Rabbit Plasminogen Research Articles

A role of streptokinase in experimental post-streptococcal glomerulonephritis

Post-streptococcal glomerulonephritis (PSGN) refers to the sequela of the acute infection, caused by Streptococcus pyogenes (group A streptococcus, GAS). This pathology has been studied for a long time, and today attempts are being made to identify the products of their life activity, able to initiate an immunopathological process in kidneys. Most attention has been paid to streptokinase, the enzyme transforming blood plasminogen into plasmin, capable, together with the plasmin receptor (NAPlr), of damaging the glomerular tissue, as well as activating the complement system. The aim of the study was to consider two tasks: to study the ability of the GAS-obtained enzyme to transform plasminogen of different species into plasmin as well as to study its role in the development of PSGN in rabbits having subcutaneously implanted tissue chambers. The animals were infected by inoculating GAS cultures into the chambers. Materials and methods. GAS strains of M types 1, 12, 22 and their ska– isogenic mutants were used in the study. Purified plasminogen preparations were isolated from fresh human, rabbit or mouse plasma by using chromatographic column with Lysine Sepharose 4B. To reveal the ability of streptokinase to activate plasminogen into plasmin, its preparation at a concentration of 1 mg/ml was added to 10 ìg of purified human, rabbit or mouse plasminogen. The concentration of plasmin was defined photometrically using S-2251 (Chromogenix, USA). To reproduce PSGN, four chambers were implanted under the skin in each rabbit; after the complete wound healing animals were infected and observed for three weeks. On day 14, the animals were treated with benzylpenicillin. The kidneys from survived rabbits were subjected to immunohistology analysis. Results. During in vitro experiments, M1, M12 and M22 GAS streptokinase showed distinct functional activity on human plasminogen, transforming it into plasmin: optical density indicators at ë = 405 nm were 0.4–0.7 compared with the negative control (ОD 0.001). Streptokinase did not activate mouse plasminogen (ОD = 0.001) and exerted quite a weak effect on transformation of the rabbit plasminogen into plasmin (ОD = 0.002). In experiments on PSGN induction in rabbits, we failed to detect streptokinase involvement, because no differences between initiation of glomerulonephritis by wild strains or ska– isogenic mutants were identified. Mutant strains deficient in the gene responsible for streptokinase synthesis but retained ability to bind rabbit and human IgG, caused morphological changes in kidney tissue, specific for PSGN. In addition, a comparative analysis of PSGN “rabbit” and “mouse” models developed by the same technology, was carried out and led to opposing conclusions regarding a role of streptokinase in pathogenesis of experimental glomerulonephritis. The role of IgG Fc-binding activity of GAS in development of experimental PSGN is discussed.

Immunological Quantitation of Rabbit Plasminogen Activator Inhibitor-1 in Biological Samples

Two immunoassays for the specific quantitation of rabbit plasminogen activator inhibitor-1 (PAI-1) antigen and activity in biological samples were developed and applied for the evaluation of PAI-1 in rabbits. Levels of PAI-1 antigen in rabbit plasma were 9.8+/-4.6 ng/ml (mean +/- SD, n = 6), with a corresponding value of 20.5+/-13.5 ng/ml for PAI-1 activity. In rabbit serum PAI-1 antigen was 11.8+/-4.9 ng/ml (n = 6) and PAI-1 activity was 2.9+/-2.0 ng/ml (n = 6). Endotoxin injection (20 microg/kg, i.v.) induced a time-dependent increase of both PAI-1 antigen and PAI-1 activity levels in rabbit plasma, eventually resulting in a 40- to 90-fold increase (p<0.0001 vs. baseline). A linear correlation was found between PAI-1 antigen and activity levels in normal plasma (r = 0.90, n = 6, p<0.05) and in plasma from endotoxin-treated rabbits (r = 0.98, n = 20, p<0.001). Analysis of PAI-1 antigen and activity in lysates of washed rabbit platelets revealed the absence of PAI-1 (i.e. <0.03 ng/10(8) platelets). In conclusion, development of specific immunological assays allowed the quantitation of PAI-1 in rabbit samples. In striking contrast to other species (human, rat, mouse, pig) rabbit platelets lack detectable amounts of PAI-1 (i.e. >100-1000 fold lower vs other species studied). This observation may have important implications for the use of experimental rabbit models especially in studies on the role of platelets in various pathological conditions including thrombosis and atherosclerosis.

