Plasminogen-free Fibrinogen Research Articles

Decreased plasmin resistance by clots of a homophenotypic Aα R 16H fibrinogen (Kingsport, slower fibrinopeptide A than fibrinopeptide B release)

Reported evidence of a role in fibrinolysis by fibrinopeptide (Fp)B-dependent intermolecular fibrin polymerization contacts and of reversed FpA/FpB release sequence from fibrinogen Kingsport led us to investigate the fibrinolytic properties of Kingsport clots. Clot lysis was induced by either plasmin (pH 7.4) or by a mixture of plasminogen and recombinant tissue plasminogen activator and measured by lysis time and by turbidity (350 nm) time course. Clots were formed by thrombin from plasminogen-free fibrinogen (pH 7.4, 8 mmol/l CaCl2), with or without 40 nmol/l factor XIII or 20% afibrinogenemic plasma. Displaying no differences from corresponding normal controls were (a) lysis of repolymerized fibrin clots, and (b) chromogenic measurements of fibrin-stimulated Glu-plasminogen activation by recombinant tissue plasminogen activator. By contrast, thrombin-induced fine and coarse network clots (n = 7) displayed faster turbidity loss than corresponding normal controls and shorter lysis times ranging 31-55% of controls. Comparison of clots of fibrinogen fractions lacking approximately 90% of their alpha chain carboxyl terminal regions, n = 2, also displayed faster plasmin-induced lysis than corresponding controls. To assess the role of FpB release-dependent intermolecular polymerization contacts, clots were prepared in the presence of three molar excess antibeta 15-42 immunoglobulin G, n = 2, and displayed no differences in plasmin-induced lysis from nonimmune immunoglobulin G controls. The reversed FpA/FpB release sequence from Kingsport fibrinogen resulted in clots with decreased resistance to plasmin. We suggest that both markedly slow polymerization and decreased plasmin resistance played causative roles in the hemorrhagic diathesis associated with this dysfibrinogen.

Expression and functional characterization of chitribrisin, a thrombin-like enzyme, in the venom of the Chinese green pit viper ( Trimeresurus albolabris)

In the present study, functionally active, recombinant chitribrisin, which is a thrombin-like enzyme in the venom of the Chinese green pit viper ( Trimeresurus albolabris), was expressed and purified using a prokaryotic system. The fusion protein of chitribrisin, together with TrxA and 6× His via an E. coli expression vector pET-32a(+), was successfully expressed in E. coli BL21(DE3) cells. After the fusion protein was isolated and purified by chelated Ni 2+ resin and specifically cleaved by enterokinase, the recombinant chitribrisin showed a strong fibrinogenolytic activity against the α and β chains of human plasminogen-free fibrinogen and weak fibrinogen clotting activity. In addition, multiple sequence alignment revealed that the expressed chitribrisin was homologous to GPV-TL1 and GPV-TL2 from the snake venom of T. albolabris from central Thailand in terms of the amino acid sequence identities. However, there were some differences in the amino acid sequences of the proteins from the same species from different geographical locations. The causes for the geographical variation in TELs in the same species remain to be investigated. Mutagenesis of chitribrisin should be performed in future studies to study the structural and functional relationship and to identify the critical residues responsible for the properties of the thrombin-like enzyme.

Enhanced Fibrinolysis of Coarse Fibrin Networks Reflects Cleavage of a B Knob Containing Fragment(s)

