This research was aimed at isolating and identifying the predominant lactic acid bacteria (LAB) in the traditional Chinese salt-fermented soybean food, douchi, from Yunnan, China. The predominant LAB present were isolated and identified by conventional culture-dependent methods combined with molecular biological methods. Two hundred and sixty isolates were obtained from thirty kinds of traditional fermented douchi from six cities and counties in Yunnan, and those strains were divided into twelve groups by their morphological and biochemical characteristics. Based on 16S ribosomal DNA (rDNA) sequencing and analysis, 56 representative strains were identified as belonging to 6 genera and 14 species: Lactobacillus (4 spp.), Weissella (3 spp.), Pediococcus (2 spp.), Staphylococcus (2 spp.), Enterococcus (1 sp.), and Bacillus (2 spp.). The results show that douchi contains a large natural population of LAB of diverse composition from which some strains could be selected as starters for functional fermented foods. This is the first study on the original douchi from Yunnan, and the results suggest that it may be a useful source for the isolation of LAB. This study has also laid a foundation for further research on developing functional douchi products.
Fermented food; Douchi; Lactic acid bacteria; Culture-dependent methods; 16S rDNA
To compare the effects of a tailored and targeted print intervention in promoting dilated fundus examinations (DFEs) in older African Americans, and to determine if other factors are associated with getting a DFE.
African Americans, 65 years of age or older, who had not had a DFE in at least two years were recruited from community settings. Participants were randomized to receive either a tailored or targeted newsletter. Telephone follow-up was conducted at one, three, and six months to ascertain eye examination status. All self-reported DFEs were confirmed by contacting their eye doctor by telephone.
Main outcome measure
Doctor-confirmed DFE at six months.
Of the 329 participants enrolled, 128 (38.9%) had a doctor-confirmed DFE. There was no difference in doctor-confirmed DFEs by intervention group (RR=1.07, 0.82–1.40 CI), with 66 participants in the tailored group (40.2%) and 62 (37.6%) participants in the targeted group having a doctor-confirmed DFE. Based on logistic regression analysis, reading the newsletter (OR=1.76, 1.08–2.87 CI) and planning on making an appointment for a DFE (OR=2.46, 1.42–4.26 CI) were significant predictors for getting a DFE.
The tailored and targeted interventions were equally effective in promoting doctor-confirmed DFEs at six months. Given the increases cost and effort associated with tailoring, our results suggest that well-designed targeted print messages can motivate older African Americans to get DFEs.
Theoretical studies of adaptation emphasize the importance of understanding the distribution of fitness effects (DFE) of new mutations. We report the isolation of 100 adaptive mutants—without the biasing influence of natural selection—from an ancestral genotype whose fitness in the niche occupied by the derived type is extremely low. The fitness of each derived genotype was determined relative to a single reference type and the fitness effects found to conform to a normal distribution. When fitness was measured in a different environment, the rank order changed, but not the shape of the distribution. We argue that, even with detailed knowledge of the genetic architecture underpinning the adaptive types (as is the case here), the DFEs remain unpredictable, and we discuss the possibility that general explanations for the shape of the DFE might not be possible in the absence of organism-specific biological details.
mutation; adaptation; evolution
Cardiovascular complications are consistently observed in diabetic patients across all age groups. The objective of the present study was to investigate the effect of aqueous extract of the fruit pulp of Hylocereus undatus (DFE) on aortic stiffness and oxidative stress in streptozotocin (STZ)-induced diabetes in rats. Twenty-four male, Sprague-Dawley rats were randomized into four groups: I (control), II (diabetic), III (DFE, 250 mg/kg) and IV (DFE 500 mg/kg). Diabetes was induced in groups II, III and IV by intraperitoneal (i.p.) injection of STZ (40 mg/kg). After confirmation of diabetes, group III and IV received DFE for 5 weeks. Pulse wave velocity (PWV) was used as a marker of aortic stiffness and was determined at the end of 5 weeks. DFE significantly decreased (P < 0.05) the fasting blood glucose levels in diabetic rats, but not to normal levels. Systolic blood pressure, pulse pressure and PWV were significantly increased (P < 0.05) in diabetic rats at the end of 5 weeks in comparison with control group. DFE treatment significantly decreased (P < 0.05) these elevations. Oxidative damage was observed in group II after 5 weeks. Plasma malondialdehyde levels significantly decreased (P < 0.05), while superoxide dismutase and total antioxidant capacity significantly increased (P < 0.05) with DFE treatment in comparison with group II. These data demonstrate that DFE treatment was effective in controlling oxidative damage and decreasing the aortic stiffness measured by PWV in STZ-induced diabetes in rats.
