390 ± 0.020, 0.492 ± 0.019) was significantly higher than that of control group (0.115 ± 0.014, 0.145 ± 0.015, 0.190 ± 0.015, 0.230 ± 0.026) (P < 0.05). The integrated absorbance of the calcium nodus in experimental group at 14th, 21st d (22.119 ± 3.694, 31.528 ± 3.162) was significantly higher than in control group (8.498 ± 2.041, 15.162 ± 2.526) (P < 0.05). The Cbfα1 and OCN gene expression in experimental group was higher than in control group (P < 0.05). CONCLUSIONS: PRFe could enhance the proliferation and differentiation of osteoblasts and promote the spread of F-actin cytoskeleton.

Biological effects of static electric field: Plasma/serum proteome analysis of rats.

The external static electric field (SEF) of man-made origin brings to the substantially increased SEF background in a human environment the biological activity of which is a moot question. The paper reports on rats blood plasma/serum proteome modifications by means of 1D polyacrilamide gel electrophoresis and clotting process alterations after the short- and long-term SEF exposures of 200 kV/m. The results indicate decrease of fast α1 and α2 globular proteins in plasma coinciding with clotting acceleration after the short-term SEF, and attenuation of clotting-dependent proteome modifications reflected with incomplete coagulation after the long-term SEF exposure. Increased lysozyme activity in serum unlike plasma was observed after both SEF exposures. Applied model of the high-voltage SEF environment indicates dependence of biological systems functioning on the external SEF.

[Stored autologous blood from a pregnant woman showing a considerable amount of agglutinates].

A 30-year-old woman was admitted to our hospital to undergo simultaneous cesarean section and radical hysterectomy when the pregnancy became 30 weeks. An ultrasonic examination had found hypoechoic region at the cervix uteri. Because she wished autologous blood transfusion, 100 ml each of her own blood was obtained 3 times preoperatively. All the stored blood was returned to the patient through a filtering system (40 microm in the pore size) during surgery. However, we found paste-like agglutinates floating in the bags. We transfused the blood carefully while confirming that there were no agglutinates in the reservoir below the filter. The paste-like agglutinates were also found on the filter. By microscopic observation fibrin-like substances attached by blood cells were detected. When the autologous blood from pregnant women is returned, special care should be taken because the blood is likely to form agglutinates even though the blood test data are within normal ranges.

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.

Fibrin sealant produced by the CryoSeal FS System: product chemistry, material properties and possible preparation in the autologous preoperative setting.

BACKGROUND AND OBJECTIVES: The CryoSeal FS has been introduced as an automated device for the production of fibrin sealant from small volumes of plasma. We tested this device and compared the product with commercially available fibrin sealants and with the requirements of the European Pharmacopoeia. MATERIALS AND METHODS: The CP3 program and disposables required were used to manufacture fibrin sealant. The chemistry and mechanical properties of the product were investigated. RESULTS: The cryoprecipitate generated with CryoSeal contains concentrated fibrinogen and critical clotting factors. The efficiency of the production process is poor, but the production procedure itself is simple and not time-consuming. The volume of plasma required allows application in the preoperative autologous setting. CONCLUSIONS: The CryoSeal FS is an automated device for cryoprecipitation and production of thrombin. It can be implemented easily in the clinical routine, although, owing to product specifications, the efficacy of the CryoSeal fibrin sealant requires further clinical trials.

[Immunofluorescent profile of patients with different kinds of cataract (in vitro study)]. / Obraz immunofluorescencyjny soczewek chorych z róznymi rodzajami zacmy (badania in vitro).

The aim of the study is to determine the influence of some selected immunological factors upon the pathogenesis of senile cataract and cataract in diabetic people. Immunofluorescent pictures of opacities lenses in senile cataract patients and in diabetic cataract patients were compared. The fluorescent microscopic examination of these preparations revealed green colored concrements. The concrements thus observed proved the presence of a given class immunoglobulin in the lenticular nucleus sections. In all preparations (100%), the presence of IgG was found. None concrements' of C4 and the fibrin were spotted in the observed preparations.

