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Indian Journal of Biochemistry &... Feb 1991The plasminogen activator was purified to the extent of 150-fold from 20,000 x g supernatant of Yoshida ascites Sarcoma by ammonium sulphate precipitation at 33%...
The plasminogen activator was purified to the extent of 150-fold from 20,000 x g supernatant of Yoshida ascites Sarcoma by ammonium sulphate precipitation at 33% saturation followed by affinity chromatography on p-aminobenzamidine-Sepharose 4B. The specific activity of the purified activator was 10,260 IU/mg expressed in terms of International units of urokinase, the known activator of plasminogen. The activator was homogeneous by polyacrylamide slab gel electrophoresis with an apparent molecular weight 75 kDa by gel filtration on Sephadex G-100. Analysis by SDS-polyacrylamide gel electrophoresis under reducing conditions, revealed the presence of two subunits of about 48 and 29 kDa. The activator displayed binding preference to fibrin and was immunologically distinguishable from urokinase, indicating that it could be of non-urokinase origin. The preparation further revealed similarity to standard tissue plasminogen activator with respect to fibrin binding and immunological cross reactivity.
Topics: Animals; Ascites; Chromatography, Affinity; Cross Reactions; Electrophoresis, Polyacrylamide Gel; Fibrin; Immunodiffusion; Male; Plasminogen Activators; Rats; Rats, Inbred Strains; Sarcoma, Yoshida; Urokinase-Type Plasminogen Activator
PubMed: 1905268
DOI: No ID Found -
Blood Jul 1991Plasminogen activator inhibitor 1 (PAI-1), an essential regulatory protein of the fibrinolytic system, harbors interaction sites for plasminogen activators (tissue-type...
The interaction of plasminogen activator inhibitor 1 with plasminogen activators (tissue-type and urokinase-type) and fibrin: localization of interaction sites and physiologic relevance.
Plasminogen activator inhibitor 1 (PAI-1), an essential regulatory protein of the fibrinolytic system, harbors interaction sites for plasminogen activators (tissue-type [t-PA] and urokinase-type [u-PA]) and for fibrin. In this study, anti-PAI-1 monoclonal antibodies (MoAbs) were used to identify interaction sites of PAI-1 with these components. The binding sites of 18 different MoAbs were established and are located on five distinct "linear" areas of PAI-1. MoAbs, binding to two distinct areas of PAI-1, are able to prevent the inhibition of t-PA by PAI-1. In addition, two interaction sites for fibrin were identified on PAI-1. The area located between amino acids 110 and 145 of PAI-1 contains a binding site for both components and its significance is discussed in the context of the t-PA inhibition by fibrin-bound PAI-1. Subsequently, the MoAbs were used to assess the role of platelet-PAI-1 in clot-lysis. An in vitro clot-lysis system was used to demonstrate that clot-lysis resistance is dependent on the presence of activated platelets and that PAI-1 is a major determinant for lysis-resistance. We propose that, upon activation of platelets, PAI-1 is fixed within the clot by binding to fibrin and retains its full capacity to inhibit t-PA and u-PA.
Topics: Antibodies, Monoclonal; Binding Sites; Chromosome Deletion; Enzyme Precursors; Epitopes; Fibrin; Fibrinolysis; Humans; Plasminogen Activators; Plasminogen Inactivators; Tissue Plasminogen Activator; Urokinase-Type Plasminogen Activator
PubMed: 1712649
DOI: No ID Found -
Blood Dec 1990The vampire bat salivary plasminogen activator (Bat-PA) is a potent PA that exhibits remarkable selectivity toward fibrin-bound plasminogen (Gardell et al, J Biol Chem... (Comparative Study)
Comparative Study
The vampire bat salivary plasminogen activator (Bat-PA) is a potent PA that exhibits remarkable selectivity toward fibrin-bound plasminogen (Gardell et al, J Biol Chem 256: 3568, 1989). Herein, we describe the activity of recombinant DNA-derived Bat-PA (rBat-PA) in a human plasma milieu. rBat-PA and recombinant human single-chain tissue plasminogen activator (rt-PA) are similarly efficacious at lysing plasma clots. In stark contrast to rt-PA, the addition of 250 nmol/L rBat-PA to plasma in the absence of a clot failed to deplete plasminogen, alpha 2-antiplasmin and fibrinogen. The lytic activities exhibited by finger-domain minus Bat-PA (F- rBat-PA) and finger and epidermal growth factor-like domains minus Bat-PA (FG- rBat-PA) were less than rBat-PA, especially at low concentrations of PA; nevertheless, these truncated forms also possessed a strict requirement for a fibrin cofactor. The loss of PA activity following the addition of rBat-PA to plasma was slower than that observed when either rt-PA or two-chain rt-PA was added. The efficacy, fibrin selectivity, and decreased susceptibility to inactivation exhibited by rBat-PA in vitro in a human plasma milieu suggests that rBat-PA may be superior to rt-PA for the treatment of thrombotic complications.