Arterial stiffness; diabetes; Hylocereus undatus; pulse wave velocity; streptozotocin
Background. To study the effects of an aqueous extract of date fruit (Phoenix dactylifera L. Arecaceae) diet on diabetic polyneuropathy (DPN) in streptozotocin- (STZ-) induced diabetic rats.
Methods. The effects of a date fruit extract (DFE) diet on diabetic neuropathy in STZ-induced diabetic rats were evaluated and compared with a nondiabetic control group, diabetic control group (sham), and vehicle group with respect to the following parameters: open field behavioral test, motor nerve conduction velocity (MNCV), and morphological observations.
Results. In the model of STZ-induced of diabetic neuropathy, chronic treatment for 6 weeks with DFE counteracted the impairment of the explorative activity of the rats in an open field behavioral test and of the conduction velocity of the sciatic nerve (MNCV). In addition, pretreatment with DFE significantly reversed each nerve diameter reduction in diabetic rats.
Conclusion. DFE treatment shows efficacy for preventing diabetic deterioration and for improving pathological parameters of diabetic neuropathy in rats, as compared with control groups.
Reperfusion therapy using thrombolytic agents has been shown to be a safe and effective treatment strategy for arterial ischemia, venous thrombosis, massive pulmonary embolism, and acute stroke. Thrombolytic agents have evolved over the course of a few decades, from nonfibrin selective to fibrin-selective agents. The development and modification of these agents have resulted in improved understanding of their pharmacologic attributes, and their effects on the complex molecular events that occur during thrombolysis goal-directed therapies. The current review focuses on the physiology and pharmacology of the thrombolytic agents that have been or are currently in use for interventional thrombolysis interventions. Attention is also given to the particular role that thrombolytic agents play in the current management of peripheral vascular disease and acute stroke.
Catheter-directed thrombolysis; pharmacology; physiology; acute limb ischemia; stroke
Iron (Fe) is an essential micronutrient for many processes in all living cells. Dissolved Fe (dFe) concentrations in the ocean are of the order of a few nM, and Fe is often a factor limiting primary production. Bioavailability of Fe in aquatic environments is believed to be primarily controlled through chelation by Fe-binding ligands. Marine microbes have evolved different mechanisms to cope with the scarcity of bioavailable dFe. Gradients in dFe concentrations and diversity of the Fe-ligand pool from coastal to open ocean waters have presumably imposed selection pressures that should be reflected in the genomes of microbial communities inhabiting the pelagic realm. We applied a hidden Markov model (HMM)-based search for proteins related to cellular iron metabolism, and in particular those involved in Fe uptake mechanisms in 164 microbial genomes belonging to diverse taxa and occupying different aquatic niches. A multivariate statistical approach demonstrated that in phototrophic organisms, there is a clear influence of the ecological niche on the diversity of Fe uptake systems. Extending the analyses to the metagenome database from the Global Ocean Sampling expedition, we demonstrated that the Fe uptake and homeostasis mechanisms differed significantly across marine niches defined by temperatures and dFe concentrations, and that this difference was linked to the distribution of microbial taxa in these niches. Using the dN/dS ratios (which signify the rate of non-synonymous mutations) of the nucleotide sequences, we identified that genes encoding for TonB, Ferritin, Ferric reductase, IdiA, ZupT, and Fe2+ transport proteins FeoA and FeoB were evolving at a faster rate (positive selection pressure) while genes encoding ferrisiderophore, heme and Vitamin B12 uptake systems, siderophore biosynthesis, and IsiA and IsiB were under purifying selection pressure (evolving slowly).