Fibrin as a matrix for grafting 2-hydroxyethyl methacrylate: preparation and characterization of the graft and its in vivo evaluation for wound healing.

In this work, fibrin was used as a substrate to graft 2-hydroxyethyl methacrylate (HEMA) by free radical polymerization using potassium persulfate and sodium metabisulfite as redox initiators. The extent of grafting the synthetic polymer on the biopolymer was studied under various experimental conditions, and the optimum factors for affording maximum grafting were standardized. The graft, fib-g-p[HEMA], was characterized by Fourier transform infrared, scanning electron microscopy, and X-ray diffraction studies. The graft exhibited a higher shelf life than native fibrin. The biocompatibility of the graft has been tested by in vivo studies and the results, in terms of collagen formation and wound size, proved its suitability for wound healing.

Defibrination of blood plasma for use in serological tests for syphilis.

Syphilitic plasma can be salvaged from discarded blood donations and converted to serum by defibrination. Sixty-nine units of plasma were treated with a stock solution of 100 U of thrombin per ml in 1 M calcium chloride and then with a 10% (wt/vol) solution of kaolin. Fibrinogen concentrations detected in initial plasma samples ranged from 94 to 4970 mg/liter (mean, 2532 mg/liter) for samples that were reactive by the rapid plasma reagin circle card test (RPR) and from 314 to 2742 mg/liter (mean 1528 mg/liter) for samples that were not reactive by the RPR. The treated samples showed no measurable fibrinogen remaining after the defibrination process. In the nontreponemal RPR for syphilis, 86% of the treated plasma samples retained the same endpoint titer as that of the initial plasma sample. When the Treponema pallidum passive-particle-agglutination test was used, 98% retained the same reactivity. In the Captia Syphilis-G enzyme immunoassay, 89% of the treated samples demonstrated no change in reactivity index, and in the fluorescent treponemal antibody absorption test, 96% showed no reduction in fluorescence. Human sera containing antibodies to syphilis are used at the Centers for Disease Control and Prevention for the preparation of reference controls or as samples for proficiency testing. Finding reactive sera is becoming more difficult due to the general decline of syphilis cases in the United States. The decreasing availability of these sera can be alleviated by salvaging plasma and converting it to serum.

Molecular basis for the susceptibility of fibrin-bound thrombin to inactivation by heparin cofactor ii in the presence of dermatan sulfate but not heparin.

Although fibrin-bound thrombin is resistant to inactivation by heparin.antithrombin and heparin.heparin cofactor II complexes, indirect studies in plasma systems suggest that the dermatan sulfate.heparin cofactor II complex can inhibit fibrin-bound thrombin. Herein we demonstrate that fibrin monomer produces a 240-fold decrease in the heparin-catalyzed rate of thrombin inhibition by heparin cofactor II but reduces the dermatan sulfate-catalyzed rate only 3-fold. The protection of fibrin-bound thrombin from inhibition by heparin.heparin cofactor II reflects heparin-mediated bridging of thrombin to fibrin that results in the formation of a ternary heparin.thrombin.fibrin complex. This complex, formed as a result of three binary interactions (thrombin.fibrin, thrombin.heparin, and heparin.fibrin), limits accessibility of heparin-catalyzed inhibitors to thrombin and induces conformational changes at the active site of the enzyme. In contrast, dermatan sulfate binds to thrombin but does not bind to fibrin. Although a ternary dermatan sulfate. thrombin.fibrin complex forms, without dermatan sulfate-mediated bridging of thrombin to fibrin, only two binary interactions exist (thrombin.fibrin and thrombin. dermatan sulfate). Consequently, thrombin remains susceptible to inactivation by heparin cofactor II. This study explains why fibrin-bound thrombin is susceptible to inactivation by heparin cofactor II in the presence of dermatan sulfate but not heparin.

Utility and quality of autologous fresh frozen plasma and autologous fibrin glue for surgical patients.

