Revisiting the Asian Buffalo Leech (Hirudinaria manillensis) Genome: Focus on Antithrombotic Genes and Their Corresponding Proteins.

Leeches are well-known annelids due to their obligate blood-feeding habits. Some leech species secrete various biologically active substances which have important medical and pharmaceutical value in antithrombotic treatments. In this study, we provided a high-quality genome of the Asian buffalo leech (Hirudinaria manillensis), based on which we performed a systematic identification of potential antithrombotic genes and their corresponding proteins. Combining automatic and manual prediction, we identified 21 antithrombotic gene families including fourteen coagulation inhibitors, three platelet aggregation inhibitors, three fibrinolysis enhancers, and one tissue penetration enhancer. A total of 72 antithrombotic genes, including two pseudogenes, were identified, including most of their corresponding proteins forming three or more disulfide bonds. Three protein families (LDTI, antistasin, and granulin) had internal tandem repeats containing 6, 10, and 12 conserved cysteines, respectively. We also measured the anticoagulant activities of the five identified hirudins (hirudin_Hman1 ~ hirudin_Hman5). The results showed that three (hirudin_Hman1, hirudin_Hman2, and hirudin_Hman5), but not the remaining two, exhibited anticoagulant activities. Our study provides the most comprehensive collection of antithrombotic biomacromolecules from a leech to date. These results will greatly facilitate the research and application of leech derivatives for medical and pharmaceutical purposes in the treatment of thrombotic diseases.

Thrombolytic and anticoagulant efficiencies of purified fibrinolytic enzyme produced from Cochliobolus hawaiiensis under solid-state fermentation.

Cochliobolus hawaiiensis Alcorn Assiut University Mycological Centre 8606 was chosen from the screened 20 fungal species as the potent producer of fibrinolytic enzyme on skimmed-milk agar plates. The greatest enzyme yield was attained when the submerged fermentation (SmF) conditions were optimized, and it was around (39.7 U/mg protein). Moreover, upon optimization of fibrinolytic enzyme production under solid-state fermentation (SSF), the maximum productivity of fibrinolytic enzyme was greatly increased recorded a bout (405 U/mg protein) on sugarcane bagasse, incubation period of 5 days, moisture level of 100%, initial pH of salt basal medium 7.8, incubation temperature at 35°C, and supplementation of the salt basal medium with corn steep liquor (80％, v/v). The yield of fibrinolytic enzyme by C. hawaiiensis under SSF was higher than that of SmF with about 10.20-fold. The purification procedures of fibrinolytic enzyme by ammonium sulfate (70%), gel filtration, and ion-exchange columns chromatography caused a great increase in its specific activity to 2581.6 U/mg protein with an overall yield of 55.89%, 6.37 purification fold and molecular weight of 35 kDa. Maximal activity was recorded at pH 7 and 37°C. Significant pH stability was recorded at pH 6.6-7.2, and thermal stability was recorded at 33-41°C. The enzyme showed the highest affinity toward fibrin, with Vmax of 240 U/mL and an apparent Km value of 47.61 mmol. Mg2+ and Ca2+ moderately induced fibrinolytic activity, whereas Cu2+ and Zn2+ greatly suppressed the enzyme activity. The produced enzyme is categorized as serine protease and non-metalloprotease. The purified fibrinolytic enzyme showed efficient thrombolytic and antiplatelet aggregation activities by completely prevention and dissolution of the blood clot which confirmed by microscopic examination and amelioration of blood coagulation assays. These findings suggested that the produced fibrinolytic enzyme is a promising agent in management of blood coagulation disorders.

Affinity Purification of a Fibrinolytic Enzyme from Sipunculus nudus.

The fibrinolytic enzyme from Sipunculus nudus (sFE) is a novel fibrinolytic agent that can both activate plasminogen into plasmin and degrade fibrin directly, showing great advantages over traditional thrombolytic agents. However, due to the lack of structural information, all the purification programs for sFE are based on multistep chromatography purifications, which are too complicated and costly. Here, an affinity purification protocol of sFE is developed for the first time based on a crystal structure of sFE; it includes preparation of the crude sample and the lysine/arginine-agarose matrix affinity chromatography column, affinity purification, and characterization of the purified sFE. Following this protocol, a batch of sFE can be purified within 1 day. Moreover, the purity and activity of the purified sFE increases to 92% and 19,200 U/mL, respectively. Thus, this is a simple, inexpensive, and efficient approach for sFE purification. The development of this protocol is of great significance for the further utilization of sFE and other similar agents.

