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Thrombosis Research Oct 1986Clot lysis and non-specific plasminogen activation in human plasma by tissue tissue plasminogen activator (t-PA) and/or pro-urokinase (pro-UK) were studied. The... (Comparative Study)
Comparative Study
A comparative study of the efficacy and specificity of tissue plasminogen activator and pro-urokinase: demonstration of synergism and of different thresholds of non-selectivity.
Clot lysis and non-specific plasminogen activation in human plasma by tissue tissue plasminogen activator (t-PA) and/or pro-urokinase (pro-UK) were studied. The fibrinolytic activity of pro-UK was expressed as latent units, i.e. measured after activation with plasmin on a fibrin plate against the reference standard. The t-PA unitage was assigned on a weight basis of a similar equivalence of 100,000 IU/mg. To simplify comparison, both activators were expressed in IU (1 IU = approximately 10 ng). At low concentration (1-50 IU/ml), t-PA induced more effective and more linear clot lysis, whereas pro-UK induced lysis was preceded by a lag phase. The two activators were equivalently effective at higher concentrations and saturated at the same lysis rate. Clots made from platelet rich plasma or whole blood were more responsive to lysis by pro-UK but not t-PA than corresponding platelet poor clots. At very low concentrations (2.5-5 IU/ml) of t-PA combined with moderate concentrations (25-50 IU/ml) of pro-UK, a synergistic effect on clot lysis, which was fibrin-specific, was observed. Plasminogen and fibrinogen and the appearance of plasmin-inhibitor complexes in plasma were measured after incubation with either activator with and without a clot present. Non-specific plasminogen activation occurred above a certain concentration of either activator but was found at lower concentrations of t-PA than pro-UK. In the absence of a clot, plasmin generation occurred with t-PA at about 30% of the concentration at which pro-UK induced a corresponding effect. It is concluded that there are important differences in the fibrinolytic and clot selective properties of t-PA and pro-UK, and that some of these properties may be complementary resulting in a fibrin specific, synergistic fibrinolytic effect.
Topics: Blood Platelets; Drug Synergism; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Fibrinolysis; Humans; Iodine Radioisotopes; Kinetics; Plasminogen; Plasminogen Activators; Tissue Plasminogen Activator; Urokinase-Type Plasminogen Activator
PubMed: 3097872
DOI: 10.1016/0049-3848(86)90137-4 -
Experimental and Molecular Pathology Aug 1985Hypersensitivity granulomas induced by infection with Schistosoma mansoni were isolated from the livers of BALB/c mice after 7, 8, 10, and 12 weeks. The parasite...
Hypersensitivity granulomas induced by infection with Schistosoma mansoni were isolated from the livers of BALB/c mice after 7, 8, 10, and 12 weeks. The parasite egg-granulomas were sequentially extracted with a Tris-buffered saline (soluble fraction) and 2 M KSCN (bound fraction). Fibrinolytic enzyme activity measured with both synthetic substrates and fibrin plates demonstrated an elevated level of plasminogen activator activity in the bound fraction 7-8 weeks after infection when mature granulomas first began to appear, followed by a gradual decrease 10-12 weeks after infection. An electrophoretic enzymography technique revealed multiple molecular species of plasminogen activator at Mr = 95K, 74K, 60K, 45K, and 24K. The bands with Mr = 45K and 24K were found compatible with the electrophoretic pattern of macrophage-plasminogen activator. When the granulomas reached maximum size after 10 to 12 weeks, the plasminogen activator with 45K and 24K diminished, while plasminogen activator activity at Mr = 95K, 74K, and 60K remained unchanged suggesting the presence of both vascular and tissue types of plasminogen activators. There was no urokinase-type plasminogen activator detectable in granulomas at any time. In the soluble fraction no enzymatic activity was found, whereas regulating inhibitor activity for plasminogen activator was consistently detectable.
