Potent Plasminogen Activator Research Articles

Cloning and expression of native streptokinase in E. coli BL21(DE3)

Streptokinase (SK) is a potent plasminogen activator naturally produced by beta-hemolytic streptococcus bacteria. Streptokinase is one of the most important thrombolytic agents that was frequently employed treatment of myocardial infarction. In this study, we amplified the SK gene isolated from Streptococcus pyogenes. The sequence of SK was recorded in the NCBI database. The Accession number of the SK sequence was LC625519.1. The SK gene was digested by EcoR1 and Hindlll, ligated with the linear pGEM®-3Zf (+) vector, and then introduced into an expression host E. coli BL21(DE3). For the production of the SK protein, we induced by 1mM IPTG. SDS-PAGE was used for the evaluation of the streptokinase synthesis with a molecular weight of around 47 kDa. Using the casein lysis method and the in vitro clot lysis assay, the activity of native SK was assessed. The native SK has clot lysis activity and a clear zone in the casein plate.

Optimization of high expression and purification of recombinant streptokinase and in vitro evaluation of its thrombolytic activity

Streptokinase (SK) is a potent plasminogen activator naturally produced by beta-hemolytic streptococcus bacteria and used as a thrombolytic drug. Optimize high yield production of recombinant streptokinase (rSK) in Escherichia coli and evaluate its thrombolytic activity . Synthetic gene encoding mature SK protein with optimization for rare codons and mRNA secondary structure was cloned into the expression vector pET-3a and transformed into Escherichia coli BL21 (DE3). Seed banks were established for high rSK expression clones. The native rSK protein expression was optimized using IPTG induction. The nonsoluble rSK inclusion bodies were purified, denatured in 6 M guanidinium chloride, and refolded using the rapid dilution method. The refolded rSK protein was purified using anion exchange chromatography and evaluated with ELISA. The activity of rSK was evaluated using the casein digestion method and in vitro blood clot lysis assay with reference drug Sedonase as standard. Seed banks with high stable expression of native rSk (MW 47 kDa) were established. High rSK expression was optimized using 1 mM IPTG at bacterial OD600 0.6. The refolded rSK was prepared and purified successfully with high productivity (494 mg purified rsk/L culture). Using ELISA, the purified rSK molecular identity and conservation of native SK epitopes were confirmed. The enzymatic activity of the purified rSK was 1.945x10 6 IU/mg with 62.94 ± 2.3% clot lysis efficiency. A high yield production of proper rSK protein with in vitro thrombolytic activity similar to commercial SK has been achieved, suggesting a more cost-effective industrial production of its biosimilar drug.

Tenecteplase versus Alteplase for Stroke Thrombolysis Evaluation Trial in the Ambulance (Mobile Stroke Unit—TASTE-A): protocol for a prospective randomised, open-label, blinded endpoint, phase II superiority trial of tenecteplase versus alteplase for ischaemic stroke patients presenting within 4.5 hours of symptom onset to the mobile stroke unit

IntroductionMobile stroke units (MSUs) equipped with a CT scanner are increasingly being used to assess and treat stroke patients’ prehospital with thrombolysis and transfer them to the most appropriate hospital...

Biosíntesis del agente fibrinolítico uroquinasa por Enterococcus gallinarum aislado de sardine

Fibrinogen degradation by thrombin leads to the formation of fibrin clots in the blood which results in cardiovascular diseases due to which certain fibrinolytic agents are used to treat cardiovascular complications. Urokinase is being favored as a fibrinolytic agent due to its high fibrin specificity and fewer side effects. Therefore, the main objective of this research was the isolation of potent urokinase producing bacteria from soil, seawater, and fermented food samples. Thirty-five isolates that were capable of producing urokinase were isolated using sixty samples and subjected to secondary screening for urokinase estimation. Bacterial isolate QGA-20 that showed maximum enzyme activity 52.6±0.03 FU/ml/min was identified by 18s RNA sequencing as Enterococcus gallinarum which has never been reported before for urokinase production. Different physical and cultural parameters such as fermentation medium, incubation time, temperature, pH, carbon source, nitrogen source, inoculum size, and trace elements were optimized for submerged fermentation to synthesize urokinase. Maximum production of urokinase 81.3±0.02 FU/ml/min was obtained with M-5 fermentation media at 96 hrs of incubation, 37oC temperature, 7 pH, sucrose as carbon, and soya flour as a nitrogen source, 3% inoculum size and CaCl2 as a trace element. In in vitro studies, urokinase enzyme was also applied for the disintegration of clot successfully. This study revealed a potent urokinase producer, reported first time, with appreciable clot lysis ability.

