Salvaging intraluminal peritoneal dialysis catheter obstruction from blood clot with a targeted thrombolytic agent: an innovation.

Peritoneal dialysis (PD) catheter malfunction commonly leads to the removal of the catheter and eventually to a transfer to hemodialysis. The most common cause is intraluminal obstruction caused by blood and fibrin clots. Recommended interventions include irrigation of the catheter with heparinized saline; if this method fails, thrombolytic agents may be used. Mechanical methods such as intraluminal brushing are also utilized, typically after medical treatment fails. Here, we present a case of a patient who developed an intraluminal blood clot that persisted despite attempts with intraluminal thrombolytic drugs and intraluminal brushing. To salvage the catheter, targeted thrombolysis was performed using an endoscopic retrograde cholangiopancreatography (ERCP) guidewire to reinforce the coiled PD catheter and puncture the clots. Additionally, a Swing Tip cannula was employed for direct injection of the thrombolytic agent. These interventions successfully preserved the catheter, resolving the clot and ensuring continued functionality.

Split intein-mediated backbone cyclization enhances the stability and activity of staphylokinase, a potent fibrin-selective plasminogen activator.

Staphylokinase (Sak), a small 15 kDa globular protein that is secreted by certain strains of Staphylococcus aureus, shows a potent fibrin-selective thrombolytic activity. Earlier work has shown that Sak could potentially become a low-cost alternative to currently used thrombolytic agents, such as tissue plasminogen activator (tPA). In attempts to improve its potential for clinical applications, numerous modifications of Sak have already been investigated. Here, we have characterized a novel Sak modification, cyclized Sak (cyc-Sak), which was prepared through split-intein mediated protein backbone cyclization. We have characterized the structure, stability and the activity of cyc-Sak using biophysical techniques, limited proteolysis studies and plasminogen (PG)-activation assays. Our results show that cyc-Sak possesses an identical structure, enhanced stability, resistance to proteolysis by exoproteases and improved PG-activation properties compared to its linear counterpart. It can be over-expressed with high yield in the cytoplasm of Escherichia coli and is easily purified in a two-step process. The intein-mediated cyclization occurs spontaneously in vivo during protein expression and does not necessitate further modification steps after purification of the protein. Furthermore, covalent Sak cyclization could be readily combined with other Sak modifications previously proposed, to generate an effective thrombolytic agent with lower immunogenicity and improved stability and activity.

An in vitro Model for Experimental Evaluation of Sonothrombolysis under Tissue-mimicking Material Conditions.

Background: The mechanical properties of therapeutic ultrasound (US) have attracted scientific interest for thrombolysis enhancement in combination with thrombolytic agents and microbubbles (MBs). The aim of the study was to develop an in vitro model to observe how the effects of sonothrombolysis change in the case where a tissue-mimicking material (TMM) is placed in the path of the US beam before the clot. Methods: Fully retracted blood clots were prepared and pulse sonicated for 1 h under various conditions. The system was in a state of real circulating flow with a branch of an open bypass and an occluded tube containing a blood clot, thus mimicking the case of ischemic stroke. The effectiveness of thrombolysis was quantified in milligrams of clots removed. An agar-based TMM was developed around the occluded tube. Results: The clot breakdown in a TMM was found to be more pronounced than in water, presumably due to the retention of the acoustic field. A higher level of acoustic power was required to initiate clot lysis (>76 W acoustic power) using only focused US (FUS). The greatest thrombolysis enhancement was observed with the largest chosen pulse duration (PD) and the use of MBs (150 mg clot mass lysis). The synergistic effect of FUS in combination with MBs on the enzymatic fibrinolysis enhanced thrombolysis efficacy by 260% compared to thrombolysis induced using only FUS. A reduction in the degree of clot lysis was detected due to the attenuation factor of the intervening material (30 mg at 1 and 4 ms PD). Conclusion: In vitro thrombolytic models including a TMM can provide a more realistic evaluation of new thrombolytic protocols. However, higher acoustic power should be considered to compensate for the attenuation factor. The rate of clot lysis is slow and the clinical use of this method will be challenging.

Production, purification and characterization of a novel antithrombotic and anticoagulant serine protease from food grade microorganism Neurospora crassa.

