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British Journal of Experimental... Oct 1988We determined plasminogen activator (PA) and PA inhibitor (PAI) activities in the intra- and extracellular compartments of an experimental pancreatic ascites tumour with...
We determined plasminogen activator (PA) and PA inhibitor (PAI) activities in the intra- and extracellular compartments of an experimental pancreatic ascites tumour with indirect and direct functional assays, and partially characterized these activities on SDS-polyacrylamide gels coupled with fibrin and reverse fibrin autography. Intact tumour cells caused lysis of plasminogen-rich but not plasminogen-free fibrin clots, and the extent of lysis of the former was related to tumour cell count. Direct assay of PA with a synthetic substrate yielded an equivalent of 109 urokinase units per 10(9) tumour cells. No PAI activity was demonstrated in tumour cells with functional assays. Contrary to tumour cells, cell-free ascitic fluids caused no lysis of fibrin clots. Instead, it inhibited tumour cell- and urokinase-induced, but not plasmin-induced, clot lysis in a dose-dependent fashion. Although functional assays failed to demonstrate PA in ascitic fluid and PAI in tumour cells, both activities were detected in electrophoresed samples of cell lysates and fluids by fibrin and reverse fibrin autography. In tumour cells, a mixture of tissue-type PA (tPA) and urokinase-type PA (uPA) were present. In the fluid, uPA together with two other PAs with greater molecular weights than tPA were detected.
Topics: Animals; Ascitic Fluid; Cell Count; Cricetinae; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Fibrinolysis; Glycoproteins; Mesocricetus; Pancreatic Neoplasms; Plasminogen Activators; Plasminogen Inactivators; Tissue Plasminogen Activator; Urokinase-Type Plasminogen Activator
PubMed: 3143395
DOI: No ID Found -
Kidney International Mar 1989In the course of some pathological and experimental nephropathies, intraglomerular fibrin deposits develop, possibly as a consequence of inefficient fibrinolysis. In...
In the course of some pathological and experimental nephropathies, intraglomerular fibrin deposits develop, possibly as a consequence of inefficient fibrinolysis. In vitro human glomeruli exhibit fibrinolytic activity due to the synthesis of plasminogen activators (PAs) such as, tissue-type PA (t-PA) and urokinase-type PA (u-PA). Immunofluorescence studies have previously shown that t-PA is localized in the capillary tufts and u-PA in the visceral epithelial cells. We have now investigated the fibrinolytic activity of cultured human mesangial cells. Inhibitory activity towards u-PA or t-PA but not plasmin was found in both conditioned medium and cellular extracts. Analysis of the conditioned medium by zymography revealed a single band of PA-activity (Mr: 110 to 120 kDa). Immunoneutralization with anti-t-PA and anti-plasminogen activator inhibitor (PAI-1) IgG but not anti-u-PA or anti-PAI-2 removed this band. Reverse fibrin autography demonstrated the presence of PAI-1 in both cellular extracts and in conditioned medium. Western Blot analysis showed that two bands (50 kD and 120 kD) were recognized by the anti-PAI-1 antibody. By ELISA t-PA and PAI-1 antigens were found to increase progressively with time in the culture medium but not in cellular extracts. Both t-PA and PAI-1, but not u-PA and PAI-2, were also detected by immunofluorescence studies. Thus human glomerular mesangial cells synthesize and secrete t-PA and PAI-1 in vitro. PAI-1 is produced in excess, therefore t-PA is only found in the form of a complex with PAI-1.
Topics: Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Fluorescent Antibody Technique; Glomerular Mesangium; Glycoproteins; Humans; Immunoblotting; In Vitro Techniques; Plasminogen Activators; Plasminogen Inactivators; Tissue Plasminogen Activator
PubMed: 2496257
DOI: 10.1038/ki.1989.56 -
Journal of the American College of... Nov 1987Since streptokinase and urokinase became available for clinical use, numerous attempts have been made to improve these useful thrombolytic agents. To decrease its... (Review)
Review
Since streptokinase and urokinase became available for clinical use, numerous attempts have been made to improve these useful thrombolytic agents. To decrease its antigenicity, streptokinase has been fragmented or coupled to human plasminogen or polyethylene glycols. With a plasmin B chain-streptokinase complex a more potent agent was obtained. To prolong their half-life, streptokinase and urokinase were immobilized with water-soluble carriers. Coupling urokinase with fibrin-specific antibodies increases its thrombolytic efficacy, at least in vitro. The only thrombolytic agents with a relative fibrin specificity available for clinical purposes are tissue-type plasminogen activator and single chain urokinase-type plasminogen activator. Mutants and hybrids of these molecules are being constructed and may further improve their fibrin specificity and therapeutic potential.