marine microbes; eukaryotic phytoplankton; Fe limitation; Fe- binding ligands; multivariate statistics; metagenomes; dN/dS ratio; aquatic niches
In a clinical trial, we have previously shown that a telephone intervention can significantly increase participation in dilated fundus examination (DFE) screening among low-income adults with diabetes. Here the costs and cost-effectiveness ratio of this intervention are calculated. Intervention effectiveness was estimated as the difference in DFE utilization between the telephone intervention and print groups from the clinical trial multiplied by the size of the telephone intervention group. A micro-costing approach was used. Personnel time was aggregated from logs kept during the clinical trial of the intervention. Wage rates were taken from a commercial compensation database. Telephone charges were estimated based on prevailing fees. The cost-effectiveness ratio was calculated as the ratio of total costs of the intervention to the number of DFEs gained by the intervention. A sensitivity analysis estimated the cost-effectiveness of a more limited telephone intervention. A probabilistic sensitivity analysis using bootstrap samples from the clinical trial results quantified the uncertainties in resource utilization and intervention effectiveness. Net intervention costs were US$18,676.06, with an associated gain of 43.7 DFEs and 16.4 new diagnoses of diabetic retinopathy. The cost-effectiveness ratio is US$427.37 per DFE gained. A restricted intervention limiting the number of calls to 5, as opposed to 7, would achieve the same results, but would cost approximately 17% less. In the probabilistic sensitivity analysis, the 5th and 95th percentiles of the cost-effectiveness ratio were US$304.05 and US$692.52 per DFE gained, respectively. Our telephone intervention is more expensive than simple mail or telephone reminders used in other settings to promote preventive care; it is, however, also considerably more effective, and is effective in a low-income minority population at greater risk for diabetes complications. The costs are dominated by labor costs, and may be substantially defrayed, without loss of effectiveness, by restricting the number of telephone calls to 5 per patient.
cost-effectiveness; diabetes mellitus; dilated fundus examination
Acute stent thrombosis remains one of the most important concerns in clinical cardiology. The mechanism is not fully understood but a prothrombotic state is a key component. We describe a case of acute stent thrombosis, within an hour of rescue angioplasty, despite use of full dose fibrinolytic (reteplase) and antiplatelet therapy (aspirin and clopidogrel). Risk of acute stent thrombosis was predicted an hour earlier, when the patient was clinically well, by a novel near-patient test of thrombotic and thrombolytic status (in vitro). Subsequent stent thrombosis was visualised angiographically (in vivo) and confirmed by extraction of the thrombus (ex vivo). The near-patient test sensitively detected reversal of the prothrombotic state after abciximab treatment. We believe this is the first description of the clinical use of a near-patient test within the cardiac catheterisation laboratory to predict risk of imminent stent thrombosis.
Previous inhalation toxicity studies from our laboratory have shown that 1,1-dichloroethylene (1,1-DCE), 1,1-dibromoethylene (1,1-DBE), and 2-chloro-1,3,-butadiene (2-CBD) are more toxic to fasted rats than to fed rats. Vinyl chloride monomer (VCM) and 1,1-difluoroethylene (1,1-DFE) were not acutely hepatotoxic at 46,500 and 82,000 ppm, respectively, in normal male rats, whether fed or fasted. On a molar basis, 1,1-DBE and 1,1-DCE have similar toxicities while 2-CBD is less toxic. All three compounds produce similar elevation of serum transaminase and bloody ascites, although at differing times following differing exposure concentrations. 1,1-DCE produces massive midzonal hepatic necrosis with hepatic thrombosis and chromatolysis within 2 hr after a 4 hr exposure of fasted rats to 200 ppm. Subsequent to formation of this midzonal lesion, the central portion of the lobule collapses, accompanied by congestion, ascites, and in increased hematocrit in the rat. Serum transaminase and sorbital dehydrogenase are greatly elevated at 6 hr. This effect in fasted rats is associated with glutathione (GSH) depletion. Diethyl maleate (DEM) which depletes GSH in fed rats potentiates the injury associated with 1,1-DCE exposure as well as that produced by 2-CBD. Rats fed ad libitum and exposed to 1,1-DCE or 2-CBD at night, a time of low hepatic GSH concentration, exhibit enhancement of hepatotoxic response when compared to animals exposed during the day when GSH is high.