A novel thrombolytic enzyme was produced by food grade microorganism Neurospora crassa using agro-industrial by-products as substrates. Process parameters were optimized using Plackett-Berman and Box-Benhken design. Under the optimized fermentation conditions, high fibrinolytic activity of 403.59 U/mL was obtained. It was purified with a specific activity of 3572.4 U/mg by ammonium sulfate precipitation and SP Sepharose chromatography. The molecular weight of the enzyme was approximately 32 kDa. It exhibited maximum activity at 40 °C and pH 7.4. Its activity was enhanced by Cu2+, Na+, Zn2+, and completely inhibited by phenylmethanesulfonyl fluoride, soybean trypsin inhibitor, aprotinin, which indicates it could be a serine protease. The enzyme could degrade fibrin clot directly without the need of plasminogen activator, and effectively cleaved Aα, Bß, γ chains of fibrinogen. It could inhibit the formation of blood clots in vitro and acts as an anticoagulant. Compared to heparin the purified enzyme showed extended anticoagulant activity. Blood clots were dissolved effectively and dissolution rate was increased with time. Based on these results, this novel enzyme has the potential to be developed as a thrombolytic agent.

Tenecteplase Thrombolysis in Posterior Circulation Stroke.

One in five ischaemic strokes affects the posterior circulation. Basilar artery occlusion is a type of posterior circulation stroke associated with a high risk of disability and mortality. Despite its proven efficacy in ischaemic stroke more generally, alteplase only achieves rapid reperfusion in ~4% of basilar artery occlusion patients. Tenecteplase is a genetically modified variant of alteplase with greater fibrin specificity and longer half-life than alteplase, which can be administered by intravenous bolus. The single-bolus administration of tenecteplase vs. an hour-long alteplase infusion is a major practical advantage, particularly in "drip and ship" patients with basilar artery occlusion who are being transported between hospitals. Other practical advantages include its reduced cost compared to alteplase. The EXTEND-IA TNK trial demonstrated that tenecteplase led to higher reperfusion rates prior to endovascular therapy (22 vs. 10%, non-inferiority p = 0.002, superiority p = 0.03) and improved functional outcomes (ordinal analysis of the modified Rankin Scale, common odds ratio 1.7, 95% CI 1.0-2.8, p = 0.04) compared with alteplase in large-vessel occlusion ischaemic strokes. We recently demonstrated in observational data that tenecteplase was associated with increased reperfusion rates compared to alteplase prior to endovascular therapy in basilar artery occlusion [26% (n = 5/19) of patients thrombolysed with TNK vs. 7% (n = 6/91) thrombolysed with alteplase (RR 4.0 95% CI 1.3-12; p = 0.02)]. Although randomized controlled trials are needed to confirm these results, tenecteplase can be considered as an alternative to alteplase in patients with basilar artery occlusion, particularly in "drip and ship" patients.

Recombinant Expression of Thrombolytic Agent Reteplase in Marine Microalga Tetraselmis subcordiformis (Chlorodendrales, Chlorophyta).

Tetraselmis subcordiformis, a unicellular marine green alga, is used widely in aquaculture as an initial feeding for fish, bivalve mollusks, penaeid shrimp larvae, and rotifers because of its rich content of amino acids and fatty acids. A stable nuclear transformation system using the herbicide phosphinothricin (PPT) as a selective reagent was established previously. In this research, the recombinant expression in T. subcordiformis was investigated by particle bombardment with the rt-PA gene that encodes the recombinant human tissue-type plasminogen activator (Reteplase), which is a thrombolytic agent for acute myocardial infarction treatment. Transgenic algal strains were selected by their resistance to PPT, and expression of rt-PA was validated by PCR, Southern blotting, and Western blotting, and bioactivity of rt-PA was confirmed by the fibrin agarose plate assay for bioactivity. The results showed that rt-PA was integrated into the genome of T. subcordiformis, and the expression product was bioactive, indicating proper post-transcriptional modification of rt-PA in T. subcordiformis. This report contributes to efforts that take advantage of marine microalgae as cell factories to prepare recombinant drugs and in establishing a characteristic pathway of oral administration in aquaculture.

Biosynthesis and Characterization of a Novel Fibrinolytic Alkaline Serine Protease from Newly Isolated Bacillus flexus BF12 for Biomedical Applications.