Topics: Animals; Granuloma; Mice; Mice, Inbred BALB C; Plasminogen Activators; Schistosoma mansoni; Schistosomiasis
PubMed: 4040036
DOI: 10.1016/0014-4800(85)90057-7 -
European Journal of Biochemistry Mar 1984Human peripheral monocytes stimulated by either muramyl dipeptide [N-acetyl-muramoyl-L-alanyl-D-isoglutamine], bacterial lipopolysaccharide or lymphokine-containing... (Comparative Study)
Comparative Study
Human peripheral monocytes stimulated by either muramyl dipeptide [N-acetyl-muramoyl-L-alanyl-D-isoglutamine], bacterial lipopolysaccharide or lymphokine-containing supernatants of human lymphocytes, could be shown to produce and secrete appreciable activities of a 52 000-Mr plasminogen activator. This enzyme was suppressed in control and stimulated cultures by dexamethasone (0.1 microM). Monocyte plasminogen activator could only be assayed under conditions of low ionic strength and had no detectable activity at 0.15 M NaCl. Intracellular enzyme was present as a proenzyme, requiring activation by preincubation with plasminogen containing traces of plasmin, before its activity could be seen on sodium dodecyl sulphate/polyacrylamide gel electrophoresis by a fibrin overlay method. Secreted enzyme was in the active form. Further incubation of lysate or supernatant plasminogen activator with plasminogen did not produce any active enzyme species of Mr 36 000, unlike incubations of urokinase with plasminogen. Moreover, comparisons with other plasminogen activators of Mr 52 000 from transformed cell lines showed that the monocyte activator was unique in its resistance to monocyte minactivin, a specific inactivator of urokinase-type plasminogen activators, and in its sensitivity to human alpha 2-macroglobulin. It was therefore concluded that human monocyte plasminogen activator, although sharing an Mr of 52 000 in common with other such activators, is not identical to the high Mr form of urokinase or the plasminogen activators of transformed cells. On present evidence it is the least likely of these enzymes to be active extracellularly under normal physiological conditions.
Topics: Cells, Cultured; Chemical Phenomena; Chemistry; Diffusion; Electrophoresis, Polyacrylamide Gel; Humans; Monocytes; Plasminogen Activators; Plasminogen Inactivators; Spectrophotometry; Substrate Specificity; alpha-Macroglobulins
PubMed: 6199200
DOI: 10.1111/j.1432-1033.1984.tb08001.x -
Thrombosis and Haemostasis Oct 1981The plasminogen activator secreted by a cultured rat brain tumor cell line (RT4-71-1) (Imada M. and Sueoka N., Develop. Biol. 69, 97-107, 1978) was purified by...
The plasminogen activator secreted by a cultured rat brain tumor cell line (RT4-71-1) (Imada M. and Sueoka N., Develop. Biol. 69, 97-107, 1978) was purified by chromatography on zinc chelate-agarose, concanavalin A-agarose and Sephadex G-150 in the presence of 0.01% (vol/vol) Tween 80. Aprotinin was added to the culture medium to a concentration of 20 KIU per ml and to the buffers in the first two chromatographic steps to a concentration of 10 KIU per ml. Approximately 90 microgram purified material was obtained from 11 of culture medium with a yield of 39% and a purification factor of 200. Sodium dodecylsulfate-polyacrylamide gel electrophoresis in the presence of reducing agents showed one main band with Mr of about 60,000, and a minor band with Mr about 30,000. Fibrinolytic activity was associated with the main band. The rat brain tumor plasminogen activator bound to a fibrin clot to a similar extent as human tissue plasminogen activator, whereas urokinase did not bind. In quenching experiments of the fibrinolytic activities the purified rat brain tumor plasminogen activator appeared to be immunologically related to the human tissue plasminogen activator but unrelated to urokinase.
Topics: Animals; Brain Neoplasms; Cells, Cultured; Chromatography; Electrophoresis, Polyacrylamide Gel; Fibrinolysis; In Vitro Techniques; Neoplasms, Experimental; Plasminogen Activators; Rats
PubMed: 7198301
DOI: No ID Found -
The Journal of Biological Chemistry Jan 1986A urokinase-type plasminogen activator was purified from conditioned media of several human cell cultures, but preferably from the human lung adenocarcinoma line CALU-3...