Production and characterization of specific bovine plasminogen monoclonal antibodies.

A collection of monoclonal antibodies was raised against bovine plasminogen by immunizing mice with purified plasminogen. More than 300 positive clones were obtained in 1 fusion experiment (1300 hybrids tested). Four types of antibodies were characterized through their relative binding to plasmin and plasminogen in ELISA. About one-third was strictly specific to plasminogen; the selectivity of this group was confirmed by immunoblots and BIA-core analysis. Cross-reactivity against horse, human, pig, sheep, rabbit, and bovine plasminogens was tested; 41% were strictly bovine specific.

Catabolism of plasminogen glycoforms I and II in rabbits: Relationship to plasminogen synthesis by the rabbit liver in vitro

The metabolisms of the two glycoforms of rabbit plasminogen have been compared in rabbits. Plasminogen I and II (ratio in plasma, 1:2.2) differ only in glycan content: plasminogen I probably possesses one N-glycan and one O-glycan, and plasminogen II only one O-glycan. New Zealand White (NZW) rabbits were injected intravenously with 125I-plasminogen I and 131I-plasminogen II, and blood samples were taken at regular intervals over 5 days. Kinetic behaviors were determined from protein-bound radioactivities using a three-compartment model. Fractional catabolic rates for plasminogen II in the vascular space (2.42 d −1) and the total body (0.56 d −1) were significantly greater than those measured for plasminogen I (1.12 and 0.45 d −1); half-lives were 1.53 and 1.23 days for plasminogen I and II, respectively ( P < .01). Fractional distributions among the vascular, noncirculating vascular, and extravascular compartments were 0.41, 0.13, and 0.46 for plasminogen I, and 0.23, 0.11, and 0.65 for plasminogen II. From these data, we determined that plasminogen II was catabolized 4.8 times more rapidly than plasminogen I and was quantitatively contained largely in the extravascular space. By comparison, perfusion of rabbit livers ex corpora showed that plasminogen II was synthesized and released 5.0 times faster than plasminogen I over a 5-hour period. The possible roles for these glycoforms in vivo with respect to their different turnover rates and compartmental distributions are discussed.

The state of tryptophan-containing sites of human, bovine and rabbit plasminogens with changing solution pHs

1. 1. The state of tryptophan-containing sites is proved to be stable by intrinsic tryptophan fluorescence with pH 5–8, 7–9, and 6–9 in human, rabbit and bovine plasminogen molecules, respectively. 2. 2. With pH < 5.0 tryptophan-containing sites of human zymogen (in contrast to rabbit and bovine ones) undergo conformational transitions. 3. 3. With the shift of solution pH from 9 to 12 tryptophan-containing sites of human and rabbit plasminogens are partially disorganized, while tryptophanyls become more available for solvent. 4. 4. Tryptophan-containing sites of bovine plasminogen molecules are less mobile in structure during changes of solution pH.

Plasminogen activator production and enhanced development in medium containing plasminogen or plasmin by rabbit embryos in vitro

A series of experiments was conducted to evaluate plasminogen activator production and effects of supplementing culture medium with plasminogen or plasmin on development of rabbit embryos in vitro. In Expt 1, 495 one- to two-cell embryos were cultured in Ham's F-12 with 15 mg BSA ml-1 containing 0, 30, 60 or 120 micrograms porcine plasminogen ml-1 or 0, 75, 150 or 300 micrograms rabbit plasminogen ml-1. Percentages of embryos developing to the expanded blastocyst, hatching blastocyst and hatched blastocyst stages were greater (P < 0.05) in medium with 120 micrograms porcine plasminogen ml-1 than in the absence of plasminogen. More (P < 0.05) embryos developed to the blastocyst, expanded blastocyst and hatching blastocyst stages in medium with 300 micrograms rabbit plasminogen ml-1 than in the absence of plasminogen. In Expt 2, 216 one- to two-cell embryos were cultured in medium with 0, 30, 60 or 120 micrograms porcine plasminogen ml-1 for 96 h, fixed and strained with haematoxylin and eosin, and the number of cells determined. No differences (P > 0.05) were observed in number of cells of morulae but blastocysts developing in medium with 120 micrograms porcine plasminogen ml-1 had more (P < 0.05) cells (109.9 +/- 10.4) than did blastocysts in medium with either 0 (69.4 +/- 14.6) or 30 micrograms porcine plasminogen ml-1 (73.3 +/- 12.2). In Expt 3, 144 one- to two-cell embryos were cultured in medium with 0, 13 or 45 micrograms porcine plasmin ml-1 or 120 micrograms porcine plasminogen ml-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and characterisation of recombinant rabbit plasminogen activator inhibitor-1 expressed in Saccharomyces cerevisiae