BACKGROUND: Cardiovascular Diseases (CVDs) such as stroke, high blood pressure, peripheral vascular disease, ischemic heart disease and acute myocardial infarction are some of the leading causes of death. To treat CVDs, commercially available thrombolytic agents are widely used. However, these thrombolytic agents have various side effects. Alternatively, fibrinolytic enzymes from bacterial sources are highly safe and have direct blood clot lytic activity. METHODS: A fibrinolytic enzyme producing bacterial strain, Bacillus flexus BF12, was isolated from a solar saltpan in Kanyakumari District, Tamilnadu, India. Enzyme production was improved by optimizing physical factors and nutritional factors. RESULTS: A novel fibrinolytic enzyme was isolated from a strain of the studied B. flexus BF12. Enzyme production was enhanced significantly by optimizing process parameters. The critical physical factors (pH and salinity) and influencing nutritional factors (carbon, nitrogen and ions) were optimized by one variable at a time approach, followed by the statistical method. The strain BF12 was highly active at alkaline pH (>7.0) and between 4 and 6% NaCl concentration. The nutrients such as fructose (carbon source), beef extract (nitrogen source) and CaCl2 significantly influenced enzyme production. Central composite design and response surface methodology improved 3.2-fold enzyme yield than unoptimized culture medium. Fibrinolytic protease was purified by ammonium sulphate precipitation, dialysis and gel filtration chromatography. DISCUSSION: The molecular weight of an enzyme was found to be 23 kDa. It was active at a broad temperature (40-60 °C) and pH (7.0-9.0) ranges. Enzyme activity was enhanced by Ca2+ and Co2+ ions. The purified protease retained 100% enzyme activity in the presence of ethanol and acetone. Acetonitrile, butanol, DMSO, methanol and chloroform showed enzyme activity of 63%, 92.5%, 94.7%, 92.3% and 90.4%, respectively. The purified enzyme degraded 100% of human blood clot. CONCLUSION: The Bacillus flexus BF12 fibrinolytic enzyme shows promising potentials in nutraceutical and food fortification applications. The application of fibrinolytic enzymes could prevent CVDs.

Streptokinase: An Efficient Enzyme in Cardiac Medicine.

An imbalance in oxygen supply to cardiac tissues or formation of thrombus leads to deleterious results like pulmonary embolism, coronary heart disease and acute cardiac failure. The formation of thrombus requires clinical encounter with fibrinolytic agents including streptokinase, urokinase or tissue plasminogen activator. Irrespective to urokinase and tissue plasminogen activator, streptokinase is still a significant agent in treatment of cardiovascular diseases. Streptokinase, being so economical, has an important value in treating cardiac diseases in developing countries. This review paper will provide the maximum information to enlighten all the pros and cons of streptokinase up till now. It has been concluded that recent advances in structural/synthetic biology improved SK with enhanced half-life and least antigenicity. Such enzyme preparations would be the best thrombolytic agents.

Spectrophotometric analysis of thrombolytic activity: SATA assay.

Introduction: Measurement of thrombolytic activity is crucial for research and development of novel thrombolytics. It is a key factor in the assessment of the effectiveness of conventionally used thrombolytic therapies in the clinic. Previous methods used for the assessment of thrombolytic activity are often associated with some drawbacks such as being costly, time-consuming, complex with low accuracy. Here, we introduce a simple, economic, relatively accurate and fast method of spectrophotometric analysis of thrombolytic activity (SATA) assay, standardized by tissue plasminogen activator (tPA), which can quantitatively measure in vitro thrombolytic activity. Methods: Blood clots were formed, uniformly, by mixing citrated whole blood with partial thromboplastin time (PTT) reagent, together with calcium chloride. Then, designated concentrations of tPA were added to the samples, and the released red blood cells from each clot were quantified using spectrophotometry (λmax=405nm) as an indicator of thrombolytic activity. The accuracy of the method was tested by assessment of dose-responsibility against R2 value obtained by linear equation and measurement of the limit of detection (LOD) and limit of quantification (LOQ). The SATA assay was validated in comparison with some currently used techniques. Results: A linear relationship was obtained between different concentrations of tPA versus the spectrophotometric absorbance of the related dilutions of lysed clots, at λmax=405nm. Calculated R2 values were greater than 0.9; with LOD of 0.90 µg/mL of tPA (436.50IU) and LOQ of 2.99 µg/mL of tPA (1450.15IU). Conclusion: Conclusively, the SATA assay is a very simple quantitative method with repeatable and reproducible results for estimating the potency of an unknown thrombolytic agent, and calculating the activity as delicate as 1 µg/mL of tPA (485 IU/mL of thrombolytic dose).

Thrombolytic therapy delay is independent predictor of mortality in acute pulmonary embolism at emergency service.