Coarse fibrin network clots display faster fibrinolysis than their fine network counterparts (Collet et al, Arterioscler Thromb Vasc Biol 2000, 20: 1354–61), and this is ostensibly of pathophysiologic relevance. Since relative to fine networks, coarse networks can be predicted to possess a relatively higher content of “B knob” (-”b” and/or -”a hole”) contacts, we investigated whether or not such contacts are more susceptible to plasmin than their A knob counterparts. Owing to much slower FpA than FpB release from Aα G16H fibrinogen, its clots acquire a high content of B knob contacts throughout polymerization (Galanakis et al, Bld Coag Fibrinol , 2007, 18: 731–737) and were therefore used for this purpose. Plasminogen-free fibrinogen was obtained by Lys-Sepharose chromatography, and clots of isolates from one homophenotypic (HM) and one (unrelated) heterophenotypic (HT) Aα G16H proband were compared with normal controls. Plasmin digests of fibrinogen, monitored by PAGE-SDS, disclosed no differences from controls. Thrombin was added to fibrinogen (1–5 μM), pH 7.4, 8 mM CaCl2, with or without 20% afibrinogenemic plasma so as to achieve > 96% clottability. For lysis times, clots were suspended in 2 volumes of a lysis solution. For turbidity (340 nm) measurements the lysis solution was carefully layered on the cuvette clot top. To obtain clots of similar turbidities 0.8 to 1.2 thrombin U/ml were used. The lysis solution contained either 2 μM plasmin in buffer (37º) or 80 μM rtPA with or without 50 μM plasminogen. Selected clots were prepared in solutions containing either anti-β 15–42 IgG (Mab, 3 mols/mol fibrin) or the GlyHisProArgProOH peptide (50 mols/mol fibrin). Using fibrin (70 nM)-enhanced glu-plasminogen (250 nM) activation by rtPA (10 μM) and S2251 substrate (40 mM), no differences (405 nm) among control, HM, and HT fibrin could be shown in two separate sets of experiments. Clot lysis times (n=3) were 31% (HM) and 54% (HT) of controls, respectively. Turbidity measurements (n=3) also disclosed HM and HT clot lysis onset ~2 and 1.5 fold faster than controls, but similar lysis rates. Duplicate clots lacking >90% of intact αC (i.e. fraction I-9) yielded similar differences. Also assessed by turbidity, clots formed in Mab containing buffer lacked lysis differences from non-immune IgG controls. Using turbidity measurements of normal fibrin, lysis differences between coarse and fine clots were abolished when clots had been obtained from fibrinogen/GlyHisProArgProOH solutions (n=2). We conclude that B knob contacts are constitutively more susceptible to plasmin than their A knob counterparts. This property is attributable to faster release by plasmin of at least one of the two β 15–53 (B knob containing) fragments relative to release of either or both α 17–19 (A Knob) fragments from the DDE complex (Olexa et al, Biochemistry 1981, 20:6139–45).

Bivalent Binding to γA/γ′-Fibrin Engages Both Exosites of Thrombin and Protects It from Inhibition by the Antithrombin-Heparin Complex

Thrombin exosite 1 binds the predominant gamma(A)/gamma(A)-fibrin form with low affinity. A subpopulation of fibrin molecules, gamma(A)/gamma'-fibrin, has an extended COOH terminus gamma'-chain that binds exosite 2 of thrombin. Bivalent binding to gamma(A)/gamma'-fibrin increases the affinity of thrombin 10-fold, as determined by surface plasmon resonance. Because of its higher affinity, thrombin dissociates 7-fold more slowly from gamma(A)/gamma'-fibrin clots than from gamma(A)/gamma(A)-fibrin clots. After 24 h of washing, however, both gamma(A)/gamma'- and gamma(A)/gamma(A)-fibrin clots generate fibrinopeptide A when incubated with fibrinogen, indicating the retention of active thrombin. Previous studies demonstrated that heparin heightens the affinity of thrombin for fibrin by simultaneously binding to fibrin and exosite 2 on thrombin to generate a ternary heparin-thrombin-fibrin complex that protects thrombin from inhibition by antithrombin and heparin cofactor II. In contrast, dermatan sulfate does not promote ternary complex formation because it does not bind to fibrin. Heparin-catalyzed rates of thrombin inhibition by antithrombin were 5-fold slower in gamma(A)/gamma'-fibrin clots than they were in gamma(A)/gamma(A)-fibrin clots. This difference reflects bivalent binding of thrombin to gamma(A)/gamma'-fibrin because (a) it is abolished by addition of a gamma'-chain-directed antibody that blocks exosite 2-mediated binding of thrombin to the gamma'-chain and (b) the dermatan sulfate-catalyzed rate of thrombin inhibition by heparin cofactor II also is lower with gamma(A)/gamma'-fibrin than with gamma(A)/gamma(A)-fibrin clots. Thus, bivalent binding of thrombin to gamma(A)/gamma'-fibrin protects thrombin from inhibition, raising the possibility that gamma(A)/gamma'-fibrin serves as a reservoir of active thrombin that renders thrombi thrombogenic.

Basics and practice in evaluating plasminogen.