2-aminophenol (2-AP) is a toxic nitrogen-containing aromatic pollutant. Burkholderia xenovorans LB400 possess an amn gene cluster that encodes the 2-AP catabolic pathway. In this report, the functionality of the 2-aminophenol pathway of B. xenovorans strain LB400 was analyzed. The amnRJBACDFEHG cluster located at chromosome 1 encodes the enzymes for the degradation of 2-aminophenol. The absence of habA and habB genes in LB400 genome correlates with its no growth on nitrobenzene. RT-PCR analyses in strain LB400 showed the co-expression of amnJB, amnBAC, amnACD, amnDFE and amnEHG genes, suggesting that the amn cluster is an operon. RT-qPCR showed that the amnB gene expression was highly induced by 2-AP, whereas a basal constitutive expression was observed in glucose, indicating that these amn genes are regulated. We propose that the predicted MarR-type transcriptional regulator encoded by the amnR gene acts as repressor of the amn gene cluster using a MarR-type regulatory binding sequence. This report showed that LB400 resting cells degrade completely 2-AP. The amn gene cluster from strain LB400 is highly identical to the amn gene cluster from P. knackmussi strain B13, which could not grow on 2-AP. However, we demonstrate that B. xenovorans LB400 is able to grow using 2-AP as sole nitrogen source and glucose as sole carbon source. An amnBA− mutant of strain LB400 was unable to grow with 2-AP as nitrogen source and glucose as carbon source and to degrade 2-AP. This study showed that during LB400 growth on 2-AP this substrate was partially converted into picolinic acid (PA), a well-known antibiotic. The addition of PA at lag or mid-exponential phase inhibited LB400 growth. The MIC of PA for strain LB400 is 2 mM. Overall, these results demonstrate that B. xenovorans strain LB400 posses a functional 2-AP catabolic central pathway, which could lead to the production of picolinic acid.
Inorganic carbon (IC), such as bicarbonate or carbon dioxide, stimulates the growth of Lactobacillus plantarum. At low IC levels, one-third of natural isolated L. plantarum strains are nutritionally dependent on exogenous arginine and pyrimidine, a phenotype previously defined as high-CO2-requiring (HCR) prototrophy. IC enrichment significantly decreased the amounts of the enzymes in the pyrimidine biosynthetic pathway encoded by the pyrR1BCAa1Ab1DFE operon, as demonstrated by proteomic analysis. Northern blot and reverse transcription-PCR experiments demonstrated that IC levels regulated pyr genes mainly at the level of transcription or RNA stability. Two putative PyrR regulators with 62% amino acid identity are present in the L. plantarum genome. PyrR1 is an RNA-binding protein that regulates the pyr genes in response to pyrimidine availability by a mechanism of transcriptional attenuation. In this work, the role of PyrR2 was investigated by allelic gene replacement. Unlike the pyrR1 mutant, the ΔpyrR2 strain acquired a demand for both pyrimidines and arginine unless bicarbonate or CO2 was present at high concentrations, which is known as an HCR phenotype. Analysis of the IC- and pyrimidine-mediated regulation in pyrR1 and pyrR2 mutants suggested that only PyrR2 positively regulates the expression levels of the pyr genes in response to IC levels but had no effect on pyrimidine-mediated repression. A model is proposed for the respective roles of PyrR1 and PyrR2 in the pyr regulon expression.
Thrombus formed in blood vessels lead to atherothrombotic diseases such as myocardial or cerebral infarction. Thrombolytic agents are used to dissolve the already formed clots in the blood vessels; however, these drugs sometimes cause serious and fatal consequences. Herbal preparations have been used since ancient times for the treatment of several diseases although they show little toxicity in some cases. Aqueous extracts of herbs used in thrombolysis have been reported before with cytotoxic data, however, the organic extracts of herbs have not been documented. This study aims to investigate whether organic extracts possess thrombolytic properties with minimal or no toxicity.