Famotidine as Urokinase Inhibitor in Anaphylactic Shock

A QSAR study was performed on a series of naphthalene cyclohexane amines derivate with experimentally determine constant of inhibition (Ki ) in order to indentify a potent human urokinase inhibitor. A QSAR model was build using artificial neural networks (ANN). Model was used for screening of compounds with potent urokinase inhibitory properties and to compute Ki of resulted screening compounds. Compound with best Ki was chosen for discussion. An induced fit docking (protein - ligand) and a protein –protein docking were used in results discussion. A isoquinoline –diol base derivate (Famotidione) resulted to have urokinase inhibitory properties.

Urokinase: Isolation, Purification, Characterization, New Spectrophotometric Bioassay Method and In-vitro Blood Clot Dissolving Activity from Cow Urine

Urokinase, a potent plasminogen activator is an enzyme present in cowurine attributing to the thromboembolic properties of cow urine but its presence in cow urine has never been convincingly documented. In our work, we did isolation, purification, partial characterization and quantitation of urokinase in cowurine using newly developed spectrophotometric bioassay method and studied its invitro blood clot dissolving activity. The cow urine was collected from cows of Indian origin ( Bos indicus ). Isolation of Urokinase was done by ammonium sulfate precipitation and dialysis. Purification was done by using Sephadex G-200 Gel Filtration Chromatography. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), Gelatin Zymography, HPLC and TLC of tryptic digest of the purified urokinase was carried out for partial characterization studies which conformed the molecular weight of 59 kD of the purified Urokinase. Quantification with the help of new spectrophotometric bioassay method showed 956 IU/ml and with the help of HPLC it was 910 IU/ml of urokinase in cow urine. The invitro blood clot dissolving activity on a cotton cloth confirmed the activity and efficacy of the isolated and purified urokinase.

Desmoteplase: discovery, insights and opportunities for ischaemic stroke

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.

Production of Recombinant Streptokinase in E. coli and Reactivity with Immunized Mice

Streptokinase (SK) is a potent plasminogen activator with widespread clinical use as a thrombolytic agent. In this study, we produce high level expression of recombinant streptokinase in E. coli by expression vector pET32a. Genomic DNA of streptokinase gene (SKC) was extracted, then amplified by polymerase chain reaction (PCR) method and sub-cloned to prokaryotic expression vector pET32a. Escherichia coli BL21 (DE3) pLysS were transformed with pET32a-skc and gene expression was induced by IPTG. The expressed protein was purified by affinity chromatography by Ni-NTA resin. High concentration of the recombinant protein obtained from the single-step purification by affinity-chromatography (Ni-NTA). The yield of recombinant streptokinase was nearly 470 mg L(-1) of initial culture. Our data showed that production of recombinant streptokinase improved by pET32a in Escherichia coli.

Spectrophotometric Bio assay method for Urokinase

Introduction Urokinase is a potent plasminogen activator. Present Bio assay methods of Urokinase are very tedious. So a new simple spectrophotometric Bio assay method was developed to estimate thrombolytic activity of Urokinase. Methods and Results This Bio assay is designed for the quantitative in vitro spectrophotometric determination of Urokinase activity by utilizing its thrombolytic activity to carry out the lysis of plasma clots. The initial concentration of the plasma clots was adjusted to 0.2 ± 0.01 absorbance and the linear decrease in the absorbance by addition of Urokinase concentration from 200 to 1200 IU/ml at λ max 530 nm was studied. The activity of sample Urokinase can be quantified by comparing the absorbance of sample Urokinase with Urokinase standard Bio assay calibration curve and predicted in IU. The r 2 value of standard calibration curve was found out to be 0.993. The repeatability of the Bio assay was studied by performing the experiment six times with fresh plasma samples and the results showed significant similarity. Discussion We can conclude that the novel Bio assay method was found to be simple, economical, reproducible and accurate than the present Bio assay methods.

Evaluation of pyrrolin-2-one derivatives synthesized by a new practical method as inhibitors of plasminogen activator inhibitor-1 (PAI-1)

We describe in this Letter a new synthetic method for pyrrolin-2-ones as potent plasminogen activator inhibitor-1 (PAI-1) inhibitors. Pyrrolin-2-one derivatives synthesized from N-2-oxoethylamides and aldehydes in aqueous NaOH by one-pot were evaluated for their PAI-1 inhibitory activity. Among these derivatives, compounds 16 and 18 were found to possess potent PAI-1 inhibitory activity (compound 16: IC 50: 0.69 μM, compound 18: IC 50: 0.65 μM).