A urokinase-type plasminogen activator was purified from conditioned media of several human cell cultures, but preferably from the human lung adenocarcinoma line CALU-3 (ATCC, HTB-55), using a combination of chromatography on zinc chelate-Sepharose, SP-Sephadex C-50, and Sephadex G-100. Final yields of 65-100 micrograms/liter of starting material were obtained with a 290-fold purification factor and a recovery of 30%. The purified plasminogen activator consists of a single polypeptide chain with Mr 54,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and is very similar or identical to single-chain urokinase-type plasminogen activator on the basis of immunodiffusion, amino acid composition, and the lack of specific binding to fibrin. It has very low amidolytic activity on Pyroglu-Gly-Arg-rho-nitroanilide and is converted to two-chain urokinase by limited exposure to plasmin. It has a specific activity of 60,000 IU/mg on fibrin plates and directly activates plasminogen following Michaelis-Menten kinetics with Km = 1.1 microM and kappa cat = 0.0026 S-1. It is concluded that the plasminogen activator purified from CALU-3-conditioned media is physically and kinetically identical to single-chain urokinase-type plasminogen activator. With the present straightforward purification method and a readily available source, sufficient amounts of single-chain urokinase-type plasminogen activator can be obtained for more detailed investigations of its biochemical, biological, and thrombolytic properties.
Topics: Cells, Cultured; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Humans; Immunodiffusion; Kinetics; Plasminogen Activators; Urokinase-Type Plasminogen Activator
PubMed: 3080423
DOI: No ID Found -
Biochimica Et Biophysica Acta Apr 1982A plasminogen activator was purified from the serum-free conditioned medium of bovine aortic endothelial cell cultures by chromatography on zinc chelate-agarose and...
A plasminogen activator was purified from the serum-free conditioned medium of bovine aortic endothelial cell cultures by chromatography on zinc chelate-agarose and benzamidine-CH-Sepharose. The final material consisted of a main fibrinolytically active component with Mr 30,000 and a minor component with Mr 41,000. It was obtained with a yield of 60%, a purification factor of 35 and a purity of 25-50%. The activity of this plasminogen activator was completely neutralized by antibodies to human urokinase but not by antibodies against human tissue plasminogen activator. Purified tissue plasminogen activator from bovine heart, however, was completely neutralized by antibodies against human tissue plasminogen activator but unaffected by antibodies to human urokinase. These findings indicate that bovine aortic endothelial cells in culture secrete mainly a urokinase-like. These findings indicate that bovine aortic endothelial cells in culture secrete mainly a urokinase-like plasminogen activator, and not a tissue-type plasminogen activator as was generally assumed.
Topics: Animals; Antibodies; Aorta; Cattle; Cells, Cultured; Chromatography, Affinity; Endothelium; Macromolecular Substances; Molecular Weight; Myocardium; Plasminogen Activators; Urokinase-Type Plasminogen Activator
PubMed: 7200373
DOI: 10.1016/0167-4838(82)90018-8 -
Journal of Cellular Physiology Feb 1988An established cell line (OC-1) was obtained from human ovarian tissue, which yielded a high concentration of plasminogen activator (PA) in the culture medium. The PA...
An established cell line (OC-1) was obtained from human ovarian tissue, which yielded a high concentration of plasminogen activator (PA) in the culture medium. The PA (OC-1-PA) produced by the cell line was purified and compared with urokinase (UK), proform of UK (pro-UK), and tissue-type PA (t-PA) purified from human melanoma cells (Bowes). OC-1-PA was purified by Zn chelate-Sepharose affinity chromatography followed by high-performance liquid chromatography with a Zn chelate-5PW column and with a p-amino-benzamidine-5PW column, giving a yield of 58.3% and a purification factor of 15,439. This purified material revealed a single band of Mr 55,000 on sodium dodecylsulfate polyacrylamide gel electrophoresis in the presence or absence of reducing agents. Electrophoretic enzymography demonstrated that the Mr 55,000 protein band had a plasminogen-dependent fibrinolytic activity. Treatment with plasmin did not change the Mr even in the presence of reducing agents. These results suggest that OC-1-PA has a single-chain structure protected from protease degradation, which is completely different from UK. The activator had higher affinities for lysine and fibrin than those of UK or pro-UK. An immunological study demonstrated that OC-1-PA cross-reacted with anti-UK IgG but not with anti-t-PA IgG. All these findings indicate that OC-1-PA belongs immunologically to the UK type, but its structure differs from that of UK.