Acute pulmonary embolism (PE) carries a high risk of morbidity and mortality. Delays in diagnosis or therapy may result in sudden, fatal deterioration; therefore, rapid diagnosis and an appropriate therapeutic approach are needed. We aimed to investigate the effect of delaying thrombolytic administration on the mortality rate in a suspected PE. We retrospectively analyzed 49 consecutive patients who were aged 18 years or older and received thrombolysis for a high-risk PE without a major contraindication. All patients were classified according to the time of onset of the thrombolytic therapy. Patients experiencing cardiopulmonary arrest were analyzed from the time of admission to thrombolytic administration with 10-minute cutoff values. Data were analyzed by a regression analysis and a receiver operating characteristic (ROC) analysis for significant and independent associated risk factors and in-hospital mortality. Mortality was seen in 17 of the 49 cases. Thirteen of these had received thrombolytic therapy 1 hour after their emergency department (ED) admission. Among all cases, the mortality rate was 35%. The ROC analysis indicated that a > 97-second delayed thrombolytic administration time was associated with mortality with 53% sensitivity and 91% specificity (area under the curve, 0.803; 95% confidence interval, 0.668-0.938). In the logistic regression, a 5-minute delay in thrombolytic therapy (beta = 1.342; 95% confidence interval, 1.818-2.231; p = 0.001) was associated with in-hospital mortality in the multivariable model. No major bleeding complications were seen in PE survivors. We conclude that early onset thrombolytic therapy in the ED for high-risk and hemodynamically worsening patients appears safe and life-saving.

Bioconversion of agro-industrial wastes for the production of fibrinolytic enzyme from Bacillus halodurans IND18: Purification and biochemical characterization

Background: Agro-wastes were used for the production of fibrinolytic enzyme in solid-state fermentation. The process parameters were optimized to enhance the production of fibrinolytic enzyme from Bacillus halodurans IND18 by statistical approach. The fibrinolytic enzyme was purified, and the properties were studied. Results: A two-level full factorial design was used to screen the significant factors. The factors such as moisture, pH, and peptone were significantly affected enzyme production and these three factors were selected for further optimization using central composite design. The optimum medium for fibrinolytic enzyme production was wheat bran medium containing 1% peptone and 80% moisture with pH 8.32. Under these optimized conditions, the production of fibrinolytic enzyme was found to be 6851 U/g. The fibrinolytic enzyme was purified by 3.6-fold with 1275 U/mg specific activity. The molecular mass of fibrinolytic enzyme was determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis, and it was observed as 29 kDa. The fibrinolytic enzyme depicted an optimal pH of 9.0 and was stable at a range of pH from 8.0 to 10.0. The optimal temperature was 60°C and was stable up to 50°C. This enzyme activated plasminogen and also degraded the fibrin net of blood clot, which suggested its potential as an effective thrombolytic agent. Conclusions: Wheat bran was found to be an effective substrate for the production of fibrinolytic enzyme. The purified fibrinolytic enzyme degraded fibrin clot. The fibrinolytic enzyme could be useful to make as an effective thrombolytic agent.

The effects of dabigatran etexilate on fracture healing in rats: An experimental study.

BACKGROUND: Deep vein thrombosis leading to pulmonary embolism is one of the major complication after fracture. After a fracture occurs, the coagulation cascade activates thrombin, a protease that finally generates clotting. Dabigatran etexilate reduce clot formation by inhibiting thrombin. Dabigatran etexilate is a widely used drug for thromboprophylaxis. There is no study of the effects of dabigatran etexilate on fracture healing in the literature, so we aimed to evaluate the effects of dabigatran etexilate on fracture healing. MATERIALS AND METHODS: Thirty-six female Sprague Dawley rats were divided into 6 groups, each consisting of 6 rats. In all rats, right tibias were used for the fracture model. An oral regimen of dabigatran etexilate suspension in 0.5% hydroxyethylcellulose was administered to the rats. Although the first and second groups received 10 mg/kg daily doses, the third and fourth groups received 50 mg/kg daily doses. The fifth and sixth groups were assigned as sham groups and only hydroxyethylcellulose solution was administered. The first, third and fifth groups were sacrificed on 14(th) days; whereas the second, fourth and sixth groups were sacrificed on 28(th) days. Results were evaluated radiologically and histologically. RESULTS: Radiologically and histologically no statistically significant differences were observed on the 14(th) day between the first, third and fifth groups; and on the 28(th) days between the second, fourth and sixth groups. CONCLUSION: Radiological and histological evaluations revealed that fracture healing was not affected by dabigatran etexilate. We think that dabigatran etexilate can be used for the prophylaxis of thromboembolism in patients with fractures, but further clinical studies are mandatory.

Antiplatelet and antithrombotic activity of a fibrin(ogen)olytic protease from Bacillus cereus strain FF01.