Urokinase-a Very Popular Cardiovascular Agent

Urokinase (UK) [EC 3.4.99.26] is a serine protease that activates plasminogen to plasmin, which in turn degrades fibrin clots. Hence, UK finds its value as an important anti-thromboembolic drug. Plasmin has diverse physiological roles apart from its fibrinolytic role in the regulation of blood clotting. It has been implicated in complement activation, cell migration, wound healing, and generation of localized extracellular proteolysis during tissue remodelling, pro-hormone conversion, carcinogenesis and neoplasia. Among the plasminogen activators, UK provides a superior alternative for the simple reasons of its being more potent as compared to tissue-plasminogen activator and non-antigenic by virtue of its human origin unlike streptokinase. Based on these observations, UK is a very popular cardiovascular agent. Hence, UK, as one of the most potent plasminogen activators is attracting a great deal of attention. We will summarize recent patents related to the occurrence, mechanism of action, structure and function, physico-chemical properties, in vitro production, cloning and expression, purification and applications of UK.

Recombinant production of Streptococcus equisimilis streptokinase by Streptomyces lividans

BackgroundStreptokinase (SK) is a potent plasminogen activator with widespread clinical use as a thrombolytic agent. It is naturally secreted by several strains of beta-haemolytic streptococci. The low yields obtained in SK production, lack of developed gene transfer methodology and the pathogenesis of its natural host have been the principal reasons to search for a recombinant source for this important therapeutic protein. We report here the expression and secretion of SK by the Gram-positive bacterium Streptomyces lividans. The structural gene encoding SK was fused to the Streptomyces venezuelae CBS762.70 subtilisin inhibitor (vsi) signal sequence or to the Streptomyces lividans xylanase C (xlnC) signal sequence. The native Vsi protein is translocated via the Sec pathway while the native XlnC protein uses the twin-arginine translocation (Tat) pathway.ResultsSK yield in the spent culture medium of S. lividans was higher when the Sec-dependent signal peptide mediates the SK translocation. Using a 1.5 L fermentor, the secretory production of the Vsi-SK fusion protein reached up to 15 mg SK/l. SK was partially purified from the culture supernatant by DEAE-Sephacel chromatography. A 44-kDa degradation product co-eluted with the 47-kDa mature SK. The first amino acid residues of the S. lividans-produced SK were identical with those of the expected N-terminal sequence. The Vsi signal peptide was thus correctly cleaved off and the N-terminus of mature Vsi-SK fusion protein released by S. lividans remained intact. This result also implicates that the processing of the recombinant SK secreted by Streptomyces probably occurred at its C-terminal end, as in its native host Streptococcus equisimilis. The specific activity of the partially purified Streptomyces-derived SK was determined at 2661 IU/mg protein.ConclusionHeterologous expression of Streptococcus equisimilis ATCC9542 skc-2 in Streptomyces lividans was successfully achieved. SK can be translocated via both the Sec and the Tat pathway in S. lividans, but yield was about 30 times higher when the SK was fused to the Sec-dependent Vsi signal peptide compared to the fusion with the Tat-dependent signal peptide of S. lividans xylanase C. Small-scale fermentation led to a fourfold improvement of secretory SK yield in S. lividans compared to lab-scale conditions. The partially purified SK showed biological activity. Streptomyces lividans was shown to be a valuable host for the production of a world-wide important, biopharmaceutical product in a bio-active form.

Inhibitors of the protease domain of urokinase-type plasminogen activator.