An in vitro thrombolytic model was used to check the clot lysis effect of six Bangladeshi herbal extracts viz., Ageratum conyzoides L., Clausena suffruticosa, Leea indica (Burm.f.) Merr., Leucas aspera Willd., Senna sophera L. Roxb., and Solanum torvum Swartz. using streptokinase as a positive control and water as a negative control. Briefly, venous blood drawn from twenty healthy volunteers was allowed to form clots which were weighed and treated with the test plant materials to disrupt the clots. Weight of clot after and before treatment provided a percentage of clot lysis. Cytotoxicity was screened by brine shrimp lethality bioassay using vincristine sulfate as positive control.
Using an in vitro thrombolytic model, Ageratum conyzoides, Clausena suffruticosa, Leea indica, Leucas aspera, Senna sophera and Solanum torvum showed 18.12 ± 2.34%, 48.9 ± 2.44%, 39.30 ± 0.96%, 37.32 ± 2.00%, 31.61 ± 2.97% and 31.51 ± 0.57% and clot lysis respectively. Among the herbs studied Clausena suffruticosa, Leea indica and Leucas aspera showed very significant (p < 0.0001) percentage (%) of clot lysis compared to reference drug streptokinase (75.00 ± 3.04%). In brine shrimp cytotoxic assay, the extracts Ageratum conyzoides, Clausena suffruticosa, Leea indica, Leucas aspera, Senna sophera and Solanum torvum showed LC50 values 508.86 ± 6.62,41.16 ± 1.26, 2.65 ± 0.16, 181.67 ± 1.65, 233.37 ± 7.74 and 478.40 ± 3.23 μg/ml, respectively, with reference to vincristine sulfate (LC50 0.76 ± 0.04).
Through our study it was found that Clausena suffruticosa, Leea indica and Leucas aspera possessed effective thrombolytic properties whereas Senna sophera and Solanum torvum showed moderate to mild thrombolytic effects while Ageratum conyzoides showed no significant effect. No extract was found cytoxic compared to positive control. Clausena suffruticosa, Leea indica and Leucas aspera could be incorporated as a thrombolytic agent with in vivo effects to improve the atherothrombotic patients. However, Clausena suffruticosa could be the best one to use in this purpose.
Thrombolysis; Clausena suffruticosa; Leea indica; Leucas aspera; Streptokinase
Nattokinase, a protein found in high levels in the traditional Japanese food natto, has been reported to have high thrombolytic activity. In the present study, the crystallization of native nattokinase and the collection of X-ray diffraction date from a nattokinase crystal to a resolution of 1.74 Å are reported.
Nattokinase is a single polypeptide chain composed of 275 amino acids (molecular weight 27 724) which displays strong fibrinolytic activity. Moreover, it can activate other fibrinolytic enzymes such as pro-urokinase and tissue plasminogen activator. In the present study, native nattokinase from Bacillus subtilis natto was purified using gel-filtration chromatography and crystallized to give needle-like crystals which could be used for X-ray diffraction experiments. The crystals belonged to space group C2, with unit-cell parameters a = 74.3, b = 49.9, c = 56.3 Å, β = 95.2°. Diffraction images were processed to a resolution of 1.74 Å with an R
merge of 5.2% (15.3% in the highest resolution shell) and a completeness of 69.8% (30.0% in the highest resolution shell). This study reports the first X-ray diffraction analysis of nattokinase.
nattokinase; Bacillus subtilis natto; fibrinolysis
Choline is a required nutrient with roles in liver and brain function, lipid metabolism, and fetal development. Recent data suggest that choline requirements may be altered by polymorphisms in the phosphatidylethanolamine N-methyltransferase (PEMT) gene (i.e., 5465G→A; rs7946 and -744G→C; rs12325817) and in the methylenetetrahydrofolate dehydrogenase (MTHFD1) gene (i.e., 1958G→A; rs2236225). This controlled feeding study, conducted in 2000–2001, examined the effects of the PEMT and MTHFD1 genetic variants on biomarkers of choline metabolism in pre-menopausal Mexican American women (n=43) after a 7-wk period of folate restriction (135μg as dietary folate equivalents, DFE) and after a 7-wk period of folate treatment (400 and 800μg DFE/d combined). Throughout the 14-wk study choline intake remained constant at 349mg/d. The genotype frequencies of the women were 3GG, 19GA, and 21AA for PEMT G5465A; 9GG, 17GC and 17CC for PEMT G-744C; and 9GG, 21GA and 13AA for MTHFD1 G1958A. During folate restriction, homocysteine was adversely influenced by PEMT 5465AA (P=0.001 relative to the G allele) and by MTHFD1 1958AA (P=0.085 relative to 1958GG); whereas the decline in phosphatidylcholine was attenuated by PEMT -744CC (P=0.017 relative to -744GG). During folate treatment, no effects of the genotypes on the response of the measured variables were detected. These data suggest that polymorphisms in genes relevant to choline metabolism modulate parameters of choline status when folate intake is restricted. Additional studies with larger samples sizes are needed to examine the relationship between these genetic variants and varied choline intake in populations with increased demands for choline (i.e., pregnant women).