Piericones A and B as Potent Antithrombotics: Nanomolar Noncompetitive Protein Disulfide Isomerase Inhibitors with an Unexpected Chemical Architecture.

Extracellular protein disulfide isomerase (PDI) is a promising target for thrombotic-related diseases. Four potent PDI inhibitors with unprecedented chemical architectures, piericones A-D (1-4), were isolated from Pieris japonica. Their structures were elucidated by spectroscopic data analysis, chemical methods, quantum 13C nuclear magnetic resonance DP4+ and electronic circular dichroism calculations, and single-crystal X-ray diffraction analysis. Piericones A (1) and B (2) were nanomolar noncompetitive PDI inhibitors possessing an unprecedented 3,6,10,15-tetraoxatetracyclo[7.6.0.04,9.01,12]pentadecane motif with nine contiguous stereogenic centers. Their biosynthetic pathways were proposed to include a key intermolecular aldol reaction and an intramolecular 1,2-migration reaction. Piericone A (1) significantly inhibited in vitro platelet aggregation and fibrin formation and in vivo thrombus formation via the inhibition of extracellular PDI without increasing the bleeding risk. The molecular docking and dynamics simulation of 1 and 2 provided a novel structure basis to develop PDI inhibitors as potent antithrombotics.

Heterologous expression of recombinant nattokinase in Escherichia coli BL21(DE3) and media optimization for overproduction of nattokinase using RSM.

Nattokinase, a serine protease, was discovered in Bacillus subtilis during the fermentation of a soybean byproduct. Nattokinase is essential for the lysis of blood clots and the treatment of cardiac diseases including atherosclerosis, thrombosis, high blood pressure, and stroke. The demand for thrombolytic drugs rises as the prevalence of cardiovascular disease rises, and nattokinase is particularly effective for the treatment of cardiovascular diseases due to its long duration of action. In this study, we cloned the nattokinase gene from the Bacillus subtilis strain into the pET32a vector and expressed the protein in the E. coli BL21(DE3) strain. The active recombinant nattokinase was purified using Ni-NTA affinity chromatography and then evaluated for fibrinolytic and blood clot lysis activity. Physiological parameters for optimizing protein production at optimal pH, temperature, IPTG concentration, and incubation time were investigated. A statistical technique was used to optimize media components for nattokinase overproduction, and Central Composite Design-Response Surface Methodology-based optimization was used to select significant components for protein production. The optimized media produced 1805.50 mg/L of expressed nattokinase and 42.80 gm/L of bacterial mass. The fibrinolytic activity obtained from refolded native protein was 58FU/mg, which was five times higher than the available orokinase drug (11FU/mg). The efficiency with which a statistical technique for media optimization was implemented improved recombinant nattokinase production and provides new information for scale - up nattokinase toward industrial applications.

Extraction, Characterization, and Platelet Inhibitory Effects of Two Polysaccharides from the Cs-4 Fungus.

Cardiovascular diseases are associated with platelet hyperactivity, and downregulating platelet activation is one of the promising antithrombotic strategies. This study newly extracted two polysaccharides (purified exopolysaccharides, EPSp and purified intercellular exopolysaccharides, IPSp) from Cordyceps sinensis Cs-4 mycelial fermentation powder, and investigated the effects of the two polysaccharides and their gut bacterial metabolites on platelet functions and thrombus formation. EPSp and IPSp are majorly composed of galactose, mannose, glucose, and arabinose. Both EPSp and IPSp mainly contain 4-Galp and 4-Glcp glycosidic linkages. EPSp and IPSp significantly inhibited human platelet activation and aggregation with a dose-dependent manner, and attenuated thrombus formation in mice without increasing bleeding risk. Furthermore, the EPSp and IPSp after fecal fermentation showed enhanced platelet inhibitory effects. The results have demonstrated the potential value of Cs-4 polysaccharides as novel protective ingredients for cardiovascular diseases.

Development of antithrombotic peptides based on the molecular interactions between von Willebrand factor and GPIbα.