Human urokinase-type plasminogen activator (uPA or uPA) has been implicated in the regulation and control of basement membrane and interstitial protein degradation. Since Urokinase plays a role in tissue remodeling, it may be responsible, in part, for the disease progression of cancer. Inhibitors of urokinase may then be useful in the treatment of cancer by retarding tumor growth and metastasis. Urokinase is a multidomain protein, two regions of the protein are most responsible for the observed proteolytic activity in cancer disease and progression. The N-terminal domain or ATF binds to a Urokinase receptor (uPAR) on the cell surface and the C-terminal serine protease domain, then, activates plasminogen to plasmin, beginning a cascade of events leading to the progression of cancer. Investigations of urokinase inhibition has been an area of ongoing research for the past 3 decades. It began with the discovery of small natural and unnatural amino acid derivatives or peptide analogs which exhibited weak inhibition of uPA. The last decade has seen the generation of several classes of potent and selective Urokinase inhibitor directed to the serine protease domain of the protein which have shown potential anti-cancer effects. The availability of structural information of enzyme-inhibitor complexes either by nuclear magnetic spectroscopy (NMR) or crystallography has allowed a detailed analysis of inhibitor protein interactions that contribute to observed inhibitor potency. Structural studies of specific inhibitor-uPA complexes will be discussed as well as the contributions of specific inhibitor protein interactions that are important for overall inhibitor potency. These data were used to discover a class of urokinase inhibitor based on the 2-Naphthamidine template that exhibits potent urokinase inhibition and excellent selectivity for urokinase over similar trypsin family serine proteases.

Towards safer thrombolytic therapy

Plasminogen activators (PA) are unique agents that are currently applied as thrombolytic therapy to achieve rapid vascular reperfusion. Regimens of PA plus anticoagulants and antiplatelet drugs have attained a high degree of sophistication and predictable rates of positive clinical outcomes for acute myocardial infarction (MI), ischemic stroke, pulmonary embolism (PE), deep vein thrombosis (DVT), and thrombosed catheters. Included in the repertoire are newly approved mutants of tissue plasminogen activator (TPA), which have biochemical advantages that allow for bolus administration. Yet, despite tremendous effort devoted to enormous trials to establish the clinical efficacy of these agents in acute MI, mortality results are not superior to those with native TPA or streptokinase (SK). Furthermore, all PAs have the potential for hemorrhagic complication, most critically intracranial hemorrhage (ICH), occurring in 0.9% of patients treated with native or mutant TPA. It is possible that a limit of clinical effectiveness has been reached, beyond which more potent PAs do not achieve greater benefit without a serious increase in risk of bleeding. A breakthrough is possible, however, if the risk of ICH could be avoided. One solution is the application of the direct-acting thrombolytic enzyme, plasmin. While intravenous plasmin is not effective when administered systemically, regional infusion to a thrombus induces local thrombolysis. Unlike the PAs, plasmin treatment should not cause hemorrhage from vascular trauma sites, as it is neutralized by antiplasmin in the blood. Animal studies are fully consistent with this approach, which offers potential for achieving a truly regional thrombolytic treatment. Semin Hematol 39:206-216. Copyright 2002, Elsevier Science (USA). All rights reserved.

ChemInform Abstract: Design and Synthesis of 4,5‐Disubstituted‐thiophene‐2‐amidines as Potent Urokinase Inhibitors.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Vampire Bat Plasminogen Activator DSPA-Alpha-1 (Desmoteplase): A Thrombolytic Drug Optimized by Natural Selection

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.

Design and Synthesis of 4,5-Disubstituted-thiophene-2-amidines as Potent Urokinase Inhibitors

A study of the S1 binding of lead 5-methylthiothiophene amidine 3, an inhibitor of urokinase-type plasminogen activator, was undertaken by the introduction of a variety of substituents at the thiophene 5-position. The 5-alkyl substituted and unsubstituted thiophenes were prepared using organolithium chemistry. Heteroatom substituents were introduced at the 5-position using a novel displacement reaction of 5-methylsulfonylthiophenes and the corresponding oxygen or sulfur anions. Small alkyl group substitution at the 5-position provided inhibitors equipotent with 3 but possessing improved solubility.

Structure-directed discovery of potent non-peptidic inhibitors of human urokinase that access a novel binding subsite

Background: Human urokinase-type plasminogen activator has been implicated in the regulation and control of basement membrane and interstitial protein degradation. Because of its role in tissue remodeling, urokinase is a central player in the disease progression of cancer, making it an attractive target for design of an anticancer clinical agent. Few urokinase inhibitors have been described, which suggests that discovery of such a compound is in the early stages. Towards integrating structural data into this process, a new human urokinase crystal form amenable to structure-based drug design has been used to discover potent urokinase inhibitors. Results: On the basis of crystallographic data, 2-naphthamidine was chosen as the lead scaffold for structure-directed optimization. This co-crystal structure shows the compound binding at the primary specificity pocket of the trypsin-like protease and at a novel binding subsite that is accessible from the 8-position of 2-napthamidine. This novel subsite was characterized and used to design two compounds with very different 8-substituents that inhibit urokinase with K i values of 30–40 nM. Conclusions: Utilization of a novel subsite yielded two potent urokinase inhibitors even though this site has not been widely used in inhibitor optimization with other trypsin-like proteases, such as those reported for thrombin or factor Xa. The extensive binding pockets present at the substrate-binding groove of these other proteins are blocked by unique insertion loops in urokinase, thus necessitating the utilization of additional binding subsites. Successful implementation of this strategy and characterization of the novel site provides a significant step towards the discovery of an anticancer clinical agent.