PEMT; MTHFD1; choline; folate; genetic variants
We investigate several questions concerning the inference of the distribution of fitness effects (DFE) of new mutations from the distribution of nucleotide frequencies in a population sample. If a fixed sequencing effort is available, we find that the optimum strategy is to sequence a modest number of alleles (approx. 10). If full genome information is available, the accuracy of parameter estimates increases as the number of alleles sequenced increases, but with diminishing returns. It is unlikely that the DFE for single genes can be reliably estimated in organisms such as humans and Drosophila, unless genes are very large and we sequence hundreds or perhaps thousands of alleles. We consider models involving several discrete classes of mutations in which the selection strength and density apportioned to each class can vary. Models with three classes fit almost as well as four class models unless many hundreds of alleles are sequenced. Large numbers of alleles need to be sequenced to accurately estimate the distribution's mean and variance. Estimating complex DFEs may therefore be difficult. Finally, we examine models involving slightly advantageous mutations. We show that the distribution of the absolute strength of selection is well estimated if mutations are assumed to be unconditionally deleterious.
mutation; selection; distribution of effects; parameter inference
The medicinal value of earthworm has been widely known since the history of Asian ancient medicine. This present study aims to determine the mechanism of action and effect of a standardized extract of Lumbricus rubellus named as DLBS1033. The fibrinogen degradation, antiplatelet aggregation, and ex vivo antithrombotic assay using human blood were performed to study antithrombotic activity. Fibrin plate and clot lysis assay were also done to examine thrombolytic properties. DLBS1033 was found to possess fibrinogenolytic activity on α-, β-, and γ-chain of fibrinogen. It also induced antiplatelet aggregation and prolonged blood clotting time, which further confirmed its antithrombotic properties. In addition, thrombolytic properties of DLBS1033 were shown with its fast and long-acting fibrinolytic activity, as well as its effective blood clot lysis activities. In conclusion, DLBS1033 conferred antithrombotic and thrombolytic action which could be used as a safe and promising oral thrombolytic drug.
The purpose of the present study is to determine whether lumbrokinase has an in vivo thrombolytic effect in a rabbit cerebral embolism model. In our previous studies, we found that lumbrokinase, an extract from Korean earth worms, has a strong in vitro fibrinolytic effect without the presence of plasminogen and significant in vivo thrombolytic effects of lumbrokinase in a rat human-clot-induced cerebral embolism model. We established the cerebral embolism model in rabbits by injecting a piece of human clot into the internal carotid artery via the external carotid artery and confirmed the occlusion with angiography. Twenty one rabbits were divided into three groups and 5cc of saline, urokinase of 50,000 u/ml, and equipotent LK were injected intraarterially for 30 minutes into each group of 7 animals. Ten minutes after the end of infusion, an angiogram was performed to confirm the recanalization. Clot lysis occurred in one, six, and one animals in the saline, urokinase and lumbrokinase treated groups respectively. With regard to its in vitro effect, lumbrokinase is not as potent in vivo. Further investigation should be performed to determine the cause of its weakened in vivo effect and to develop a method to potentiate it.
