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Journal of Dairy Science Jun 1996Several tissue remodeling events that require extracellular proteolysis are thought to be mediated by plasminogen activators that convert the inactive proenzyme... (Review)
Review
Several tissue remodeling events that require extracellular proteolysis are thought to be mediated by plasminogen activators that convert the inactive proenzyme plasminogen to active plasmin. The involvement of plasminogen activator in many biological phenomena reflects the ubiquitous presence of plasminogen and the ability of numerous cell types to synthesize plasminogen activator in a highly regulated manner. Increased plasmin and plasminogen activator in bovine milk are correlated with gradual involution (the declining phase of lactation). Treatment with bST prevented the increase in plasmin during gradual involution, indicating that bST interferes with conversion of plasminogen to plasmin. Concentrations of plasminogen activator in mammary tissue are high after cessation of milking. These results reinforce the association of the plasmin-plasminogen system with gradual involution postlactation. Recently, a role has been proposed for plasminogen activator in cell proliferation in several cellular systems. Insulin and IGF-I increased synthesis of urokinase plasminogen activator and enhanced proliferation of cultured bovine mammary epithelial cells. In contrast, phorbol myristate acetate, which increased expression of urokinase plasminogen activator mRNA by mammary epithelial and myoepithelial cells, stimulated proliferation of myoepithelial cells, but not epithelial cells. Thus, expression of plasminogen activator is not simply related to mitogenesis but is likely to serve multiple functions in bovine mammary epithelial cells.
Topics: Animals; Cattle; Cell Division; Epithelial Cells; Female; Fibrinolysin; Mammary Glands, Animal; Plasminogen; Plasminogen Activators; Pregnancy; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator
PubMed: 8827475
DOI: 10.3168/jds.S0022-0302(96)76463-9 -
The Journal of Clinical Investigation Jun 2002
Review
Topics: Animals; Cell Death; Hippocampus; Humans; Neurons; Plasminogen; Plasminogen Activators; Seizures; Tissue Plasminogen Activator
PubMed: 12070298
DOI: 10.1172/JCI15961 -
Journal of the American College of... Dec 1986Streptokinase and urokinase have proved to be useful in a limited number of clinical conditions. Mainly because of the risk and unpredictability of bleeding with this... (Review)
Review
Streptokinase and urokinase have proved to be useful in a limited number of clinical conditions. Mainly because of the risk and unpredictability of bleeding with this first generation of thrombolytic agents, thrombolysis has not been ingrained in medical practice. In the interim, more fibrin-specific thrombolytic agents have been developed such as acylated streptokinase-human plasminogen complex, tissue-type plasminogen activator (t-PA) and single chain urokinase-type plasminogen activator (scu-PA or pro-urokinase). Only the latter two drugs do not induce major systemic fibrinogenolysis at thrombolytic effective doses. These two agents, obtained by recombinant techniques, as well as acylated streptokinase-plasminogen complex are available for clinical investigations. The first results of systemic administration of recombinant tissue-type plasminogen activation (t-PA) in patients with acute myocardial infarction were published and are promising. Continued experimentation with t-PA and pro-urokinase in evolving myocardial infarction and other thrombotic disorders is essential to better delineate their therapeutic index.
Topics: Drug Combinations; Fibrinolytic Agents; Humans; Plasminogen; Plasminogen Activators; Streptokinase; Tissue Plasminogen Activator; Urokinase-Type Plasminogen Activator
PubMed: 3097100
DOI: 10.1016/s0735-1097(86)80005-5 -
Clinical Cardiology Jun 1990Thrombotic disorders such as myocardial infarction and stroke are the leading causes of death and disability in industrialized nations. Timely institution of... (Review)
Review
Thrombotic disorders such as myocardial infarction and stroke are the leading causes of death and disability in industrialized nations. Timely institution of thrombolytic therapy can achieve a reduction of infarct size, a preservation of left ventricular function, and a reduction in mortality. The administration of streptokinase, urokinase, and acylated plasminogen-streptokinase activator complex (APSAC) can be associated with a complete breakdown of the hemostatic system. Tissue-type plasminogen activator (t-PA) and single-chain urokinase-type plasminogen activator (scu-PA, prourokinase) are more fibrin specific; however, at the large dosages of activator needed for therapeutic efficacy, bleeding complications are still a problem. New approaches to optimizing the risk/benefit ratio for the patient by improving efficacy without sacrificing specificity include the use of synergistic combinations of plasminogen activators, mutants of t-PA and scu-PA, chimeric molecules, and antibody-targeted thrombolytic agents. The last approach opens the possibility of targeting several different components of the clot with either fibrinolytic or antiplatelet effector functions in one optimized molecule.
Topics: Animals; Antibodies, Monoclonal; Drug Synergism; Humans; Myocardial Infarction; Plasminogen Activators; Thrombolytic Therapy
PubMed: 2188764
DOI: 10.1002/clc.4960130602 -
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 Chemical Society Nov 2018Although the functional specificity and catalytic versatility of enzymes have been exploited in numerous settings, controlling the spatial and temporal activity of...
Although the functional specificity and catalytic versatility of enzymes have been exploited in numerous settings, controlling the spatial and temporal activity of enzymes remains challenging. Here we describe an approach for programming the function of streptokinase (SK), a protein that is clinically used as a blood "clot buster" therapeutic. We show that the fibrinolytic activity resulting from the binding of SK to the plasma proenzyme plasminogen (Pg) can be effectively regulated (turned "OFF" and "ON") by installing an intrasteric regulatory feature using a DNA-linked protease inhibitor modification. We describe the design rationale, synthetic approach, and functional characterization of two generations of intrasterically regulated SK-Pg constructs and demonstrate dose-dependent and sequence-specific temporal control in fibrinolytic activity in response to short predesignated DNA inputs. The studies described establish the feasibility of a new enzyme-programming approach and serves as a step toward advancing a new generation of programmable enzyme therapeutics.
Topics: DNA; Drug Design; Humans; Plasminogen Activators; Protease Inhibitors; Streptokinase
PubMed: 30347143
DOI: 10.1021/jacs.8b10166 -
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 -
British Journal of Pharmacology Jan 2012Nature has provided a vast array of bioactive compounds that have been exploited for either diagnostic or therapeutic use. The field of thrombosis and haemostasis in... (Review)
Review
Nature has provided a vast array of bioactive compounds that have been exploited for either diagnostic or therapeutic use. The field of thrombosis and haemostasis in particular has enjoyed much benefit from compounds derived from nature, notably from snakes and blood-feeding animals. Indeed, the likelihood that blood-feeding animals would harbour reagents with relevant pharmacology and with potential pharmaceutical benefit in haemostasis was not too far-fetched. Blood-feeding animals including leeches and ticks have evolved a means to keep blood from clotting or to at least maintain the liquid state, and some of these have been the subject of clinical development. A more recent example of this has been the saliva of the common vampire bat Desmodus rotundus, which has proven to harbour a veritable treasure trove of novel regulatory molecules. Among the bioactive compounds present is a fibrinolytic compound that was shown over 40 years ago to be a potent plasminogen activator. Studies of this vampire bat-derived plasminogen activator, more recently referred to as desmoteplase, revealed that this protease shared a number of structural and functional similarities to the human fibrinolytic protease, tissue-type plasminogen activator (t-PA) yet harboured critically important differences that have rendered this molecule attractive for clinical development for patients with ischaemic stroke.
Topics: Animals; Chiroptera; Fibrinolytic Agents; Humans; Plasminogen Activators; Saliva; Stroke
PubMed: 21627637
DOI: 10.1111/j.1476-5381.2011.01514.x -
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 -
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