Topics: Drug Combinations; Enzymes, Immobilized; Fibrinolysin; Fibrinolysis; Fibrinolytic Agents; Humans; Mutation; Plasminogen; Plasminogen Activators; Polyethylene Glycols; Recombinant Proteins; Streptokinase; Tissue Plasminogen Activator; Urokinase-Type Plasminogen Activator
PubMed: 2959714
DOI: 10.1016/s0735-1097(87)80421-7 -
The Journal of Clinical Investigation Dec 1993
Review
Topics: Child; Female; Fibrinolysin; Humans; Plasminogen Activator Inhibitor 1; Plasminogen Activators
PubMed: 8254010
DOI: 10.1172/JCI116866 -
The Israel Medical Association Journal... Nov 2006Acute ischemic stroke is one of the leading causes of mortality and chronic disability in the western world. Yet, despite the enormous socioeconomic burden that it... (Review)
Review
Acute ischemic stroke is one of the leading causes of mortality and chronic disability in the western world. Yet, despite the enormous socioeconomic burden that it imposes, therapies to combat AIS are not widely available. Moreover, revascularization of the ischemic tissue with tissue plasminogen activator, the only FDA-approved therapy for AIS, is hampered by a very narrow therapeutic time window and is only used in a minority of patients. Cerebral ischemia leads to brain damage caused by several pathologic mechanisms that can potentially be blocked by neuroprotective drugs that aim to salvage the ischemic penumbra. However, despite numerous clinical trials, no single drug candidate has proved efficacious in AIS. The current situation calls for novel therapeutic strategies to be used in acute ischemic stroke. This review surveys some of these novel and promising cutting edge therapies.
Topics: Benzenesulfonates; Humans; Membrane Proteins; Myocardial Revascularization; Neuroprotective Agents; Plasminogen Activators; Randomized Controlled Trials as Topic; Stroke; Tetraspanins; Tissue Plasminogen Activator
PubMed: 17180832
DOI: No ID Found -
The Journal of Biological Chemistry Jul 1981The plasminogen activator secreted by a cultured human melanoma cell line was purified and compared with urokinase and with tissue plasminogen activator from human...
The plasminogen activator secreted by a cultured human melanoma cell line was purified and compared with urokinase and with tissue plasminogen activator from human uterus. The purification procedure consisted of chromatography on zinc chelate-agarose, concanavalin A-agarose, and Sephadex G-150 in the presence of 0.01% (v/v) Tween 80. The purified material was obtained from the culture medium with a yield of 46% and a purification factor of 263. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed one main band with a molecular weight of about 72,000, and in the presence of reducing agents, two bands of 33,000 and 39,000. Addition of the protease inhibitor Aprotinin to the culture media and column buffers yielded a one-chain plasminogen activator with a molecular weight of about 72,000. One molecule of activator reacted with about one molecular of [3H]diisopropylfluorophosphate. The melanoma plasminogen activator and the uterine tissue plasminogen activator appeared to be very similar on dodecyl sulfate-polyacrylamide gel electrophoresis, amino acid analysis, and amidolytic properties. Both activators bound to fibrin clots, while urokinase did not. In immunodiffusion, as well as in quenching experiments of the fibrinolytic activities, the melanoma plasminogen activator appeared to be immunologically identical with the uterine tissue plasminogen activator, but unrelated to urokinase. All these findings indicate that the plasminogen activator secreted by human melanoma cells in culture is very similar to, or identical with, the plasminogen activator found in normal tissue, but different from urokinase.
Topics: Amino Acids; Cell Line; Female; Fibrinolysis; Humans; Immune Sera; Immunodiffusion; Kinetics; Melanoma; Plasminogen Activators; Uterus
PubMed: 6787058
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 -
Haemostasis 2001Plasminogen activators are enzymes found in all vertebrate species investigated so far. Their physiological function is the generation of localized proteolysis in the... (Comparative Study)
Comparative Study Review
Plasminogen activators are enzymes found in all vertebrate species investigated so far. Their physiological function is the generation of localized proteolysis in the context of tissue remodeling, wound healing and neuronal plasticity. The common vampire bat (Desmodus rotundus) is a New World species that feeds exclusively on blood. Its saliva contains highly potent plasminogen activators, specialized in rapid lysis of fresh blood clots. Biochemical and pharmacological evidence indicates that these plasminogen activators represent a new class of thrombolytics with pharmacological and toxicological properties superior to human tissue-type plasminogen activator, the clot dissolving agent now most frequently used in medicine. A form of the enzyme produced by recombinant DNA technology is currently employed to test this hypothesis in clinical studies.