Fibrin(ogen)olytic enzymes offer great promise for the treatment of thrombosis associated disorders. The present study describes the characterization of an extracellular fibrin(ogen)olytic serine protease (named Bacethrombase) purified from the Bacillus cereus strain FF01. The molecular mass of the Bacethrombase was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and matrix assisted laser desorption/ionization-time-of-flight-mass spectroscopy analyses at 39.5 kDa and 38,450.51 Da, respectively. The peptide mass fingerprinting and analyses of the composition of the amino acids revealed the similarity of the Bacethrombase to the bacterial serine proteases. The secondary structure of the Bacethrombase was composed of 14% helix, 6.6% beta-sheet, and 79.4% random coil. Bacethrombase was found to contain 48% sialic acid and it preferentially degraded the Aα-chain of fibrinogen, as well as fibrin. The anticoagulant potency of the Bacethrombase was comparable with that of warfarin and heparin, and was corroborated by its fibrinogenolytic activity rather than the inhibition of thrombin, prothrombin or FXa. Bacethrombase demonstrated antiplatelet activity, and dose-dependently inhibited the ADP-induced platelet aggregation. Bacethrombase (10 mg/kg) did not show toxicity after i.v. administration in Wistar rats; however, it revealed an in vivo anticoagulant effect and significantly inhibited the carrageenan-induced in vivo thrombus formation in rats.

A low cost fermentation medium for potential fibrinolytic enzyme production by a newly isolated marine bacterium, Shewanella sp. IND20.

Agro-residues were used as the substrate for the production of fibrinolytic enzyme in solid state fermentation. In this study, two-level full factorial design (25) and response surface methodology were applied to optimize a fermentation medium for the production of fibrinolytic enzyme from the marine isolate Shewanella sp. IND20. The 25 factorial design demonstrated that the physical factors (pH and moisture) and nutrient factors (trehalose, casein, and sodium dihydrogen phosphate) had significant effect on fibrinolytic enzyme production. Central composite design was employed to search for the optimal concentration of the three factors, namely moisture, pH, and trehalose, and the experimental results were fitted with a second-order polynomial model at 99% level (p < 0.0001). The optimized medium showed 2751 U/mL of fibrinolytic activity, which was 2.5-fold higher than unoptimized medium. The molecular weight of fibrinolytic enzyme was found to be 55.5 kDa. The optimum pH and temperature were 8.0 and 50 °C, respectively.

Targeted thrombolysis strategies for neuroprotective effect.

Stroke is usually treated by systemic thrombolytic therapy if the patient presents within an appropriate time window. There is also widespread interest in the development of thrombolytic agents that can be used in cases of delayed presentation. Current agents that can be used in cases of delayed presentation of nerve damage by thrombus. Current systemic thrombolytic therapy is associated with adverse effects such as fibrinogenolysis and bleeding. In an attempt to increase the efficacy, safety, and specificity of thrombolytic therapy, a number of targeted thrombolytic agents have been studied in recent years. This review focuses on the concepts underlying targeted thrombolytic therapy and describes recent drug developments in this field.

Development of an immunoaffinity method for purification of streptokinase.

BACKGROUND: Streptokinase is a potent activator of plasminogen to plasmin, the enzyme that can solubilize the fibrin network in blood clots. Streptokinase is currently used in clinical medicine as a thrombolytic agent. It is naturally secreted by ß-hemolytic streptococci. METHODS: To reach an efficient method of purification, an immunoaffinity chromatography method was developed that could purify the streptokinase in a single step with high yield. At the first stage, a CNBr-Activated sepharose 4B-Lysine column was made to purify the human blood plasminogen. The purified plasminogen was utilized to construct a column that could purify the streptokinase. The rabbit was immunized with the purified streptokinase and the anti-streptokinase (IgG) purified on another streptokinase substituted sepharose-4B column. The immunoaffinity column was developed by coupling the purified anti-Streptokinase (IgG) to sepharose 6MB-Protein A. The Escherichia coli (E.coli) BL21 (DE3) pLysS strain was transformed by the recombinant construct (cloned streptokinase gene in pGEX-4T-2 vector) and gene expression was induced by IPTG. The expressed protein was purified by immunoaffinity chromatography in a single step. RESULTS: The immunoaffinity column could purify the recombinant fusion GST-SK to homogeneity. The purity of streptokinase was confirmed by SDS-PAGE as a single band of about 71 kD and its biological activity determined in a specific streptokinase assay. The yield of the purification was about 94%. CONCLUSION: This method of streptokinase purification is superior to the previous conventional methods.