Binding of platelets on vascular endothelia at the damaged site using von Willebrand factor (vWF) as a bridge is of great significance for platelet adhesion and subsequent arterial thrombosis. Molecular interactions between vWF and a receptor on a platelet surface, GPIbα, were studied by molecular dynamics (MD) simulations and molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) analysis. Key amino acid residues were identified based on the contribution to the binding of GPIbα and the vWF A1 domain. A vWF-targeting inhibitor library with the amino acid sequence EXEXXDXD (where X represents any of the 20 natural amino acid residues) was then established based on the molecular interactions between GPIbα and the vWF A1 domain, subject to subsequent screening using docking, MD simulations, etc. Two efficient inhibitors including EGEPWDGD and EAEPWDPD were obtained, with experimental validation on their abilities to bind on the vWF and inhibiting platelet adhesion.

Discovery of 7, 4'-dimethoxy-3-hydroxyflavone as a protease-activated receptor 4 antagonist with antithrombotic activity and less bleeding tendency in mice.

There is growing evidence of the importance of protease-activated receptor 4 (PAR4), one of thrombin receptors, as a therapeutic target in thrombotic cardiovascular diseases. In the present study, we utilized ligand-based virtual screening, bioassay, and structure-activity relationship study to discover PAR4 antagonists with new chemical scaffolds from natural origin, and examined their application as antiplatelet agents. By using these approaches, we have identified a flavonoid, 7, 4'-dimethoxy-3-hydroxyflavone, that exhibits anti-PAR4 activity. 7, 4'-Dimethoxy-3-hydroxyflavone inhibited PAR4-mediated human platelet aggregation, GPIIb/IIIa activation, and P-selectin secretion. Also, it inhibited PAR4 downstream signaling pathways, including Ca2+/protein kinase C, Akt, and MAP kinases ERK and p38, in human platelets, and suppressed PAR4-mediated ß-arrestin recruitment in CHO-K1 cells exogenously expressed human PAR4. In a microfluidic system, 7, 4'-dimethoxy-3-hydroxyflavone reduced thrombus formation on collagen-coated chambers at an arterial shear rate in recalcified whole blood. Furthermore, mice treated with 7, 4'-dimethoxy-3-hydroxyflavone were significantly protected from FeCl3-induced carotid arterial occlusions, without significantly affecting tail bleeding time. In conclusion, 7, 4'-dimethoxy-3-hydroxyflavone represents a new class of nature-based PAR4 antagonist, it shows effective in vivo antithrombotic properties with less bleeding tendency, and could be a potential candidate for developing new antiplatelet agents.

In vivo evaluation and molecular docking studies of Schinus molle L. fruit extract protective effect against isoproterenol-induced infarction in rats.

The aim of the current study was to assess the potential cardiopreventive effect of the methanolic extract of S. molle L. (MESM) on isoproterenol-induced infarction in rats. The biomolecules content was evaluated using HPLC-DAD-ESI-QTOF-MS/MS analysis. On the 29th and 30th days, two successive injections of isoproterenol (ISO) were given to Wistar rats to provoke myocardial infarction following pretreatment with either MESM (60 mg/kg b.w) or Pidogrel (Pid; 2 mg/kg b.w.). A total of sixteen phenolics were identified with masazino-flavanone as the most prevalent compound (1726.12 µg/g dm). Results showed that MESM offered cardioprevention by normalizing the ST segment and reducing the elevated cardiac risk parameters. The altered lipid biomarkers together with the plasma ionic levels were improved. Additionally, MESM inhibited the cardiac oxidative stress generated by ISO injection though enhancing antioxidant enzymes (GSH, CAT, SOD and GPX) which reduced lipid peroxidation and protein oxidation. MESM reduced myocardial apoptosis by significantly repressing mRNA expressions of Caspase-3 and Bax, with an upregulated Bcl-2 expression. Moreover, MESM reduced DNA fragmentation as well as the infarct size observed by TTC staining. In addition, MESM exhibited an antifibrotic effect by downregulating TGF-1ß expression and reducing collagen deposition in myocardial tissue, as confirmed by Trichrom Masson analysis. The histopathological findings revealed less muscle separation and fewer inflammatory cells in the ISO + MESM-treated rats. Results of the docking simulation indicated that catechin in MESM was inhibitory mainly due to hydrogen bonding interactions with PDI, ACE and TGF-ß1 proteins which could highlight the antithrombotic and antifibrotic capacity of MESM.

Double-Gene Copromoting Expression Analysis in tPA/GH Transgenic Goat Mammary Epithelial Cells and Thrombolytic Activity of tPA In Vitro.