The quality of autologous fresh frozen plasma and autologous fibrin glue prepared in our hospital was evaluated. The Auto-FFP was separated from whole blood or collected using a cell separator and the Auto-FG was then prepared from the Auto-FFP. The Auto-FFP showed significantly higher levels of fibrinogen, Factor-V and Factor-VIII than commercially available FFP. The Auto-FG contained approximately 10 times the concentration of fibrinogen, Factor-VIII, Factor-XIII, von Willbrand factor and fibronectin and 1.5 times that of alpha 2-Plasminogen inhibitor compared to those of the Auto-FFP. When the Auto-FG was sprayed over diffusely bleeding sites from surgical wounds, hemostasis occurred within 5 s. These results suggest that Auto-FFP and Auto-FG may have sufficient utility and quality to reduce the use of allogeneic blood products.

Flow and antibody binding properties of hydrated fibrins prepared from plasma, platelet rich plasma and whole blood.

Previous studies, using cross-linked fibrin prepared from purified fibrinogen, showed low binding of a fibrin-specific monoclonal antibody designated T2G1 (Procyk et al., Blood 77:1469-75, 1991). In this study we investigated the binding of T2G1 and one other antibody to clots prepared from platelet poor plasma (PPP), platelet rich plasma (PRP) and whole blood. In contrast to our previous study, we used unlabelled antibodies and quantitated the level bound by ELISA, measuring antibody concentration in the non-adsorbed fraction. Antibody T2G1 bound 1.35 +/- 0.10 pmol/pmol fibrin (n = 11) to whole blood columns, 1.64 +/- 0.18 (n = 10) to PRP columns and 1.58 +/- 0.13 (n = 8) to PPP columns. The binding of T2G1 to columns made from purified fibrinogen was 0.78 +/- 0.05 pmol/pmol fibrin (n = 15). An antibody to a conformation-dependent epitope on Fragment D (Fd4-7B3) bound in comparable amounts to the different fibrins. Flow data show that whole blood columns, and also, but to a lesser extent those made with plasma, had a higher flow rate, permeability and fiber mass-length ratio than columns prepared from fibrinogen indicating a more coarse fibrin network. These data show that the presence of other proteins and blood cells, similar to what might occur in vivo, not only lead to an increase in the permeability of gels but also allow for better exposure of some epitopes.

Characterization of the murine plasma fibrinolytic system.

The main components of the murine plasma fibrinolytic system, including fibrinogen, plasminogen, alpha 2-antiplasmin, tissue-type plasminogen activator and plasminogen activator inhibitor-1, were purified to homogeneity and their interactions were quantitated and compared with those of the human counterparts. Initial activation rates of murine and human plasminogen by autologous tissue-type plasminogen activator were comparable (catalytic efficiencies, k2/Km, of 0.4 and 0.6 mM-1 s-1, respectively), but murine plasminogen appeared to be resistant to activation by human tissue-type plasminogen activator (k2/Km = 0.01 mM-1 s-1). Plasminogen activation by tissue-type plasminogen activator was stimulated 100- and 160-fold in autologous murine and human systems, respectively, with saturating concentrations of 0.45 and 0.32 microM, respectively, of CNBr-digested fibrinogen. Nearly quantitative binding (85-90%) of tissue-type plasminogen activator to fibrin was observed both in autologous and heterologous systems. Murine and human plasmin were very rapidly inhibited by autologous and heterologous alpha 2-antiplasmin (second-order inhibition rate constants, k1,app, of 2.1-2.3 x 10(7) M-1 s-1) and murine and human tissue-type plasminogen activator were very rapidly inhibited by autologous or heterologous plasminogen activator inhibitor-1 (k1,app of 1.8-4.9 x 10(7) M-1 s-1). Two-chain murine tissue-type plasminogen activator (added at a concentration of 1 microgram/ml) was inhibited in normal or plasminogen activator inhibitor-1-deficient murine plasma with half-lives of 6.5 min and 4.2 min, respectively, as compared to 80 min for human tissue-type plasminogen activator, suggesting that murine plasma contains proteinase inhibitors other than plasminogen activator inhibitor-1 which efficiently inhibit autologous tissue-type plasminogen activator. Clot lysis experiments in autologous plasma revealed that the murine plasma fibrinolytic system is more resistant to activation than the human system (20-30% clot lysis in 2 h with 100 nM tissue-type plasminogen activator in the murine system, as compared to 50% clot lysis in 2 h with 3.5 nM tissue-type plasminogen activator in the human system). Several mechanisms appear to be involved in this relative resistance observed in the murine system, including resistance of murine plasminogen to quantitative activation and short plasma half-life of murine tissue-type plasminogen activator. Thus, although these quantitative interactions between purified components of the murine fibrinolytic system appear to be comparable to those between the human counterparts, murine plasma clots are > 30-fold more resistant to lysis with autologous tissue-type plasminogen activator than human plasma clots.