Topics: Animals; Chiroptera; Clinical Trials as Topic; Fibrinolytic Agents; Humans; Plasminogen Activators; Selection, Genetic; Tissue Plasminogen Activator
PubMed: 11910176
DOI: 10.1159/000048054 -
Journal of the American College of... Nov 1987Recombinant tissue-type plasminogen activator (rt-PA) and single chain urokinase-type plasminogen activator (scu-PA) are thrombolytic agents, characterized by a high but... (Review)
Review
Recombinant tissue-type plasminogen activator (rt-PA) and single chain urokinase-type plasminogen activator (scu-PA) are thrombolytic agents, characterized by a high but not absolute degree of fibrin specificity that is mediated through different molecular mechanisms. Both activators are still under clinical investigation but it has become apparent that their therapeutic dose in humans is high and associated with a variable degree of systemic activation of the fibrinolytic system and fibrinogen breakdown. Therefore, the quest for further improvement of agents and therapeutic schemes continues. Research is being pursued in this area along the following lines: 1) tissue-type plasminogen activator (t-PA) and single chain urokinase-type plasminogen activator in molar ratios of 4:1 to 1:4 do not act synergistically on thrombolysis in a plasma environment in vitro, but display significant synergism in animal models of thrombosis. In pilot studies in patients with coronary artery occlusion, rt-PA and scu-PA are markedly synergistic and efficient thrombolysis can be obtained with a fivefold lower combined dose than that of the separate agents. The combined dose does not seem to induce systemic fibrinogen breakdown. 2) Deletion mutants of rt-PA can be constructed with a significantly prolonged half-life in vivo, and a better thrombolytic potential after bolus intravenous injection. 3) Cleavage site-specific mutants of scu-PA that abolish the conversion to urokinase may have a higher fibrin specificity. The mutants constructed thus far, however, seem to have a lower specific thrombolytic activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Topics: Animals; Drug Synergism; Fibrinolysis; Fibrinolytic Agents; Humans; Mutation; Plasminogen Activators; Recombinant Proteins; Structure-Activity Relationship; Tissue Plasminogen Activator; Urokinase-Type Plasminogen Activator
PubMed: 3117858
DOI: 10.1016/s0735-1097(87)80422-9 -
The Journal of Biological Chemistry Apr 2001The therapeutic properties of plasminogen activators are dictated by their mechanism of action. Unlike staphylokinase, a single domain protein, streptokinase, a 3-domain... (Comparative Study)
Comparative Study
The therapeutic properties of plasminogen activators are dictated by their mechanism of action. Unlike staphylokinase, a single domain protein, streptokinase, a 3-domain (alpha, beta, and gamma) molecule, nonproteolytically activates human (h)-plasminogen and protects plasmin from inactivation by alpha(2)-antiplasmin. Because a streptokinase-like mechanism was hypothesized to require the streptokinase gamma-domain, we examined the mechanism of action of a novel two-domain (alpha,beta) Streptococcus uberis plasminogen activator (SUPA). Under conditions that quench trace plasmin, SUPA nonproteolytically generated an active site in bovine (b)-plasminogen. SUPA also competitively inhibited the inactivation of plasmin by alpha(2)-antiplasmin. Still, the lag phase in active site generation and plasminogen activation by SUPA was at least 5-fold longer than that of streptokinase. Recombinant streptokinase gamma-domain bound to the b-plasminogen.SUPA complex and significantly reduced these lag phases. The SUPA-b.plasmin complex activated b-plasminogen with kinetic parameters comparable to those of streptokinase for h-plasminogen. The SUPA-b.plasmin complex also activated h-plasminogen but with a lower k(cat) (25-fold) and k(cat)/K(m) (7.9-fold) than SK. We conclude that a gamma-domain is not required for a streptokinase-like activation of b-plasminogen. However, the streptokinase gamma-domain enhances the rates of active site formation in b-plasminogen and this enhancing effect may be required for efficient activation of plasminogen from other species.
Topics: Amino Acid Sequence; Animals; Antifibrinolytic Agents; Binding Sites; Cattle; Cloning, Molecular; Fibrinolysin; Humans; Kinetics; Metalloendopeptidases; Molecular Sequence Data; Plasminogen; Plasminogen Activators; Polymerase Chain Reaction; Recombinant Proteins; Sequence Alignment; Sequence Homology, Amino Acid; Streptococcus; Streptokinase
PubMed: 11278483
DOI: 10.1074/jbc.M009265200