For many decades, intravenous (IV) thrombolytics have been delivered to treat acute thrombosis. Although these medications were originally effective for coronary thrombosis, their mechanisms have proven beneficial for many other disease processes, including ischemic stroke. Treatment paradigms for acute ischemic stroke have largely followed those of cardiology. Specifically, the aim has been to recanalize the occluded artery and to restore perfusion to the brain that remains salvageable. To that end, rapid clot lysis was sought using thrombolytic medicines already proven effective in the coronary arteries. IV-thrombolysis for ischemic stroke began its widespread adoption in the late 1990s after the publication of the National Institute of Neurological Disorders and Stroke study. Since that time, other promising IV-thrombolytics have been developed and tested in human trials, but as of yet, none have been proven better than a placebo. Adjunctive treatments are also being evaluated. The challenge remains balancing reperfusion and salvaging brain tissue with the potential risks of brain hemorrhage.
Electronic supplementary material
The online version of this article (doi:10.1007/s13311-011-0049-x) contains supplementary material, which is available to authorized users.
Intravenous thrombolysis; Thrombolytics; Stroke; Treatment; Fibrinolytic; Ischemia
The coagulation and fibrinolytic pathways regulate hemostasis and thrombosis, and an imbalance in these pathways may result in pathologic hemophilia or thrombosis. The plasminogen system is the primary proteolytic pathway for fibrinolysis, but also has important proteolytic functions in cell migration, extracellular matrix degradation, metalloproteinase activation, and hormone processing. Several studies have demonstrated plasmin cleavage and inactivation of several coagulation factors, suggesting plasmin may be not only be the primary fibrinolytic enzyme, but may have anticoagulant properties as well. The objective of this review is to examine both in vitro and in vivo evidence for plasmin inactivation of coagulation, and to consider whether plasmin may act as a physiological regulator of coagulation. While several studies have demonstrated strong evidence for plasmin cleavage and inactivation of coagulation factors FV, FVIII, FIX, and FX in vitro, in vivo evidence is lacking for a physiologic role for plasmin as an anticoagulant. However, inactivation of coagulation factors by plasmin may be useful as a localized anticoagulant therapy or as a combined thrombolytic and anticoagulant therapy.
thrombosis; anticoagulant; cardiovascular disease; plasminogen; protease; blood
The plasminogen activator staphylokinase (SAK) is a promising thrombolytic agent for treatment of myocardial infarction. It can specifically stimulate the thrombolysis of both erythrocyte-rich and platelet-rich clots. However, SAK lacks fibrin-binding and thrombin inhibitor activities, two functions which would supplement and potentially improve its thrombolytic potency. Creating a recombinant fusion protein is one approach for combining protein domains with complementary functions. To evaluate SAK for use in a translational fusion protein, both N- and C-terminal fusions to SAK were constructed by using hirudin as a fusion partner. Recombinant fusion proteins were secreted from Bacillus subtilis and purified from culture supernatants. The rate of plasminogen activation by SAK was not altered by the presence of an additional N- or C-terminal protein sequence. However, cleavage at N-terminal lysines within SAK rendered the N-terminal fusion unstable in the presence of plasmin. The results of site-directed mutagenesis of lysine 10 and lysine 11 in SAK suggested that a plasmin-resistant variant cannot be created without interfering with the plasmin processing necessary for activation of SAK. Although putative plasmin cleavage sites are located at the C-terminal end of SAK at lysine 135 and lysine 136, these sites were resistant to plasmin cleavage in vitro. Therefore, C-terminal fusions represent stable configurations for developing improved thrombolytic agents based on SAK as the plasminogen activator component.
The strong fibrin affinity of recombinant tissue plasminogen activator (rt-PA) theoretically obviates continuous infusion or replacement of t-PA after direct intrathrombic injection. This hypothesis led the authors to evaluate single daily catheter-directed injection of rt-PA as a thrombolytic treatment for acute deep vein thrombosis of the lower extremity. Once-daily injection of rt-PA was performed in large thrombosed veins (popliteal or larger) with use of pulse-spray catheters and in small thrombosed veins in patients' calves with use of 3−4-F coaxial catheters. Patients received only full systemic anticoagulation on his/her patient care unit. This dosing regimen has been tested in 10 patients (12 legs) with a maximum dose of 50 mg per leg per day. Extensive thrombolysis was achieved in nine patients and partial thrombolysis was achieved in one patient, at an average total dose of 106 mg of rt-PA per leg. Minor bleeding was seen in three patients and no transfusions were needed. Our technique and the rationale for this pilot study is the focus of this article.
