Exploring a Hirudin variant from nonhematophagous leeches: Unraveling full-length sequence, alternative splicing, function, and potential as a novel anticoagulant polypeptide.

ETHNOPHARMACOLOGICAL RELEVANCE: Leeches exhibit robust anticoagulant activity, making them useful for treating cardiovascular diseases in traditional Chinese medicine. Whitmania pigra, the primary source species of leech-derived medicinal compounds in China, has been demonstrated to possess formidable anticoagulant properties. Hirudin-like peptides, recognized as potent thrombin inhibitors, are prevalent in hematophagous leeches. Considering that W. pigra is a nonhematophagic leech, the following question arises: does a hirudin variant exist in this species? AIM OF THE STUDY: In this study we identified the hirudin-encoding gene (WP_HV1) in the W. pigra genome. The goal of this study was to assess its anticoagulant activity and analyze the related mechanisms. MATERIALS AND METHODS: In this study, a hirudin-encoding gene, WP_HV1, was identified from the W. pigra genome, and its accurate coding sequence (CDS) was validated through cloning from cDNA extracted from fresh W. pigra specimens. The structure of WP_HV1 and the amino acids associated with its anticoagulant activity were determined by sequence and structural analysis and prediction of its binding energy to thrombin. E. coli was used for the expression of WP_HV1 and recombinant proteins with various structures and mutants. The anticoagulant activity of the synthesized recombinant proteins was then confirmed using thrombin time (TT). RESULTS: Validation of the WP_HV1 gene was accomplished, and three alternative splices were discovered. The TT of the blank sample exceeded that of the recombinant WP_HV1 sample by 1.74 times (0.05 mg/ml), indicating positive anticoagulant activity. The anticoagulant activity of WP_HV1 was found to be associated with its C-terminal tyrosine, along with the presence of 9 acidic amino acids on both the left and right sides. A significant reduction in the corresponding TT was observed for the mutated amino acids compared to those of the wild type, with decreases of 4.8, 6.6, and 3.9 s, respectively. In addition, the anticoagulant activity of WP_HV1 was enhanced and prolonged for 2.7 s when the lysine-67 residue was mutated to tryptophan. CONCLUSION: Only one hirudin-encoding variant was identified in W. pigra. The active amino acids associated with anticoagulation in WP_HV1 were resolved and validated, revealing a novel source for screening and developing new anticoagulant drugs.

In situ formed depot of elastin-like polypeptide-hirudin fusion protein for long-acting antithrombotic therapy.

Thrombosis, induced by abnormal coagulation or fibrinolytic systems, is the most common pathology associated with many life-threatening cardio-cerebrovascular diseases. However, first-line anticoagulant drugs suffer from rapid drug elimination and risk of hemorrhagic complications. Here, we developed an in situ formed depot of elastin-like polypeptide (ELP)-hirudin fusion protein with a prodrug-like feature for long-term antithrombotic therapy. Highly secretory expression of the fusion protein was achieved with the assistance of the Ffu312 tag. Integration of hirudin, ELP, and responsive moiety can customize fusion proteins with properties of adjustable in vivo retention and controllable recovery of drug bioactivity. After subcutaneous injection, the fusion protein can form a reservoir through temperature-induced coacervation of ELP and slowly diffuse into the blood circulation. The biological activity of hirudin is shielded due to the N-terminal modification, while the activated key proteases upon thrombus occurrence trigger the cleavage of fusion protein together with the release of hirudin, which has antithrombotic activity to counteract thrombosis. We substantiated that the optimized fusion protein produced long-term antithrombotic effects without the risk of bleeding in multiple animal thrombosis models.

New anticoagulant protein from medicinal leech.