Plasmic degradation of fibrin rapidly decreases platelet adhesion and spreading.

Plasmin cleaves fibrin at or near sites involved in platelet recognition and may modulate platelet adhesion and spreading. Using an in vitro system, we have characterized the effects of limited plasmic degradation of polymerized fibrin on platelet adhesion and spreading. As shown by scanning electron microscopy, exposure to plasmin changed the tight fibrillar fibrin surface to a less dense structure with irregular and broken fibers. There was a gradient of proteolytic degradation through the fibrin clot as shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis with the most extensive degradation at the surface. Plasmic degradation resulted in a rapid and progressive decrease in platelet adhesion. Plasmin exposure for 5 minutes resulted in only 6% solubilization of the fibrin but a 56% decrease in platelet adhesion. After 30 minutes of plasmin exposure, spreading of adherent platelets on fibrin also decreased sharply to a minimum of 35% of baseline. Inhibition experiments with specific monoclonal antibodies (MoAbs) indicated that platelet adhesion to undergraded fibrin involved residues within the sequence 566 through 580 of the alpha chain (including the RGDS site), the carboxyl terminal dodecapeptide of the gamma chain, and the amino terminus of the beta chain. MoAb 7E3, reactive with alpha IIb beta 3, inhibited platelet adhesion to fibrinogen by 90% +/- 5%, and to desA fibrin, prepared with Reptilase (American Diagnostica, Greenwich, CT), by 94% +/- 6%, whereas inhibition of adhesion to undegraded desAB fibrin was significantly less (48% +/- 8%, P < .01). The addition of 7E3 to MoAb T2G1, reactive with beta 15-21, significantly increased inhibition to desAB fibrin to 69% +/- 6% (P < .025), suggesting that the newly exposed amino terminus of the beta chain contributes to platelet adhesion. The results show that plasmin exposure of fibrin markedly decreases platelet adhesion and spreading, suggesting that plasmin degradation may play a role in modulating cellular responses to fibrin.

A microtiter plate assay for factor XIII A-chain-fibrin interactions.

Factor XIII A-chain-fibrin interactions regulate factor XIIIa formation and fibrin cross-linking. A microtiter plate assay was developed for studying these interactions. Microtiter plate wells were coated with fibrinogen and converted to fibrin by thrombin. After blocking the wells with bovine serum albumin, factor XIII A-chain was added and binding was monitored by incubating first with anti-factor XIII followed by anti-rabbit IgG-alkaline phosphatase. Enzymatic hydrolysis of p-nitrophenyl phosphate was quantitated by the absorbance at 405 nm. BInding was specific, sensitive, rapid, saturable, and reversible, requiring only nanograms of either factor XIII or fibrin. Binding was time- and concentration-dependent and independent of divalent cations. The bound material was identified as factor XIII A-chain by sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblotting. Factor XIII binding was inhibited > 75% by 250 mM sodium chloride or 250 nM anti-factor XIII IgG. The method was also suitable for demonstrating binding using 0.8% plasma or with r-factor XIII expressed in Saccharomyces cerevisiae or Escherichia coli. This method is suitable for identifying the binding sites that are important for plasma factor XIII activation and factor XIIIa activity.