The rabbit plasminogen activator inhibitor-1 (PAI-1) cDNA has been isolated from a rabbit corneal cell cDNA library. The cDNA encodes a 402-amino acid (AA) protein that shares an overall 66% AA sequence identity with the rat, mouse, bovine and human forms of PAI-1 and exhibits the greatest AA sequence identity (85%) with human PAI-1. Three potential N-linked glycosylation sites and the P1, P1′ reactive centre of PAI-1 are conserved among all five species of animals. The cDNA encoding the proposed mature form of rabbit PAI-I was expressed in Saccharomyces cerevisiae as an intracellular, non-glycosylated protein. The purified, recombinant rabbit PAI-1 (R-rPAI-1) has an apparent M r of 39 100 and exists primarily in a latent form which can be activated by guanidine HCI treatment. Activated R-rPAI-1 exhibits in vitro functional properties which are virtually indistinguishable from a recombinant, non-glycosylated form of human PAI-I and from fully glycosylated, native human PAI-1.

Behavior of plasminogen at the luminal surface of the normal and deendothelialized rabbit aorta in vivo and in vitro

The behavior of purified rabbit plasminogen at the luminal surface of the uninjured and deendothelialized rabbit aorta has been studied in vivo and in vitro. After intravenous injection, 125I-plasminogen associated rapidly with the endothelium (approximately 0.1 pmol/cm2 at saturation) and passed through to accumulate in the subendothelium. At two to 15 hours after injection, 11 to 15 times more radioactivity was associated with the subendothelium than with the endothelium. Removal of the endothelium by balloon catheter led to a rapid adsorption of 125I-plasminogen by the luminal surface of the vessel; saturation (9.1 pmol/cm2) was attained at ten to 20 minutes after deendothelialization. Of the adsorbed plasminogen (radioactivity), only 2% to 4% was associated with the adherent platelet monolayer. Uptake of 125I- plasminogen by the deendothelialized vessel was not significantly inhibited by epsilon-aminohexanoic acid whether injected before or after the 125I-plasminogen. No evidence of plasmin activity at the aorta surface was found from either transmission electron microscopy studies or from amidolytic assays of plasminogen-saturated deendothelialized aorta samples before or after urokinase treatment. Balloon catheter treatment in vivo, however, generated significant antiplasmin activity of the deendothelialized aorta surface. We conclude that plasmin formed in vivo is probably inactivated by the antiplasmin activity that is associated with the subendothelium.

Behavior of Plasminogen at the Luminal Surface of the Normal and Deendothelialized Rabbit Aorta in vivo and in vitro

The behavior of purified rabbit plasminogen at the luminal surface of the uninjured and deendothelialized rabbit aorta has been studied in vivo and in vitro. After intravenous injection, 125I-plasminogen associated rapidly with the endothelium (approximately 0.1 pmol/cm2 at saturation) and passed through to accumulate in the subendothelium. At two to 15 hours after injection, 11 to 15 times more radioactivity was associated with the subendothelium than with the endothelium. Removal of the endothelium by balloon catheter led to a rapid adsorption of 125I-plasminogen by the luminal surface of the vessel; saturation (9.1 pmol/cm2) was attained at ten to 20 minutes after deendothelialization. Of the adsorbed plasminogen (radioactivity), only 2% to 4% was associated with the adherent platelet monolayer. Uptake of 125I-plasminogen by the deendothelialized vessel was not significantly inhibited by epsilon-aminohexanoic acid whether injected before or after the 125I-plasminogen. No evidence of plasmin activity at the aorta surface was found from either transmission electron microscopy studies or from amidolytic assays of plasminogen-saturated deendothelialized aorta samples before or after urokinase treatment. Balloon catheter treatment in vivo, however, generated significant antiplasmin activity of the deendothelialized aorta surface. We conclude that plasmin formed in vivo is probably inactivated by the antiplasmin activity that is associated with the subendothelium.