There exist different ways of assays of plasminogen which give information about different properties of this proenzyme. The concentration of plasminogen can be determined by its antigenicity. Since the normal concentration of plasminogen in plasma is between 15 and 25 mg/dl the test can be carried out by simple methods such as radial immunodiffusion on Partigen plates. The possibility of errors is small and there is no need of special apparatus. The disadvantages are the lapse of 24 h until the result is available and the fact that the knowledge of the concentration does not give any information about the activity. The activity can be measured by different coagulation tests. A typical assay would involve activation of plasminogen to plasmin, addition of plasminogen-free thrombin and measuring of the lysis time. The result is however, dependent on more than one variable. Plasmin is rapidly inhibited by alpha-2-antiplasmin (APL) and there is also a dependence of the lysis time on the amount of clottable fibrinogen in the test system. Better results can be obtained by the use of diluted test plasma and addition of a constant amount of plasminogen-free fibrinogen. A different way would be the use of the euglobulin fraction instead of plasma. This has however, the possible disadvantage of incomplete precipitation of plasminogen. Instead of coagulation tests the activity can also be determined when diluted activated plasma is placed on plasminogen-free fibrin plates and the amount of lysis in the plate is recorded. All assays of this group also depend on the method of activation of plasminogen.(ABSTRACT TRUNCATED AT 250 WORDS)

高濃度ｔｈｒｏｍｂｉｎの凝固・線溶に及ぼす影響について

Since high-concentrate thrombin preparations have excellent effectiveness for local hemostasis in patients with coagulopathies, the blood coagulation mechanism in the presence of high-concentrate thrombin, using purified thrombin and plasminogen-free fibrinogen was investigated.1) The blood coagulation analyzer, Coag-Stat was utilized in the kinetic study of fibrinogen to fibrin transformation.The initial velocity of fibrin formation was accelerated as the concentration increased and the amounts of fibrin were gradually decreased in relation to the given amount of sodium heparin.The general result of this investigation was that anti-thrombin III and a large amount of heparin caused neutralization of dosage of 100 units/ml thrombin with neutralization increasing with heparin dosage.2) The influence of coagulum stiffness, which depended on the thrombin dose, was investigated by thrombelastograph.The higher concentration thrombin solutions above 10 units/ml dosage had indicated a fibrinolytic activity by reason of “ma” value decrease.This was particularly remarkable with dosages of 100 units/ml and 1, 000 units/ml thrombin.3) The progress of the digestion of fibrin by high-concentrate thrombin was also monitored by SDS-polyacrylamide gel electrophoresis of the lysed fibrin at frequent intervals during the 180 minute digestion period.The gels stained with Coomassie blue, which were applied to plasminogen-free fibrinogen, showed the α, β and γ chains. After 60, 120 and 180 min of digestion the α chains were present in much smaller amounts and appear to have been extensively degraded.4) From the standpoint of prophylactic and continual effectiveness in postoperative bleeding, we also examined the diffusion state of high-concentrate thrombin from the fibrin network. In the investigation of thrombin diffusion, high-concentration thrombin was diffused on an agarose plate, containing plasminogen-free fibrinogen, and was seen to precipitate an increasing amount of fibrin.From the result of these study, the accelerative function of thrombin in blood coagulation was again confirmed.High-concentrate thrombin penetrated and diffused into the fibrin network during a period of several hours.We presume that this function produces continual hemostatic efficacy.In a practical clinical application of thrombin, it seems that the plasma inhibitor and released heparinoid affect the preparation.From this experiment about 100 units/ml of thrombin were neutralized under AT-III and extra doses of heparin.Moreover, taking many kinds of plasma inhibitors into consideration, this approach showed that clinically more than 1, 000 units/ml thrombin was more effective.

高濃度ｔｈｒｏｍｂｉｎの止血効果について

Since high-concentrate thrombin preparations have excellent effectiveness for local hemostasis in patients with coagulopathies, the blood coagulation mechanism in the presence of high-concentrate thrombin, using purified thrombin, and plasminogen-free fibrinogen was investigated. The blood coagulation analyzer, Coag-Stat was utilized in the kinetic study of fibrin transformation. The first stage of fibrin precipitation speed was accelerated as the concentration increased and amounts of fibrin were gradually decreased in relation to the amount of sodium heparin given. The general result of this investigation was that anti-thrombin III and a large amount of heparin caused neutralization of dosages of 100 units/ml thrombin with neutralization increasing with heparin dosage. In the investigation of thrombin diffusion, high-concentrate thrombin was diffused on an agarose plate, containing plasminogen-free fibrinogen, and was seen to precipitate an increasing amount of fibrin.From the result of this study, the accelerative function of thrombin in blood coagulation was confirmed again. High-concentrate thrombin penetrated and diffused into the fibrin network during scores of hours. We presume that this function brings about continual hemostatic efficacy. In a practical clinical application of thrombin, it seems that plasma inhibitor and released heparinoid affect the preparation. From this experiment about 100 units/ml of thrombin was neutralized under AT-III and extra doses of heparin. Moreover, taking many kinds of plasma inhibitors into consideration, this approach showed that clinically more than 1000 units/ml thrombin was more effective.