Human tissue-plasminogen activator (tPA) is a thrombolytic drug widely used in the treatment of stroke, pulmonary thrombosis, acute myocardial infarction, and other thrombotic diseases. The double genes cointegrated into the organisms and cells can produce a synergistic effect, which will improve the expression level of the target gene. However, the study of the integration of the GH and tPA genes to improve the expression level of tPA has not yet been reported. In order to elucidate this, we generated monoclonal goat mammary epithelial cell lines with tPA/GH double-gene integration and analyzed the tPA expression level in single- and double-gene integrated cells. We selected the mammary gland-specific expressing vectors BLC14/tPA and BLC14/GH with the ß-lactoglobulin gene as a regulatory sequence in our previous research. The tPA and GH genes were electronically cotransfected into goat mammary epithelial cells. Resistant cell lines were screened by G418, and transgenic monoclonal cell lines were confirmed by PCR. The tPA expression was induced by prolactin and detected in the cell induction solution after 48 h by ELISA and Western blotting. We detected the tPA biological activity in vitro by fibrin agarose plate assay (FAPA). The results showed that a total of 207 resistant monoclonal cells were obtained, including 126 cell lines with tPA monogenic integration and 51 cell lines with tPA/GH double-gene integration. The rate of double-gene integration was 24.6% (51/207). A total of 48 cells expressed tPA, of which 25.3% (19/75) cells expressed single gene, and 56.9% (29/51) cells expressed double genes. The concentration of tPA in single-gene-expressing cells was 8.0-64.0 µg/mL, and the tPA level in double-gene-expressing cells was significantly higher (200-7200 µg/mL). In addition, the tPA had a relatively strong in vitro thrombolytic activity determined by FAPA. The results showed that goat mammary epithelial cell lines with tPA/GH gene integration were successfully established by electrotransfection, and the expression level of tPA in double-gene integrated cell lines was significantly increased. This study provided a new way for the preparation of a transgenic goat and other animal with high tPA expression by somatic cell nuclear transfer. The findings also laid a foundation for efficient production of pharmaceutical proteins in transgenic animal mammary gland bioreactors in the future.

Development of the Antithrombotic Peptide LEKNSTY Targeting the Collagen Surface: I. Design and Validation.

Exposed collagen on the diseased vessel wall is crucial for arterial thrombosis. The currently developed antithrombotic drugs mostly target blood components such as platelets and suffer from the risk of bleeding. Therefore, anticollagen therapy of covering the collagen surface was proposed as an alternative in our previous study, and an antithrombotic peptide LWWNSYY was designed and validated. However, its application was hindered due to the poor water solubility. In the present study, in order to develop a novel antithrombotic peptide with enhanced water solubility, redesigning of LWWNSYY to LEKNSTY using the EK pattern was proposed. Improved solubility was obtained for LEKNSTY. Moreover, the binding of LEKNSTY on the collagen surface was confirmed by molecular docking, molecular dynamics simulations, and experimental validation. A Kd of 0.91 ± 0.44 µM was observed. The effective inhibition of platelet adhesion on the collagen surface by LEKNSTY was demonstrated at an IC50 of 2.48 ± 0.59 µg/mL. Therefore, the successful design of the antithrombotic peptide LEKNSTY was confirmed, which would facilitate the research into the interface involving thrombus and the development of antithrombotic agents.

Rational design of a new variant of Reteplase with optimized physicochemical profile and large-scale production in Escherichia coli.

Structural engineering of the recombinant thrombolytic drug, Reteplase, and its cost-effective production are important goals in the pharmaceutical industry. In this study, a single-point mutant of the protein was rationally designed and evaluated in terms of physicochemical characteristics, enzymatic activity, as well as large-scale production settings. An accurate homology model of Reteplase was used as the input to appropriate tools to identify the aggregation-prone sites, while considering the structural stability. Selected variants underwent extensive molecular dynamic simulations (total 540 ns) to assess their solvation profile and their thermal stability. The Reteplase-fibrin interaction was investigated by docking. The best variant was expressed in E. coli, and Box-Behnken design was used through response surface methodology to optimize its expression conditions. M72R mutant demonstrated appropriate stability, enhanced enzymatic activity (p < 0.05), and strengthened binding to fibrin, compared to the wild type. The optimal conditions for the variant's production in a bioreactor was shown to be 37 ºC, induction with 0.5 mM IPTG, for 2 h of incubation. Under these conditions, the final amount of the produced enzyme was increased by about 23 mg/L compared to the wild type, with an increase in the enzymatic activity by about 2 IU/mL. This study thus offered a new Reteplase variant with nearly all favorable properties, except solubility. The impact of temperature and incubation time on its large-scale production were underlined as well.