Structural aspects of the plasminogen of various species.

The N-terminal amino acid sequence of equine, ovine, canine, goat and rabbit plasminogen were determined and compared with those already known of the human, bovine, porcine and feline molecule. Furthermore, the kringle 4 domains of equine, ovine, canine and goat plasminogen, prepared by limited cleavage with elastase, were sequenced and compared with the known species of human, bovine, porcine and chicken plasminogen. Homology with the human kringle 4 ranges between 73% (chicken) and 90% (bovine). Comparison of sequences, fragmentation patterns with elastase and adsorption on lysine-Bio-Gel suggests the same structural and functional domains in the animal species as in human plasminogen.

Ｌ鎖（Ｐｌａｓｍｉｎ）・ＳＫｃｏｍｐｌｅｘの精製とその酵素学的特性

After human plasminogen activated by SK was partially reduced by dithioerythritol in the presence of plasmin-inhibitor leupeptin, a functionally active light (B)·SK complex was isolated by affinity chromatography with Lysine-Sepharose.This LB·SK complex shows a molecular weight of 60, 000 composed of LB 22, 000 and modified SK: 37, 000 by SDS-polyacrylamide gel electrophoresis.Results were as follows.1) Binding site to SK was located in the part of LB chain of human plasmin.2) Making complex with SK, amidolytic activity of LB·SK was increased twice to three times than that of LB itself.3) LB·SK complex showed both fibrinolytic and plasminogen activator activities and activated human, bovine and rabbit plasminogen to plasmin in spite of race difference.4) Thrombolytic activity of LB·SK complex had the most powerfull effect compared with SK, UK and human plasmin.

Interspecies cross-reactivity of monoclonal antibodies to various epitopes of human plasminogen

The immunological cross-reactivities of three conformationally specific monoclonal antibodies to distinct epitopes on human plasminogen toward plasminogens purified from 14 additional species have been examined. Antibody 10-F-1, which is produced against an epitope on the kringle 4 region of human plasminogen, shows a high degree (>80%) of cross-reactivity against baboon, goat, monkey, ovine, and rabbit plasminogens; more limited (20–50%) cross-reactivity against bovine, equine, goose, guinea pig, mouse, rat, and porcine plasminogens; and little comparable cross-reactivity against canine and chicken plasminogens. Antibody 10-H-2, generated to an epitope of the kringles 1–3 region of human plasminogen, shows extensive cross-reactivity (72%) only toward monkey plasminogen, more limited (22–35%) cross-reactivity toward equine and rabbit plasminogens, and much less cross-reactivity toward any other of the above plasminogens. Antibody 10-V-1, also produced against an epitope on the kringle 1–3 region of human plasminogen, which is distinct from the 10-H-2 epitope, shows extensive cross-reactivity (72–100%) with baboon, monkey, and rabbit plasminogens; more limited cross-reactivity with equine (48%) and mouse (28%) plasminogens; and a low level of such reactivity with the remaining plasminogens. These studies show that the extent of interspecies cross-reactivity of various plasminogens greatly depends upon the epitope in question. The K4 region of these molecules appears more extensively conserved than the K1-3 region, at least in regard to the particular epitopes examined in this study.

Effect of tunicamycin on appearance of carbohydrate variants of plasminogen in rat and rabbit plasma.