H-D-val-leu-lys-aminoisophthalic acid, dimethyl ester, is sensitive to streptokinase-activator not to plasmin.

The ability of human plasma and purified human plasmin and plasminogen to hydrolyze a new synthetic substrate, H-D-valine-leucine-lysine-5-aminoisophthalic acid, dimethyl ester, ditrifluoroacetate, was studied. Contrary to published data, we found this substrate was only minimally hydrolyzed by plasmin or urokinase-treated plasminogen or plasma. Plasminogen-free bovine fibrinogen was readily degraded by plasmin and urokinase-activated plasminogen. However, in the presence of streptokinase, the synthetic substrate was highly sensitive to human plasma and purified plasmin and plasminogen. Apparently, the substrate is specific for streptokinase-plasmin and streptokinase-plasminogen activators, not for plasmin.

Structure of alpha-polymer from in vitro and in vivo highly cross-linked human fibrin.

Peptides derived from plasmic and cyanogen bromide (CNBr) cleavage of highly cross-linked fibrin were isolated and characterized by sodium dodecyl sulfate-gel electrophoresis, amino acid analyses, cyanoethylation, and NH2-terminal analyses. Extended plasmic digestions of human fibrin containing four epsilon-(gamma-glutamyl)lysine cross-links per molecule produced a peptide of alpha-chain origin (Mr congruent to 21,000) which was comprised of a small donor peptide cross-linked to the acceptor site peptide from the middle of the alpha-chain. CNBr cleavage of highly cross-linked in vitro fibrin or of fibrin from a spontaneously formed in vivo arterial embolus produced about three cross-linked species of molecular weights 30,000 to 40,000, each of which contained the largest CNBr fragment (Mr = 29,000) from the alpha-chain. The predominant cross-link-containing CNBr fragments derived their donor group from the near COOH-terminal region of the alpha-chain as judged by difference amino acid compositions and NH2-terminal analyses. Additionally, cross-linked fragments of molecular weights 68,000 to 70,000 which appeared to contain two acceptor site peptides (Mr = 29,000) were detected in minor amounts in the CNBr digests of fibrin formed from whole plasma or from purified, plasminogen-free fibrinogen. No larger polymeric cross-linked CNBr fragment was generated from any of the highly cross-linked fibrin preparations examined. A model for the predominant mode of alpha-chain polymerization is proposed.

Studies on the Fibrin-Agarose and Agar Plate Methods for Measurement of Fibrinolytic Activities

The methods for assessment of fibrinolytic activity has not been well established, although the fibrinogen plate method developed by Astrup et al. has been utilized as an assay method of fibrinolytic activity so far. There are, however, some difficulties associated with the preparation, use and interpretation of the Astrup fibrin plate. Our standard and plasminogen free fibrin agarose and agar plate method is made with a little modification of Bisohp's fibrin plate method.Standardized method is as follows. The solution is composed of 0.12% plasminogen free fibrinogen (Daiichi Seiyaku Co. Tokyo) and 1.25% agarose (1% agar) in 0.005M phosphate buffer (pH 7.5).Sodium azide is added at the rate of 0.1% to agarose solution as an antiseptic. Fibrin plate using 2.5×7.0×0.15cm plastic plate is made with 2.52ml of a above solution and added 0.5ml of thrombin solution which is adjusted to 50 unit per one ml of saline. Then washed in distilled water in a moment exactly 15 seconds after addition of thrombin. Plate is covered with a strip of filter paper and seald airtight in plastic bag. Those plates are kept at 4°C before use. Wells of 2mm of diameter are mechanically punched in the fibrin plate just before experiment.Using this method, several interesting findings are obtained in our study on the several preparatory codition, effect of pH, fibrinogen consentration, agarose consentration…etc, of the fibrin agarose plate.