Thrombolysis; Thrombosis, venous; Tissue-type plasminogen activator
Bacillus sp. strain CK 11-4, which produces a strongly fibrinolytic enzyme, was screened from Chungkook-Jang, a traditional Korean fermented-soybean sauce. The fibrinolytic enzyme (CK) was purified from supernatant of Bacillus sp. strain CK 11-4 culture broth and showed thermophilic, hydrophilic, and strong fibrinolytic activity. The optimum temperature and pH were 70 degrees C and 10.5, respectively, and the molecular weight was 28,200 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The first 14 amino acids of the N-terminal sequence of CK are Ala-Gin-Thr-Val-Pro-Tyr-Gly-Ile-Pro-Leu-Ile-Lys-Ala-Asp. This sequence is identical to that of subtilisin Carlsberg and different from that of nattokinase, but CK showed a level of fibrinolytic activity that was about eight times higher than that of subtilisin Carlsberg. The amidolytic activity of CK increased about twofold at the initial state of the reaction when CK enzyme was added to a mixture of plasminogen and substrate (H-D-Val-Leu-Lys-pNA). A similar result was also obtained from fibrin plate analysis.
This study aimed to compare the efficacy and safety of thrombolytic and anticoagulant therapy for acute submassive pulmonary embolism (PE). A retrospective evaluation was performed on 25 consecutive inpatients with acute submassive PE treated by thrombolytic therapy and 25 earlier consecutive inpatients with acute submassive PE treated by anticoagulant therapy. No statistically significant difference in clinical curative effect was identified between the thrombolysis and anticoagulation groups (P>0.05). Following 24 h of therapy, the improvement rates of dyspnea and revascularization in the thrombolysis group achieved statistical significance compared with those of the anticoagulation group (P<0.01 for each). The PO2 level of the thrombolysis group (81.18±5.66 mmHg) was notably higher than that of the anticoagulation group and the difference was statistically significant (P<0.01). The pulmonary arterial pressures of the thrombolysis group (51.21±6.86 mmHg) were significantly lower than those of the anticoagulation group (60.64±5.17 mmHg) (P<0.01). Furthermore, the difference between the hemorrhage rates of the two groups was statistically significant (P<0.05). Thrombolysis was shown to rapidly relieve dyspnea, reduce pulmonary arterial pressure and revascularize the embolized blood vessels. However, the hemorrhage rate of the thrombolysis group was higher than that of the anticoagulation group. The overall efficacies and fatality rates of the thrombolysis and anticoagulation groups were similar.
submassive; pulmonary embolism; anticoagulation; thrombolysis
Hemodialysis grafts often fail due to stenosis at the venous anastomosis and thrombotic occlusion. Percutaneous management often relies on thrombolysis with plasminogen activators, mechanical removal of thrombus and angioplasty of the stenotic lesion. This report describes a Phase I trial using purified human plasmin, a direct-acting fibrinolytic agent, to evaluate safety and secondarily, to establish effective thrombolytic dosing.
Methods and Results
Six cohorts of 5 patients with acute hemodialysis (HD) graft occlusion documented by angiography were treated with escalating dosages of plasmin (1, 2, 4, 8, 12 and 24 mg) infused over 30 minutes via criss-crossed pulse spray catheters within the graft. The primary efficacy endpoint was ≥50% thrombolysis, as determined by comparison of pre- and 30-minute post-plasmin fistulograms. Of 31 subjects who received study drug (safety analysis group), one withdrew and 30 completed the trial (evaluable for efficacy). There was a dose-response relationship for the primary efficacy endpoint, and all 5 subjects receiving 24 mg achieved >75% lysis. There was no significant change in plasma α-2 antiplasmin or fibrinogen concentration, major bleeding did not occur and there were no deaths. Serious adverse events in 4 patients were not related to study drug.
This first Phase I study of human plasmin infused into thrombosed HD grafts documents safety at dosages of 1-24 mg and an effective thrombolytic dosage of 24 mg. The results establish a foundation for further clinical study of catheter-based plasmin administration in thrombotic disorders.
Kidney; shunts; catheters; thrombolysis