Uncoupling fibrin from integrin receptors hastens fibrinolysis at the platelet-fibrin interface.

A well-characterized in vitro model system composed of thrombin-stimulated gel-filtered human platelets, fibrin-(ogen), plasminogen, and recombinant tissue plasminogen activator (rt-PA) was used to examine the relationship between platelet-fibrin adhesive interactions and the lytic resistance of a platelet-rich thrombus. Laser light scattering kinetic experiments demonstrated that the ligand-mimetic peptide D-RGDW and an anti-alpha IIb beta 3 monoclonal antibody both inhibited clot retraction, but neither integrin-targeted reagent affected the overall delay in lysis of "bulk" fibrin caused by thrombin-stimulated platelets. However, lysis of the model platelet-rich thrombus did proceed some 30% more quickly when treated with a plasminogen activator inhibitor (PAI)-resistant t-PA variant. Taken together, these results confirm that platelet-released PAI-1 is a major determinant of global lytic resistance. Next events occurring during fibrinolysis in the unique microenvironment near the platelet surface were monitored by scanning electron microscopy and quantitative fluorescence microscopy. Scanning electron micrographs of the partially lysed model thrombus in the presence of 200 mumol/L of D-RGDW showed no platelet aggregates, and fibrin was attached by fewer strands to the platelets. Quantitative fluorescence microscopy, using fluorescein-labeled fibrin, showed that fibrin adherent to the surface of thrombin-stimulated platelets lysed 20% to 50% more slowly than bulk fibrin (monitored in parallel by laser light scattering). Furthermore, this microspectroscopic technique showed that D-RGDW reduced the quantity of platelet-bound fibrin, and accelerated lysis near the platelet surface with both native rt-PA and the PAI-resistant variant. These observations suggest that the dense network of fibrin bound to the platelet surface is protected from fibrinolysis by tissue-type plasminogen activators. Further, uncoupling fibrin from its platelet receptors uniquely hastens fibrinolysis at the cell/fibrin interface.

Fibrin induction of tissue plasminogen activator expression in corneal endothelial cells in vitro.

PURPOSE: Fibrin is deposited in the anterior chamber of the eye in response to injury and can damage corneal endothelial cells (CEC). Fibrin degradation is plasmin dependent and is regulated by the balance between plasminogen activators (PA), tissue-PA (t-PA), urokinase-PA (u-PA), and their inhibitors (PAI). Although several factors can modulate PA/PAI expression in cells, the effect of fibrin is inconclusive. We hypothesized that fibrin can regulate fibrinolysis in the anterior segment by modulating PA/PAI expression in CEC. METHODS: Bovine CEC (BCEC) were treated for 3 to 72 hours with in situ polymerized fibrin (2 mg/ml) +/- 35S-methionine, cycloheximide, or actinomycin D. Polymerization was thrombin catalyzed, and control BCEC were incubated with or without thrombin or polymerization by-products. PA and PAI in conditioned medium, fibrin matrix, and cell fractions were analyzed by PA-specific zymographic and enzymatic assays. RESULTS: Fibrin treatment induced a dramatic (> 20-fold) accumulation of extracellular, fibrin-bound PA. This activity was identified as t-PA by its Mw (70 kD) affinity for fibrin and sensitivity to inhibition by Erythrina. Induction of t-PA was not observed in control BCEC under any condition. Fibrin induction of t-PA was selective because the levels of u-PA (45 kD), PAI-1 (50 kD), or protein synthesis in general were unaffected. Fibrin induction of t-PA was not accompanied by changes in cellular t-PA levels and was dependent on both RNA and protein synthesis. CONCLUSIONS: Fibrin selectively induces t-PA expression in CEC. Induced t-PA is released extracellularly and binds exclusively to the fibrin matrix. These findings suggest a role for fibrin and CEC in the regulation of fibrinolysis in the anterior segment of the eye.