Protection of ischemic white matter and oligodendrocytes in mice by 3K3A-activated protein C.

Subcortical white matter (WM) stroke accounts for 25% of all strokes and is the second leading cause of dementia. Despite such clinical importance, we still do not have an effective treatment for ischemic WM stroke, and the mechanisms of WM postischemic neuroprotection remain elusive. 3K3A-activated protein C (APC) is a signaling-selective analogue of endogenous blood protease APC that is currently in development as a neuroprotectant for ischemic stroke patients. Here, we show that 3K3A-APC protects WM tracts and oligodendrocytes from ischemic injury in the corpus callosum in middle-aged mice by activating protease-activated receptor 1 (PAR1) and PAR3. We show that PAR1 and PAR3 were also required for 3K3A-APC's suppression of post-WM stroke microglia and astrocyte responses and overall improvement in neuropathologic and functional outcomes. Our data provide new insights into the neuroprotective APC pathway in the WM and illustrate 3K3A-APC's potential for treating WM stroke in humans, possibly including multiple WM strokes that result in vascular dementia.

Recombinant Destabilase from Hirudo medicinalis Is Able to Dissolve Human Blood Clots In Vitro.

Leeches are amazing animals that can be classified as conditionally poisonous animals since the salivary cocktail they produce is injected directly into the victim, and its components have strictly defined biological purposes, such as preventing blood clot formation. Thrombolytic drugs are mainly aimed at treating newly formed blood clots. Aged clots are stabilized by a large number of isopeptide bonds that prevent the action of thrombolytics. These bonds are destroyed by destabilase, an enzyme of the leech's salivary glands. Here, we conducted a pilot study to evaluate the feasibility and effectiveness of the use of destabilase in relation to blood clots formed during real pathological processes. We evaluated the isopeptidase activity of destabilase during the formation of a stabilized fibrin clot. We showed that destabilase does not affect the internal and external coagulation cascades. We calculated the dose-response curve and tested the ability of destabilase to destroy isopeptide bonds in natural blood clots. The effect of aged and fresh clots dissolving ability after treatment with destabilase coincided with the morphological characteristics of clots during surgery. Thus, recombinant destabilase can be considered as a potential drug for the treatment of aged clots, which are difficult to treat with known thrombolytics.

Haematococcus pluvialis as a Potential Source of Astaxanthin with Diverse Applications in Industrial Sectors: Current Research and Future Directions.

Haematococcus pluvialis, a green microalga, appears to be a rich source of valuable bioactive compounds, such as astaxanthin, carotenoids, proteins, lutein, and fatty acids (FAs). Astaxanthin has a variety of health benefits and is used in the nutraceutical and pharmaceutical industries. Astaxanthin, for example, preserves the redox state and functional integrity of mitochondria and shows advantages despite a low dietary intake. Because of its antioxidant capacity, astaxanthin has recently piqued the interest of researchers due to its potential pharmacological effects, which include anti-diabetic, anti-inflammatory, and antioxidant activities, as well as neuro-, cardiovascular-, ocular, and skin-protective properties. Astaxanthin is a popular nutritional ingredient and a significant component in animal and aquaculture feed. Extensive studies over the last two decades have established the mechanism by which persistent oxidative stress leads to chronic inflammation, which then mediates the majority of serious diseases. This mini-review provides an overview of contemporary research that makes use of the astaxanthin pigment. This mini-review provides insight into the potential of H. pluvialis as a potent antioxidant in the industry, as well as the broad range of applications for astaxanthin molecules as a potent antioxidant in the industrial sector.

Embolic Stroke Model with Magnetic Nanoparticles.

Stroke models are vital tools in neuropharmacology and rehabilitation research. However, a classic and widely used model-the suture occlusion model-is not suitable for all research approaches, especially regarding thrombolysis. For embolic stroke models in thrombolytic research, the surgical procedures of thrombin injection in the middle cerebral artery or clot injection in the carotid artery involved are too sophisticated. Here, we report a new stroke model in mice that uses magnetic nanoparticle (MNP) cross-linked with thrombin to embolize. Briefly, after the magnet was positioned in the common carotid artery, MNP@Thrombin was injected from the tail vein. Within several minutes postinjection, the MNP@Thrombin accumulated in the carotid artery and induced thrombus formation. These complex clots were flushed into and subsequently blocked the cerebral artery. Collectively, these results suggested that this new method was a quick and easy stroke model that blocked hemisphere blood flow and damaged neural function. Importantly, this model had an excellent response to thrombolytic drugs. After urokinase injection, cerebral blood flow was restored and symptom scores were enhanced by nearly one. This method, including a quick synthesis of MNP and thrombin, provided an easy and minimally invasive process for a new stroke model that is usable in both pharmacological and rehabilitative research.