Topics: Animals; Chiroptera; Fibrin; Fibrinogen; Humans; Plasma; Plasminogen; Plasminogen Activators; Plasminogen Inactivators; Saliva; Tissue Plasminogen Activator
PubMed: 2124935
DOI: No ID Found -
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 -
Thrombosis and Haemostasis Oct 1988Recombinant chimaeric molecules between tissue-type plasminogen activator (t-PA) and single chain urokinase-type plasminogen activator (scu-PA) or two chain...
Inhibition in purified systems and in human plasma of chimaeric plasminogen activators consisting of the NH2-terminal region of tissue-type plasminogen activator and the COOH-terminal region of urokinase-type plasminogen activator.
Recombinant chimaeric molecules between tissue-type plasminogen activator (t-PA) and single chain urokinase-type plasminogen activator (scu-PA) or two chain urokinase-type plasminogen activator (tcu-PA) have intact enzymatic properties of scu-PA or tcu-PA towards natural and synthetic substrates (Nelles et al., J. Biol Chem 1987; 262: 10855-10862). In the present study, we have compared the reactivity with inhibitors of both the single chain and two chain variants of recombinant u-PA and two recombinant chimaeric molecules between t-PA and scu-PA (t-PA/u-PA-s: amino acids 1-263 of t-PA and 144-411 of u-PA; t-PA/u-PA-e: amino acids 1-274 of t-PA and 138-411 of u-PA). Incubation with human plasma in the absence of a fibrin clot for 3 h at 37 degrees C at equipotent concentrations (50% clot lysis in 2 h), resulted in significant fibrinogen breakdown (to about 40% of the normal value) for all two chain molecules, but not for their single chain counterparts. Preincubation of the plasminogen activators with plasma for 3 h at 37 degrees C, resulted in complete inhibition of the fibrinolytic potency of the two chain molecules but did not alter the potency of the single chain molecules. Inhibition of the two chain molecules occurred with a t1/2 of approximately 45 min.(ABSTRACT TRUNCATED AT 250 WORDS)
Topics: Antifibrinolytic Agents; Humans; Kinetics; Plasminogen Activators; Plasminogen Inactivators; Recombinant Proteins; Tissue Plasminogen Activator; Urokinase-Type Plasminogen Activator
PubMed: 3146141
DOI: No ID Found -
American Journal of Health-system... Nov 1997The molecular pharmacology of plasminogen activators and its implications for thrombolytic therapy are discussed. The benefits of coronary thrombolysis were first... (Review)
Review
The molecular pharmacology of plasminogen activators and its implications for thrombolytic therapy are discussed. The benefits of coronary thrombolysis were first demonstrated with intracoronary and i.v. streptokinase. Tissue plasminogen activator (t-PA) or recombinant t-PA (alteplase) proved to be superior to streptokinase with respect to speed of reperfusion and reperfusion efficacy. Since alteplase neither lessened the risk of bleeding found with streptokinase nor generated Thrombolysis in Myocardial Infarction (TIMI) grade 3 flow rates above about 50%, the quest for faster-acting, safer, and more effective thrombolytic agents has continued. The ideal agent would be highly efficient at converting plasminogen to plasmin, have an intermediate half-life, have a low affinity for fibrin, and be of reasonable cost. Genetic engineering of the wild-type t-PA molecule resulted in reteplase, which has a longer half-life than alteplase and may be superior in terms of lytic activity, myocardial salvage, and survival. Also under investigation are TNK-t-PA and n-PA, which have longer half-lives and, in animal models, seem to produce more rapid and complete thrombolysis, at less risk of intracranial bleeding, than alteplase. The risk of intracranial bleeding remains a problem with all thrombolytics; the risk versus the benefit will have to be assessed in large randomized trials. An understanding of the functions of various regions of the t-PA molecule has led to genetic engineering of new and promising plasminogen activators.