A simple assay method for the activity of plasma antiplasmin and SK-activated plasmin on the basis of fibrin clot lysis

A simple assay method based on the fibrin clot lysis for the activity of plasma antiplasmin and SK-activated plasmin (a potential activity of plasminogen and proactivator) was studied, and the evaluation was made on the controlled conditions, theoretical bases and the applicability of this method.The materials used here were the following, Plasmin; Midorijuzi Co. Streptokinase; Varidase of Lederle Lab. Thrombin; bovine origin, Mochida Co. Plasminogen-free fibrinogen; prepared by affinity chromatography from bovine fibrinogen (Miles Co.) using Lysine-Sepharose. Lysine-Sepharose; prepared according to the method of P. Cuatrecasas with Sepharose (Pharmacia Co.).Separation of antiplasmin fraction and SK-activated plasmin fraction from plasma was carried out by affinity chromatography using Lysine-Sepharose. In this procedure a specially designed column was used. Antiplasmin fraction was obtained from 1.0ml plasma through the column as 6.0ml sample. The plasminogen fraction was also obtained as 6.0ml sample.The assay system for determining antiplasmin is Antiplasmin fraction 0.1mlPlasmin solution (1.0cu/ml) 0.1mlPlasminogen-free fibrinogen 0.3ml (183mg/dl)Thrombin (20u/ml) 0.05mland for SK-activated plasmin is Plasminogen fraction 0.1mlSK solution (110u/ml) 0.1mlPlasminogen-free fibrinogen 0.3ml (183mg/dl)Thrombin (20u/ml) 0.05mlThe activity was measured by clot lysis time. The end point of clot lysis was taken at the point on which all the bubbles in the clot reached on the surface. The calculation of the activity value was ref eyed to the standard curve of plasmin. The value of antiplasmin was calculated from the remained activity of the standard plasmin.It was found that the antiplasmin fraction obtained by affinity chromatography contained α2-macroglobulin and α1-antitrypsin. The plasminogen fraction showed both SK and UK reactivity and also proactivator activity.When this method was applied on normal subjects and leukemic patients, the antiplasmin value was higher in patients plasma than in normal plasma, while the SK-activated plasmin value was slightly lower in the patients.

Unique Pattern of Fibrinogen Cleavage by Human Leukocyte Proteases

An extract of human leukocytes was prepared which had marked fibrinogenolytic activity, as determined by its effect upon the thrombin clotting time of plasminogen-free fibrinogen. When incubated with a 1/10 dilution of the extract of 108 leukocytes, fibrinogen became incoagulable within 30 min. The activity of the leukocyte extract was unaffected by 0.2 M epsilon-amino caproic acid and minimally affected by 100 U/ml of trasylol, but it was completely inhibited by 0.1 mg/ml soybean trypsin inhibitor and by 1% plasma. The degradation products of leukocyte protease-treated fibrinogen were clearly different from those of plasmin-treated fibrinogen when examined by polyacrylamide gel electrophoresis. The pattern of cleavage of the amino terminal ends of the Aα and Bβ chains of fibrinogen by the leukocyte extract was determined using radioimmunoassays specific for fibrinopeptides A and B. The fibrinopeptides themselves were not cleaved, but slightly larger dialyzable fragments containing the peptides were cleaved from the amino terminal ends of the Aα and Bβ chains. These larger fragments were clearly distinguished from the fibrinopeptides themselves by immunochemical means. The pattern of release of the fibrinopeptide-containing segments by the leukocyte proteases was different from the patterns described for thrombin, plasmin, trypsin, and certain snake venoms. The results suggest that the specificity of the leukocyte proteases for fibrinogen is unique, and provide a technique for further study of the role of these enzymes in normal and abnormal fibrinogenolysis.

Blood fibrinolytic activity before and after oral surgery

Blood samples were collected in cold O.1 M sodium citrate from 30 healthy young persons whose mandibular impacted molars were to be removed. Blood sampling was repeated immediately after operation. Fibrinolytic activity of the euglobulin fraction, precipitated at pH 5.9, was measured by the fibrin plate method. Three different fibrinogen solutions were used: 1) bovine fibrinogen, 2) bovine plasminogen-free fibrinogen, and 3) human fibrinogen. The differences between preoperative and postoperative fibrinolytic activity were evaluated. No increase of fibrinolytic activity was seen in general, indicating that oral surgery does not necessarily influence the fibrinolytic activity of the blood. Dividing the patients according to sex or to a long/short surgical procedure did not influence the activity either. However, in four patients who developed alveolitis sicca (dry socket), a significant rise of activity on all the fibrin plates was seen ( P<0.01) when compared with the variations measured in patients with normal healing. The result suggests that a rise of blood fibrinolytic activity of a certain magnitude during oral surgery may predispose to the development of alveolitis sicca. Four females who took oral contraceptives showed a higher postoperative fibrinolytic activity ( P<0.05) than the other females. The importance of this observation is to be investigated in further studies.