Chemical, biological and protein-receptor binding profiling of Bauhinia scandens L. stems provide new insights into the management of pain, inflammation, pyrexia and thrombosis.

Bauhinia scandens L. (Family, Fabaceae) is a medicinal plant used for conventional and societal medication in Ayurveda. The present study has been conducted to screen the chemical, pharmacological and biochemical potentiality of the methanol extracts of B. scandens stems (MEBS) along with its related fractions including carbon tetrachloride (CTBS), di-chloromethane (DMBS) and n-butanol (BTBS). UPLC-QTOF-MS has been implemented to analyze the chemical compounds of the methanol extracts of Bauhinia scandens stems. Additionally, antinociceptive and anti-inflammatory effects were performed by following the acetic acid-induced writhing test and formalin-mediated paw licking test in the mice model. The antipyretic investigation was performed by Brewer Yeast induced pyrexia method. The clot lysis method was implemented to screen the thrombolytic activity in human serum. Besides, the in silico study was performed for the five selected chemical compounds of Bauhinia scandens, found by UPLC-QTOF-MS By using Discover Studio 2020, UCSF Chimera, PyRx autodock vina and online tools. The MEBS and its fractions exhibited remarkable inhibition in dose dependant manner in the antinociceptive and antiinflammatory investigations. The antipyretic results of MEBS and DMBS were close to the standard drug indomethacin. Investigation of the thrombolytic effect of MEBS, CTBS, DMBS, and BTBS revealed notable clot-lytic potentials. Besides, the phenolic compounds of the plant extracts revealed strong binding affinity to the COX-1, COX-2, mPGES-1 and plasminogen activator enzymes. To recapitulate, based on the research work, Bauhinia scandens L. stem and its phytochemicals can be considered as prospective wellsprings for novel drug development and discovery by future researchers.

Heterologous expression of nattokinase from B. subtilis natto using Pichia pastoris GS115 and assessment of its thrombolytic activity.

BACKGROUND: Nattokinase is a fibrinolytic enzyme that has huge market value as a nutritional supplement for health promotion. In order to increase nattokinase yields, fermentation conditions, strains, cultivation media, and feeding strategies have been optimized. Nattokinase has been expressed using several heterologous expression systems. Pichia pastoris heterologous expression system was the alternative. RESULTS: This report aimed to express high levels of nattokinase from B. subtilis natto (NK-Bs) using a Pichia pastoris heterologous expression system and assess its fibrinolytic activity in vivo. Multicopy expression strains bearing 1-7 copies of the aprN gene were constructed. The expression level of the target protein reached a maximum at five copies of the target gene. However, multicopy expression strains were not stable in shake-flask or high-density fermentation, causing significant differences in the yield of the target protein among batches. Therefore, P. pastoris bearing a single copy of aprN was used in shake-flask and high-density fermentation. Target protein yield was 320 mg/L in shake-flask fermentation and approximately 9.5 g/L in high-density fermentation. The recombinant nattokinase showed high thermo- and pH-stability. The present study also demonstrated that recombinant NK-Bs had obvious thrombolytic activity. CONCLUSIONS: This study suggests that the P. pastoris expression system is an ideal platform for the large-scale, low-cost preparation of nattokinase.

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.

Phosphinanes and Azaphosphinanes as Potent and Selective Inhibitors of Activated Thrombin-Activatable Fibrinolysis Inhibitor (TAFIa).

Selective and potent inhibitors of activated thrombin activatable fibrinolysis inhibitor (TAFIa) have the potential to increase endogenous and therapeutic fibrinolysis and to behave like profibrinolytic agents without the risk of major hemorrhage, since they do not interfere either with platelet activation or with coagulation during blood hemostasis. Therefore, TAFIa inhibitors could be used in at-risk patients for the treatment, prevention, and secondary prevention of stroke, venous thrombosis, and pulmonary embolisms. In this paper, we describe the design, the structure-activity relationship (SAR), and the synthesis of novel, potent, and selective phosphinanes and azaphosphinanes as TAFIa inhibitors. Several highly active azaphosphinanes display attractive properties suitable for further in vivo efficacy studies in thrombosis models.