The saliva of the medicinal leech contains various anticoagulants. Some of them, such as hirudin, are well known. However, it is reasonable to believe that not all anticoagulant proteins from medicinal leech saliva have been identified. We previously performed a comprehensive study of the transcriptome, genome, and proteome of leech salivary gland cells, which led to the discovery of several previously unknown hypothetical proteins that may have anticoagulant properties. Subsequently, we obtained a series of recombinant proteins and investigated their impact on coagulation in in vitro assays. We identified a previously undescribed protein that exhibited a high ability to suppress coagulation. The His-tagged recombinant protein was expressed in Escherichia coli and purified using metal chelate chromatography. To determine its activity, commonly used coagulation methods were used: activated partial thromboplastin time, prothrombin time, and thrombin inhibition clotting assay. Clotting and chromogenic assays for factor Xa inhibition were performed to evaluate anti-Xa activity. We used recombinant hirudin as a control anticoagulant protein in all experiments. The new protein showed significantly greater inhibition of coagulation than hirudin at the same molar concentrations in the activated partial thrombin time assay. However, hirudin demonstrated better results in the direct thrombin inhibition test, although the tested protein also exhibited the ability to inhibit thrombin. The chromogenic analysis of factor Xa inhibition revealed no activity, whereas the clotting test for factor Xa showed the opposite result. Thus, a new powerful anticoagulant protein has been discovered in the medicinal leech. This protein is homologous to antistatin, with 28 % identical amino acid residues. The recombinant protein was expressed in E. coli. This protein is capable of directly inhibiting thrombin, and based on indirect evidence, other proteases of the blood coagulation cascade have been identified.

Mechanism of Hirudin-Mediated Inhibition of Proliferation in Ovarian Cancer Cells.

To investigate the inhibitory effect of hirudin on the cell proliferation of human ovarian cancer A2780 cells by preventing thrombin and its underlying molecular mechanism. Cell Counting Kit-8 (CCK-8) method was used to detect the effect of different concentrations of hirudin and thrombin on the cell proliferation of A2780 cells. PAR-1 wild-type overexpression plasmid was constructed utilizing enzyme digestion identification, and it was transferred to A2780 cells. Sequencing and Western blot were used to detect the changes in PAR-1 protein expression. Western blot detection of PKCα protein phosphorylation in A2780 cells was performed. We also implemented quantitative PCR to detect the mRNA expression levels of epithelial-mesenchymal transition (EMT)-related genes, CDH2, Snail, and Vimentin, in A2780 cells. 1 µg/ml hirudin treatment maximally inhibited the promotion of A2780 cell proliferation by thrombin. Hirudin inhibited the binding of thrombin to the N-terminus of PAR-1, hindered PKCα protein phosphorylation in A2780 cells, and downregulated the mRNA expression levels of CDH2, Snail, and Vimentin. In conclusion, hirudin inhibits the cell proliferation of ovarian cancer A2780 cells, and the underlying mechanism may be through downregulating the transcription level of EMT genes, CDH2, Snail, and Vimentin. This study indicates that hirudin may have a therapeutic potential as an anti-cancer agent for ovarian cancer.

Tyrosine phosphorylation of recombinant hirudin increases affinity to thrombin and antithrombotic activity.

Thrombin is one of the key enzymes of the blood coagulation system and a promising target for the development of anticoagulants. One of the most specific natural thrombin inhibitors is hirudin, contained in the salivary glands of medicinal leeches. The medicinal use of recombinant hirudin is limited because of the lack of sulfation on Tyr63, resulting in a 10-fold decrease in activity compared to native (sulfated) hirudin. In the present work, a set of hirudin derivatives was tested for affinity to thrombin: phospho-Tyr63, Tyr63(carboxymethyl)Phe, and Tyr63Glu mutants, which mimic Tyr63 sulfation and Gln65Glu mutant and lysine-succinylated hirudin, which enhance the overall negative charge of hirudin, as well as sulfo-hirudin and desulfo-hirudin as references. Using steered molecular dynamics simulations with subsequent umbrella sampling, phospho-hirudin was shown to exhibit the highest affinity to thrombin among all hirudin analogs, including native sulfo-hirudin; succinylated hirudin was also prospective. Phospho-hirudin exhibited the highest antithrombotic activity in in vitro assay in human plasma. Taking into account the modern methods for obtaining phospho-hirudin and succinylated hirudin, they are prospective as anticoagulants in clinical practice.

From haemadin to haemanorm: Synthesis and characterization of full-length haemadin from the leech Haemadipsa sylvestris and of a novel bivalent, highly potent thrombin inhibitor (haemanorm).