A Method for the Measurement of Fibrinolytic Activity Based on One Dimensional Diffusion Using Small Glass Tubes

The optimal conditions for the measurement of the fibrinolytic factors of plasma were examined using human and bovine plasminogen-rich fibrinogen or plasminogen-free fibrinogen as the substrates using the one dimensional diffusion method. The results were as follows: 1. There was no essential difference found between using human or bovine fibrinogen. 2. The levels of proactivator-plasminogen and plasminogen could be measured while using either plasminogen-rich or plasminogen-free fibrinogen. But, in using the latter, the proactivator-plasminogen level could not be measured, if a final concentration of more than 2,000 Christensen units of streptokinase were employed. 3. When using plasminogen-rich fibrinogen, anti-plasmin(s) and anti-activator(s) could be measured while using urokinase and plasmin, but not while using streptokinase. However, further study should be given to the measurement of the inhibitors, when using plasminogen-free fibrinogen.

Salivary fibrinolytic activity before and after oral surgery estimated on different types of fibrin

Mixed saliva was collected from 25 healthy young people from -whom mandibular impacted molars were to be removed. Collection of saliva was repeated immediately after operation and 5 days later. Fibrinolytic activity was measured by the fibrin plate method employing four different solutions of fibrinogen: (1) bovine plasmino-gen-rich fibrinogen; (2) bovine fibrinogen with human β-plasminogen; (3) bovine plasminogen-free fibrinogen; and (4) human fibrinogen. After 20 hours of incubation the fibrinolytic activity was measured. The preoperative samples all showed activator activity, whereas plas-min activity seldom was detectable. The measured activity was significantly higher on the human fibrin plates than on any of the other plates. No difference was detected between the fibrin made of solutions (1) and (2), indicating that the possible presence of streptokinase in saliva is unimportant for the quantitative fibrinolytic activity. Immediately after surgery the activity had decreased or disappeared on the bovine fibrin plates. Five days after operation, fibrinolytic activity had not returned to the normal preoperative level. The findings show that salivary fibrinolytic activity after oral surgery is usually controlled by the fibrinolytic inhibitors present in plasma and in the exudate of the oral ulcer.

A rapidly produced 125I labelled autologous fibrinogen: in vitro properties and preliminary metabolic studies in man.

The properties of fibrinogen extracted by a precipitation method using glycine at ambient temperatures near neutral pH are described. The simple and reproducible method gives a 73% yield of high purity plasminogen-free fibrinogen in 45 minutes from small volumes of plasma. The protein extract was labelled with 125I using chloramine-T under conditions optimal for fibrinogen stability. The extraction procedure, radio-iodination, desalting, and sterilization take only 70 minutes for completion from the time donor blood is received in the laboratory. The methods, using a specially developed extraction vessel and desalting/sterilizing column, can be used in a small hospital laboratory. Autologous fibrinogen can thus be extracted from patients' blood, eliminating the risk of transmitting hepatitis when it is re-administered. The autologous material, which is 97% clottable and contains less than 0-05% free iodide, is being routinely used as a diagnostic tool in the detection of deep vein thrombosis. The high purity of the preparation facilitates metabolic studies and in vitro experimental work. In vivo results show a mean half-life in three normal volunteers of 3-95 days and a catabolic rate of 25-23% per day with the extravascular space estimated as 24-86%. In 30 surgical patients an expected reduced half-life in plasma was determined with a mean of 3-1 days.