The in vivo incorporation of [4,5-3H]leucine and [2-3H]mannose into the lysine-Sepharose affinity chromatography-resolvable carbohydrate variants of rat and rabbit plasminogen has been studied in the absence and presence of tunicamycin (TM). When rabbits and rats were exposed to 0.5 mg of TM per kg of body weight for 30 min and 2 hr, respectively, followed by a single pulse of [4,5-3H]leucine for 2 hr, no radiolabel was found in rat or rabbit variant 1 plasminogen, whereas [4,5-3H]leucine was incorporated into variant 2 plasminogen. In similar experiments utilizing [2-3H]mannose in place of [4,5-3H]leucine, very little [2-3H]mannose was incorporated into either plasminogen variant in the presence of TM. The trichloroacetic acid-precipitable protein of rabbit plasma incorporated virtually identical quantities of [4,5-3H]leucine in the absence or presence of TM, under the above dosage regimens, while, at the same time, a large decrease in the incorporation of [2-3H]mannose was observed in the plasma proteins of rabbits treated with TM. This indicates that many of the plasma proteins may be secreted into plasma divested of asparagine-linked carbohydrate side chains. Further, we conclude that the functional difference (antifibrinolytic amino acid binding capacity) between the two plasma variants of plasminogen more than likely results from additional glycosylation of asparagine in variant 1 plasminogen that normally does not occur in variant 2 plasminogen.

The Thrombolytic Activity of Streptokinase in the Rabbit

The activity of streptokinase (SK) has been examined in a rabbit model of venous thrombosis. Significant thrombolysis was observed at low doses of SK without evidence of systemic activation of the fibrinolytic system. The origin of the specificity of SK-induced fibrinolysis in the rabbit was explored, using a chromogenic substrate coupled assay to measure the enhancement of plasmin generation in the presence of rabbit fibrin. The results obtained suggest that rabbit fibrin influences the fibrinolytic system at three points, viz: the assembly of an activator complex from rabbit plasminogen and SK; the activity of that activator complex towards further rabbit plasminogen; and the activity of the resulting rabbit plasmin in the presence of plasma inhibitors.

Immunohistochemical and Histochemical Investigations on In Vivo Thrombolysis with Urokinase in Rabbits

Using human urokinase in vivo thrombolysis was studied in autologous artificial thrombi in the pulmonary circulation of rabbits by immunofluorescence and Todd's fibrinolysis autography techniques. As compared to the control thrombi of the untreated rabbits, a small increase of thrombolysis was found in the rabbits treated with urokinase. Urokinase fluorescence was observed in the leukocytes, but not along the fibrin fibrils in the thrombi of the rabbits treated with urokinase. By Todd's fibrinolysis autography, lytic areas were observed around aggregated leukocytes in the thrombi of the rabbits treated with urokinase. The small lysis of autologous artificial thrombi of rabbits by human urokinase may be caused by (1) low affinity of human urokinase for rabbit plasminogen, (2) weak adsorption of human urokinase to rabbit fibrin, (3) phagocytosis of human urokinase by leukocytes and (4) high level of antifibrinolytic activity of rabbit plasma.

Assessment of plasminogen synthesis in vitro by mouse tumor cells using a competition radioimmunoassay for mouse plasminogen

A sensitive, specific competition radioimmunoassay for mouse plasmin(ogen) has been developed in order to determine whether mouse tumor cells can synthesize plasminogen in vitro. The rabbit anti-BALB/c mouse plasminogen antibodies used in the assay react with the plasminogen present in serum from BALB/c, C3H, AKR and C57BL/6 mice, and also recognized mouse plasmin. The competition radioimmunoassay can detect as little as 50 ng of mouse plasminogen. No competition was observed with preparations of fetal calf, human an rabbit plasminogens. A variety of virus-transformed and mouse tumor cell lines were all found to contain less than 100 ng mouse plasminogen/mg of cell extract protein. Thus, if the plasminogen activator/plasmin system is important in the growth or movement of this group of tumor cells, the cells will be dependent upon the circulatory system of the host for their plasminogen supply.

Interaction of streptokinase with plasminogen. Isolation and characterization of a streptokinase degradation product.

