Design of a thrombin inhibitory staphylokinase based plasminogen activator with anti-reocclusion potential.

In the quest of a therapeutically improved thrombolytic drug, we designed and expressed a Staphylokinase based, recombinant fusion protein with fibrin specific clot lysis and anti-thrombin activities derived from human thrombomodulin, a transmembrane glycoprotein with anticoagulant properties, known to form a complex with thrombin and then activating Protein C. The fusion construct was expressed in the yeast Pichia pastoris for cost effective and facile production. The purified construct had comparable plasminogen activation and clot lysis capability as compared to native SAK in that it showed plasmin dependent Plasminogen activation, but exhibited significantly lower fibrinogenolysis (an indicator of fibrin-specific action) even at much higher doses than SAK. In addition, its successful activation of the thrombin-mediated Protein C anticoagulant pathway was reflected in increased in vitro plasma clotting time, as well as inhibition of clot bound thrombin, which led to nearly 15-25% lesser re-formation of fibrin clots after initial successful clot lysis in a specially designed in vitro assay for re-occlusion. Thus, this novel chimeric protein could be of high therapeutic potential as a thrombolytic drug with enhanced fibrin specific clot dissolving properties along with lower bleeding and re-thrombosis complications- a dire need today, especially in the treatment of thrombotic strokes.

Cloning and purification of an anti-thrombotic, chimeric Staphylokinase in Pichia pastoris.

There has been an increasing prevalence of cardiovascular diseases such as myocardial infarction and stroke in modern societies because of multiple lifestyle related issues like sedentariness and obesity, alcohol consumption and many more "life-style"factors. The FDA-approved thrombolytics such as Tissue Plasminogen Activator, Streptokinase etc. are used to lyse the clots in thrombotic disorders such as myocardial infarction, stroke etc. but re-occlusion and bleeding that are co-incident to their clinical usage are not addressed. Hence, there is need to develop thrombolytics having properties like increased fibrin clot specificity and thrombin inhibition capability to prevent re-occlusion. In the present work, a fusion protein construct containing two components i.e. Staphylokinase (SAK) and Epidermal Growth Factor (EGF) 4, 5, 6-like domains of human thrombomodulin (THBD) was expressed in Pichia pastoris after genetic optimization. SAK isolated from Staphylococcus aureus is a fibrin-specific plasminogen activator while EGF 4, 5, 6-like domains are reported to be responsible for imparting thrombin inhibition to human thrombomodulin, and therefore, expected could help prevent re-occlusion in the novel construct - SAK_EGF, which is a 43 kDa protein. After expression, it was purified (approx. 13-fold) using two-step purification protocol involving ion-exchange followed by Gel Filtration Chromatography (GFC). The functional characterization including plasminogen activation and thrombin inhibition showed that both the fusion partners viz. SAK and 4,5,6 EGF-like domains retained their respective activities after fusion, confirming it to be a bio-active construct. Thus, this engineered protein could be clinically promising due to the combinatorial effect of fibrin-specific thrombus lysis and prevention of re-occulusion.

Formation of Se(0), Te(0), and Se(0)-Te(0) nanostructures during simultaneous bioreduction of selenite and tellurite in a UASB reactor.

Simultaneous removal of selenite and tellurite from synthetic wastewater was achieved through microbial reduction in a lab-scale upflow anaerobic sludge blanket reactor operated with 12 h hydraulic retention time at 30 °C and pH 7 for 120 days. Lactate was supplied as electron donor at an organic loading rate of 528 or 880 mg COD L-1 day-1. The reactor was initially fed with a synthetic influent containing 0.05 mM selenite and tellurite each (phase I, day 1-60) and subsequently with 0.1 mM selenite and tellurite each (phase II, day 61-120). At the end of phase I, selenite and tellurite removal efficiencies were 93 and 96%, respectively. The removal percentage dropped to 87 and 81% for selenite and tellurite, respectively, at the beginning of phase II because of the increased influent concentrations. The removal efficiencies of both selenite and tellurite were gradually restored within 20 days and stabilized at ≥ 97% towards the end of the experiment. Powder X-ray diffraction and Raman spectroscopy confirmed the formation of biogenic Se(0), Te(0), and Se(0)-Te(0) nanostructures. Scanning transmission electron microscopy coupled with energy-dispersive X-ray spectroscopy showed aggregates comprising of Se(0), Te(0), and Se-Te nanostructures embedded in a layer of extracellular polymeric substances (EPS). Infrared spectroscopy confirmed the presence of chemical signatures of the EPS which capped the nanoparticle aggregates that had been formed and immobilized in the granular sludge. This study suggests a model for technologies for remediation of effluents containing Se and Te oxyanions coupled with biorecovery of bimetal(loid) nanostructures.