Pharmacology and clinical trial results of saruplase (scuPA) in acute myocardial infarction

Saruplase is the non-glycosylated form of single chain urokinase-type plasminogen activator; it is produced by recombinant technique in Escherichia coli. Saruplase has plasminogen activating properties; it can also be cleaved to the potent plasminogen activator, double chain urokinase, resulting in the generation of plasmin. By means of a positive feedback mechanism, plasmin itself is able to cleave saruplase. At present, a bolus of 20 mg followed by an infusion of 60 mg over 1 h and preceded by a heparin bolus of 5000 U, represents the standard saruplase regimen. The PRIMI trial showed that this standard saruplase regimen achieves higher early patency rates than streptokinase. In the COMPASS trial, saruplase has been shown to be at least as effective as streptokinase, in terms of 30 day mortality. A comparative trial with urokinase (SUTAMI) resulted in similar late coronary patency. Saruplase has been tested against alteplase (100 mg over 180 min) in the SESAM trial. The early coronary patency and safety of both regimens were comparable. In a recent study, three bolus applications of saruplase have been investigated. A double bolus of 40 mg each given 30 min apart, is suggested to be more effective than standard saruplase, but concerns about safety remain. Further studies are required to assess safety of the double bolus regimen, and to compare saruplase with the gold-standard of frontloaded alteplase, or the equivalent double bolus reteplase regimen.

NH2-terminal Structural Motifs in Staphylokinase Required for Plasminogen Activation

Staphylokinase (Sak) forms an inactive 1:1 stoichiometric complex with plasminogen which requires both conversion of plasminogen to plasmin and hydrolysis of the Lys10-Lys11 peptide bond of Sak to become a potent plasminogen activator (Schlott, B., Guhrs, K.-H., Hartmann, M., Rocker, A., and Collen, D. (1997) J. Biol. Chem. 272, 6067-6072). Exposure of a positively charged NH2-terminal amino acid after hydrolysis of Sak is a major determinant of the plasminogen-activating potential, but in itself is neither necessary nor sufficient. Here, the structural motifs of the NH2-terminal region Lys11-Gly-Asp-Asp-Ala-Ser16-Tyr-Phe-Glu of processed Sak, required for plasminogen activating potential, were studied by deletion and substitution mutagenesis. Expression in Escherichia coli of variants with deletion of 11, 14, 15, or 16 NH2-terminal amino acids yielded correctly processed but inactive molecules. Expression of their homologues with the NH2-terminal amino acid substituted with Lys-generated derivatives from which the NH2-terminal initiation Met was no longer removed, yielding inactive (</= 10%) Sak42DDeltaN11(M),G12K, active (>50%) Sak42DDeltaN14(M), A15K and Sak42DDeltaN15(M),S16K, and inactive Sak42DDeltaN16(M),Y17K. Lys variants without NH2-terminal Met, generated from fusion proteins in which a His6 tag and a factor Xa recognition sequence were linked to the NH2 terminus of the Sak variants, were indistinguishable from their NH2-terminal Met-containing counterparts. All variants studied had intact affinities for plasminogen as measured by biospecific interaction analysis. The activity of Sak42DDeltaN11(M),G12K could be restored by additional substitution of both Asp13 and Asp14 with Asn, yielding active Sak42DDeltaN11(M),G12K, D13N, D14N, whereas substitution in Sak42DDeltaN16(M),Y17K of Phe18 and Glu19 with Asn yielded inactive Sak42DDeltaN16(M),Y17K,F18N,E19N. These data, in combination with the recent finding that the 20 NH2-terminal amino acids of Sak lack secondary structure, suggest that the NH2-terminal region of Sak is not required for binding to plasmin/plasminogen, but that a positively charged amino acid in the ultimate or penultimate NH2-terminal position corresponding to amino acids 11-16 of this flexible region participates in the reconfiguration of the active site of the plasmin molecule to endow it with plasminogen-activating potential.