When streptokinase is incubated with human or rabbit plasminogen, one event which occurs is a specific fragmentation of streptokinase. At least five major identifiable streptokinase fragments appear with time, and they possess molecular weights of approximately 40,000 (SK 1), 36,000 (SK 2), 31,000 (SK 3), 26,000 (SK 4), and 10,000 (SK 5) under denaturing conditions, as observed on calibrated sodium dodecyl sulfate-polyacrylamide gels, compared to native streptokinase of molecular weight 45,000. The amount of each of the fragments generated at given times of incubation of plasminogen and streptokinase depends upon the species of plasminogen employed. Utilizing rabbit plasminogen and streptokinase, the SK 4 fragment was purified. This fragment arises by proteolysis at both the NH2 and COOH regions of native streptokinase. However, when isolated utilizing dilute aqueous buffers, the SK 4 fragment contained a portion of the original NH2 terminus of native streptokinase noncovalently bound to the molecule (SK 4'). SK 4' is capable of activating human plasminogen to plasmin, albeit more slowly than native streptokinase. However, the SK 4'-human plasmin complex possess only very weak plasminogen-activating activity toward sheep plasminogen. Upon removal of the noncovalently bound small NH2-terminal peptide of native streptokinase from SK 4', SK 4 is formed. This particular fragment possesses practically no human plasminogen-activating activity and cannot be used as an activator of sheep plasminogen, even with added human plasminogen.

A kinetic characterization of the rabbit plasmin isozymes

Steady-state and presteady-state kinetic parameters for plasmins derived from the two rabbit plasminogen isozymes have been determined. Steady-state kinetic experiments with N-α-tosyl- l-arginine methyl ester indicate that each isozyme has a similar V. Plasmin isozyme 2 has a higher K m value for this substrate as well as a higher K i , for the competitive inhibitor, benzamidine-HCl. Presteady-state kinetic experiments, using the p-nitrophenyl esters of p-(methylethylsulfoniummethyl)benzoate, p-(pyridiniummethyl) benzoate, p-(thiouroniummethyl)benzoate and p-(guanidinium)benzoate, indicate that each plasmin has similar rate constants of acylation ( k 2). However, values of the dissociation constant ( K S ) indicate that plasmin isozyme 1 has a greater binding affinity for these substrates than does isozyme 2. The magnitude of this difference varies with the substrate and is the greatest for those containing analogs of the guanidino moiety of l-arginine.

23] Rabbit plasminogen and plasmin isozymes

Assays of plasminogen first require its conversion to the enzyme plasmin by common activators. The enzyme is then assayed on the basis of its esterolytic activity toward N-a-toluenesulfonyl-L-arginine methylester (TosArgOMe, TAME) utilizing a recording pH stat to titrate the amount of acid, Na-tosyl-L-arginine, liberated with time. In this chapter the Colorimetric method is described. This method is used if the equipment to perform esterolytic activity is not available. Colorimetric method is very sensitive and is used for analysis of the concentration of TosArgOMe remaining after incubation of plasmin with this substrate for a given period of time. This method is based upon the quantitative reaction of esters, such as TosArgOMe, with hydroxylamine, yielding the hydroxamic acid derivative. Upon addition of ferric chloride, a ferric ion-hydroxamic acid complex is formed, possessing spectral properties different from ferric chloride. The chapter also explains the Potentiometric Method. The purification procedure of plasminogen, chemical, physical and kinetic properties of rabbit plasminogen are also discussed.

Carbohydrate compositions of the rabbit plasminogen isozymes

The carbohydrate compositions of the two affinity-chromatography-resolved isozymes of rabbit plasminogen and plasmin as well as the isoelectric-focusing-resolved subforms of each plasminogen isozyme have been investigated in detail. The first plasminogen isozyme as well as its subforms all possess four to five residues of N-acetylglucosamine, two residues of N-acetylgalactosamine, three residues of mannose and five residues of galactose per molecule of protein. Additionally, we previously reported three residues of sialic acid present on this protein molecule. The corresponding plasmin heavy chain for this isozyme contains essentially all of the carbohydrate, and the plasmin light chain appears devoid of carbohydrate. On the other hand, the second plasminogen isozyme as well as its subforms all possess only trace amounts of N-acetylglucosamine, two residues of N-acetylgalactosamine, less than one residue of mannose and three residues of galactose per molecule of protein. In addition, we have previously reported two residues of sialic acid for this molecule. Here, also, all carbohydrate appears on the heavy chain of the plasmin, which is prepared by activation of this particular plasminogen. Thus, the carbohydrate differences which we reported earlier in rabbit plasminogen isozymes are confirmed and extended.