Topics: Animals; Fibrinolysin; Humans; Myocardial Infarction; Plasminogen; Plasminogen Activators; Recombinant Proteins; Streptokinase; Thrombolytic Therapy; Tissue Plasminogen Activator
PubMed: 9397233
DOI: 10.1093/ajhp/54.suppl_1.S17 -
Thrombosis and Haemostasis Jun 1987
Synergism of tissue-type plasminogen activator (t-PA) and single-chain urokinase-type plasminogen activator (scu-PA) on clot lysis in vitro and a mechanism for this effect.
Topics: Drug Synergism; Fibrinolysis; Fibrinolytic Agents; Humans; Plasminogen Activators; Tissue Plasminogen Activator; Urokinase-Type Plasminogen Activator
PubMed: 3116707
DOI: No ID Found -
Thrombosis and Haemostasis Jun 1989Study has been made of the influence of addition of human NH2 terminal glutamic acid plasminogen (Glu-Plg) or human NH2 terminal lysine plasminogen (Lys-Plg) to normal...
Study has been made of the influence of addition of human NH2 terminal glutamic acid plasminogen (Glu-Plg) or human NH2 terminal lysine plasminogen (Lys-Plg) to normal citrated plasma upon the rate of lysis of fully crosslinked plasma clots in the presence of single or two chain urokinase type plasminogen activator (scu-PA/tcu-PA) or tissue plasminogen activator (t-PA). The specificity of any thrombolytic property was evaluated by measurement of plasma fibrinogen levels. Lys-Plg added to a concentration of 20% of normal plasma plasminogen caused 5 to 6 fold increase in the extent of lysis observed at 6 hours by 100 units/ml of scu-PA and with a small increase in fibrinogenolysis. Glu-Plg added at 20% of normal level had no influence on thrombolysis but at 50% of normal caused increased thrombolysis with rapid depletion of plasma fibrinogen. An apparently synergistic effect of addition of tcu-PA on scu-PA activity was increased by addition of plasminogen (e.g. addition of 20% Lys-Plg increased the lysis rate 4 to 5 fold over the first hour equivalent to an increase of potency of approximately three to four fold). Addition of plasminogen up to double the normal plasma concentration was observed to have no influence on clot lysis in the presence of t-PA. Plasminogen potentiated the rate of lysis by scu-PA/t-PA synergic mixtures with an approximately 1.5 to 1.9 fold increase in potency. Potentiation occurred without increase in the depletion of plasma fibrinogen.(ABSTRACT TRUNCATED AT 250 WORDS)
Topics: Blood Coagulation Tests; Drug Combinations; Drug Synergism; Fibrinogen; Fibrinolysis; Humans; Molecular Structure; Peptide Fragments; Plasminogen; Plasminogen Activators; Structure-Activity Relationship; Tissue Plasminogen Activator; Urokinase-Type Plasminogen Activator
PubMed: 2508259
DOI: No ID Found -
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 -
Nature Mar 1994Indirect evidence suggests a crucial role for the fibrinolytic system and its physiological triggers, tissue-type (t-PA) and urokinase-type (u-PA) plasminogen activator,...
Indirect evidence suggests a crucial role for the fibrinolytic system and its physiological triggers, tissue-type (t-PA) and urokinase-type (u-PA) plasminogen activator, in many proteolytic processes. Inactivation of the t-PA gene impairs clot lysis and inactivation of the u-PA gene results in occasional fibrin deposition. Mice with combined t-PA and u-PA deficiency suffer extensive spontaneous fibrin deposition, with its associated effects on growth, fertility and survival.
Topics: Animals; Blood Coagulation; Embryonic and Fetal Development; Fibrin; Fibrinolysis; Growth; Longevity; Macrophages; Mice; Mutagenesis; Plasminogen Activators; Stem Cells; Thrombosis; Tissue Plasminogen Activator; Urokinase-Type Plasminogen Activator
PubMed: 8133887
DOI: 10.1038/368419a0