In vitro and in vivo studies on the effects of Defibrase (snake venom, Bothrops Marajoensis) on coagulation, fibrinolysis and platelets, and its defibrinogenating activity, compared with those of thrombin

In vitro and in vivo studies obtained on the effects of Defibrase on coagulation, fibrinolysis and platelets, and its defibrinogenating activity, compared with those of thrombin were performed.The following results were:1) Normal citrated human plasma clotting activity with Defibrase was weaker than that with thrombin on the basis of the same NIH units.2) There were no effects on coagulation and fibrinolysis of plasma by Defibrase, but the decrease of factor V and factor VIII activity were observed dy thrombin as a serine protease. 0.16units/ml of Defibrase and thrombin in final concentration were used in this experiment.3) There was found that the susceptibility of fibrin clotted by Defibrase by fibrinolytic enzymes (urokinase, streptokinase and plasmin) was stronger than that of fibrin clotted by thrombin.These facts were observed by fibrin plate method using plasminogen rich fibrinogen and plasminogen free fibrinogen, and platelet aggregation examined by using EL-aggregometer.4) In vivo studies using rabbit, there were observed decreases of factor VIII, fibrinogen, platelets and ratio of platelet retention to glass beads column.From these results it was concluded that Defibrase would be effective in defibrination of blood without making hypercoagulation and hyperfibrinolysis. And also decreases of factor VIII, platelet count and platelet retention ratio were observed in vivo study by infusion of Defibrase into rabbits.

Immunologic relationship among fibrinolysins secreted by cultured mammalian tumor cells

Summary Antibodies were produced in goats by immunization with highly purified fibrinolysin(s) secreted by rat ovarian and breast carcinoma tumor cells in culture. The fibrinolysin had previously been shown to be different than a secreted plasminogen activator and biochemically distinct from plasmin in human, horse, and calf sera. After fractionation of the goat antiserum to eliminate activatable fibrinolytic factors and inhibitors, the antibodies in the isolated IgG fraction were characterized as 7S heat-stable (56°C, 30 min) non-precipitating types that neutralized fibrinolytic activity. Three assay methods were used to test the inhibitory effect of the IgG fraction: fibrin-agar plates (heated and unheated); release of [125I]-peptides from plasminogen-free fibrinogen, and the hydrolysis of N-p-tosyl- l -arginine methyl ester. Antibodies to rat tumor fibrinolysin cross-reacted with fibrinolysin(s) produced by cultured human melanoma and breast carcinoma cells, to some preparations of urokinase-activated human plasmin, but had no effect on purified horse and calf plasmin. Rabbit anti-human plasminogen antibodies cross-reacted with the rat tumor fibrinolysin. Inhibition of human plasmin activity was greatly reduced after absorption of goat anti-rat tumor fibrinolysin IgG fraction with human plasminogen, but the fraction remained completely inhibitory for the rat tumor fibrinolysin. Thus, the fibrinolysin secreted by the rat tumor cells was immunologically distinct from human, horse, and calf plasmin.

Characteristics of fibrinolysin secreted by cultured rat breast carcinoma cells

Summary Fibrinolysin secreted by cultured R2426 rat breast carcinoma cells has been purified and partially characterized. Three methods of assay were used: the hydrolysis of [ 125 I]fibrinogen and N-p -tosyl- l -arginine methyl ester (TAME), and clearing of fibrin-agar plates (heated and unheated). Two types of fibrinolytic activity were identified: a plasminogen activator and a direct fibrinolysin. The fibrinolysin had direct hydrolytic action on plasminogen-free fibrinogen, fibrin, and TAME. The products of fibrinogen hydrolysis by the tumor cell fibrinolysin differed from those of human plasmin. It was heat stable (60°C, 12 h), and was inhibited by e-aminocaproic acid and soybean trypsin inhibitor. Ethylenediaminetetraacetate (EDTA) inhibited the action of the tumor cell enzyme on TAME, but not on human fibrinogen. The direct fibrinolytic activity could be separated from plasminogen activator activity by gel filtration on Sephadex G-200, and the molecular weight was estimated to be approx. 110 000 D.

A simple, large scale method for preparation of plasminogen-free fibrinogen

A simple method, involving lysine-Sepharose chromatography was developed for large scale preparation of fibrinogen free from detectable plasminogen. After incubation of the preparation with human urokinase under appropriate conditions for over 24 hours at 37°C, no evidence of degradation of the subunits of fibrinogen was detected by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Moreover, fibrin clots formed by addition of purified bovine thrombin were not lyzed during incubation for more than 72 hours. Large scale preparation of plasminogen-free fibrinogen requires only a smal column and the treatment takes only a short time, so this method seems much better than those used previously, such as Sephadex G-200 gel filtration or precipitation with ethanol-lysine or ethanol-epsilon aminocaproic acid.