Continuous removal and recovery of tellurium in an upflow anaerobic granular sludge bed reactor.

Continuous removal of tellurite (TeO32-) from synthetic wastewater and subsequent recovery in the form of elemental tellurium was studied in an upflow anaerobic granular sludge bed (UASB) reactor operated at 30°C. The UASB reactor was inoculated with anaerobic granular sludge and fed with lactate as carbon source and electron donor at an organic loading rate of 0.6g CODL-1d-1. After establishing efficient and stable COD removal, the reactor was fed with 10mg TeO32-L-1 for 42 d before increasing the influent concentration to 20mg TeO32-L-1. Tellurite removal (98 and 92%, respectively, from 10 and 20mg TeL-1) was primarily mediated through bioreduction and most of the removed Te was retained in the bioreactor. Characterization using XRD, Raman spectroscopy, SEM-EDX and TEM confirmed association of tellurium with the granular sludge, typically in the form of elemental Te(0) deposits. Furthermore, application of an extracellular polymeric substances (EPS) extraction method to the tellurite reducing sludge recovered up to 78% of the tellurium retained in the granular sludge. This study demonstrates for the first time the application of a UASB reactor for continuous tellurite removal from tellurite-containing wastewater coupled to elemental Te(0) recovery.

Amino-Terminal Fusion of Epidermal Growth Factor 4,5,6 Domains of Human Thrombomodulin on Streptokinase Confers Anti-Reocclusion Characteristics along with Plasmin-Mediated Clot Specificity.

Streptokinase (SK) is a potent clot dissolver but lacks fibrin clot specificity as it activates human plasminogen (HPG) into human plasmin (HPN) throughout the system leading to increased risk of bleeding. Another major drawback associated with all thrombolytics, including tissue plasminogen activator, is the generation of transient thrombin and release of clot-bound thrombin that promotes reformation of clots. In order to obtain anti-thrombotic as well as clot-specificity properties in SK, cDNAs encoding the EGF 4,5,6 domains of human thrombomodulin were fused with that of streptokinase, either at its N- or C-termini, and expressed these in Pichia pastoris followed by purification and structural-functional characterization, including plasminogen activation, thrombin inhibition, and Protein C activation characteristics. Interestingly, the N-terminal EGF fusion construct (EGF-SK) showed plasmin-mediated plasminogen activation, whereas the C-terminal (SK-EGF) fusion construct exhibited 'spontaneous' plasminogen activation which is quite similar to SK i.e. direct activation of systemic HPG in absence of free HPN. Since HPN is normally absent in free circulation due to rapid serpin-based inactivation (such as alpha-2-antiplasmin and alpha-2-Macroglobin), but selectively present in clots, a plasmin-dependent mode of HPG activation is expected to lead to a desirable fibrin clot-specific response by the thrombolytic. Both the N- and C-terminal fusion constructs showed strong thrombin inhibition and Protein C activation properties as well, and significantly prevented re-occlusion in a specially designed assay. The EGF-SK construct exhibited fibrin clot dissolution properties with much-lowered levels of fibrinogenolysis, suggesting unmistakable promise in clot dissolver therapy with reduced hemorrhage and re-occlusion risks.

Site-Specific Thiol-mediated PEGylation of Streptokinase Leads to Improved Properties with Clinical Potential.

Streptokinase (SK) is an efficient thrombolytic agent that dissolves fibrin blood clots with clinical efficiency comparable to the high priced drug, tissue plasminogen activator (tPA). However, being of bacterial origin, its major drawbacks are its potentially high antigenicity, and relatively short circulating half-life (approximately 10-15 min). In the present investigation, an attempt has been made to address both these shortcomings by site-specific pegylation, and to obtain longer lasting thrombolytics, which are consistent with clinical requirements. Therefore, we employed available three-dimensional structural information on SK to carry out site-specific cysteine incorporation at 'optimal' surfaceexposed sites within all the three domains in streptokinase followed by pegylation with 20KDa PEG groups, and screening for biologically active variants. Interestingly, some of these SK PEG-conjugates exhibited considerably subdued immunereactivity along with enhanced in-vitro proteolytic stability profiles and extended circulating in-vivo half-lives (2 to 20-fold compared to that of native unconjugated SK) depending upon location and number of PEG-groups per molecule obtained in homogeneous form. The obtained results are a promising approach for favorably modulating immune-reactivity and half-life by cysteine- specific PEGylation of SK to achieve therapeutic attributes desirable for the treatment of different circulatory disorders, such as ischemic stroke, myocardial infarction and pulmonary embolism.