Topics: Cell Line; Chemical Phenomena; Chemistry; Female; Fibrin; Humans; Isoelectric Point; Lysine; Ovary; Plasminogen Activators
PubMed: 3126195
DOI: 10.1002/jcp.1041340211 -
The Journal of Biological Chemistry Mar 1984Two components of the fibrinolytic system, plasminogen and the vascular plasminogen activator, have been isolated to apparent homogeneity from the post-venous occlusion...
Two components of the fibrinolytic system, plasminogen and the vascular plasminogen activator, have been isolated to apparent homogeneity from the post-venous occlusion plasma of three diabetic patients (hemoglobin A1C greater than 7%) and of one nondiabetic control person. Plasminogen activation was studied for each person separately in the absence and presence of CNBr fragments of fibrinogen. Activation of diabetic plasminogen by urokinase was not significantly altered as compared to the activation of control plasminogen. The same was found when diabetic plasminogen was activated by control vascular plasminogen activator in the presence of fibrinogen fragments but only at plasminogen concentrations below 10-30 nM; at higher substrate concentrations, however, plasminogen activation was impaired in a pattern resembling substrate inhibition. Activation of control plasminogen by diabetic vascular plasminogen activator was completely impaired in the absence of fibrinogen fragments. Addition of fibrinogen fragments stimulated plasmin formation by diabetic vascular plasminogen activator resulting in kinetic constants which were similar to the activation of control plasminogen by control vascular plasminogen activator in the absence of fibrinogen fragments (Km = 7.5 microM, kcat = 0.05 S-1). Addition of fibrinogen fragments in controls decreased Km values to less than 0.1 microM. Despite addition of fibrinogen fragments the rate of plasmin formation from diabetic plasminogen by diabetic vascular plasminogen activator isolated from the same diabetic donor was so small that kinetic constants could not be calculated.
Topics: Diabetes Mellitus; Enzyme Activation; Female; Fibrinolysin; Humans; Kinetics; Male; Plasminogen; Plasminogen Activators; Urokinase-Type Plasminogen Activator
PubMed: 6230354
DOI: No ID Found -
Heart Disease (Hagerstown, Md.) 2001This article reports the first case of a patient presenting with acute myocardial infarction in whom a repeated dose of tissue plasminogen activator (t-PA) was able to... (Review)
Review
This article reports the first case of a patient presenting with acute myocardial infarction in whom a repeated dose of tissue plasminogen activator (t-PA) was able to achieve successful thrombolysis after a first dose of t-PA itself failed to do so. This case report presents an alternative approach for the treatment of patients who fail thrombolysis after an initial dose of t-PA, an approach that might be particularly useful in hospitals that do not have immediate access to advanced interventional services.
Topics: Humans; Male; Middle Aged; Myocardial Infarction; Plasminogen Activators; Retreatment; Thrombolytic Therapy; Time Factors; Tissue Plasminogen Activator; Treatment Failure
PubMed: 11975820
DOI: 10.1097/00132580-200111000-00004 -
Thrombosis and Haemostasis Jun 1978The effect of Demulen (ethinyl estradiol 0.05 mg and ethynodiol diacetate 1 mg) and exercise on the level of plasminogen activators was studied in 25 women (12 controls...
The effect of Demulen (ethinyl estradiol 0.05 mg and ethynodiol diacetate 1 mg) and exercise on the level of plasminogen activators was studied in 25 women (12 controls and 13 contraceptive users). Plasma plasminogen activator level was increased by the use of the oral contraceptive and further increased by exercise. Urine plasminogen activator level was unchanged by the use of Demulen but, in both groups of subjects, was decreased by exercise.
Topics: Blood Coagulation; Contraceptives, Oral; Female; Fibrinolysis; Humans; Menstruation; Physical Exertion; Plasminogen Activators; Time Factors
PubMed: 705703
DOI: No ID Found