Hirudin from Hirudo medicinalis is a bivalent α-Thrombin (αT) inhibitor, targeting the enzyme active site and exosite-I, and is currently used in anticoagulant therapy along with its simplified analogue hirulog. Haemadin, a small protein (57 amino acids) isolated from the land-living leech Haemadipsa sylvestris, selectively inhibits αT with a potency identical to that of recombinant hirudin (KI = 0.2 pM), with which it shares a common disulfide topology and overall fold. At variance with hirudin, haemadin targets exosite-II and therefore (besides the free protease) it also blocks thrombomodulin-bound αT without inhibiting the active intermediate meizothrombin, thus offering potential advantages over hirudin. Here, we produced in reasonably high yields and pharmaceutical purity (>98%) wild-type haemadin and the oxidation resistant Met5 → nor-Leucine analogue, both inhibiting αT with a KI of 0.2 pM. Thereafter, we used site-directed mutagenesis, spectroscopic, ligand-displacement, and Hydrogen/Deuterium Exchange-Mass Spectrometry techniques to map the αT regions relevant for the interaction with full-length haemadin and with the synthetic N- and C-terminal peptides Haem(1-10) and Haem(45-57). Haem(1-10) competitively binds to/inhibits αT active site (KI = 1.9 µM) and its potency was enhanced by 10-fold after Phe3 → ß-Naphthylalanine exchange. Conversely to full-length haemadin, haem(45-57) displays intrinsic affinity for exosite-I (KD = 1.6 µM). Hence, we synthesized a peptide in which the sequences 1-9 and 45-57 were joined together through a 3-Glycine spacer to yield haemanorm, a highly potent (KI = 0.8 nM) inhibitor targeting αT active site and exosite-I. Haemanorm can be regarded as a novel class of hirulog-like αT inhibitors with potential pharmacological applications.

Cloning, characterization, and heterologous expression of a candidate Hirudin gene from the salivary gland transcriptome of Hirudo nipponia.

Hirudin is a pharmacologically active substance in leeches with potent blood anticoagulation properties. Although recombinant hirudin production isolated from Hirudo medicinalis Linnaeus and Hirudinaria manillensis Lesson is known, to our knowledge, this study is the first to report recombinant hirudin expression and production from Hirudo nipponia Whitman. Thus, the present study aimed to clone and characterize the full-length cDNA of a candidate hirudin gene (c16237_g1), which is localized on the salivary gland transcriptome of H. nipponia, and further evaluate its recombinant production using a eukaryotic expression system. The 489-bp cDNA possessed several properties of the hirudin "core" motifs associated with binding to the thrombin catalytic pocket. A fusion expression vector (pPIC9K-hirudin) was constructed and successfully transformed into Pichia pastoris strain GS115 via electroporation. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and western blot analysis confirmed hirudin expression. The recombinant protein was expressed with a yield of 6.68 mg/L culture. Mass spectrometry analysis further confirmed target protein expression. The concentration and antithrombin activity of purified hirudin were 1.67 mg/mL and 14,000 ATU/mL, respectively. These findings provide a basis for further elucidating the molecular anticoagulation mechanism of hirudin, and address China's growing market demand for engineered H. nipponia-derived hirudin and hirudin-based drugs.

Life without blood: Molecular and functional analysis of hirudins and hirudin-like factors of the Asian non-hematophagous leech Whitmania pigra.

BACKGROUND: Several leech species of the genera Hirudo, Hirudinaria, and Whitmania are widely used in traditional Chinese medicine (TCM) for the oral treatment of disorders associated with blood stasis. Among them, the non-hematophagous leech Whitmania pigra expresses a variety of components that have the potential to act on the vertebrate blood coagulation system. OBJECTIVE: Whether the thrombin inhibitor hirudin, probably the most prominent leech-derived anticoagulant, is actually present in Whitmania pigra, is still a matter of debate. To answer that open question was the aim of the study. METHODS: We identified several putative hirudin-encoding sequences in transcriptome data of Whitmania pigra. Upon gene synthesis and molecular cloning the respective recombinant proteins were expressed in Escherichia coli, purified, processed, and eventually functionally characterized for thrombin-inhibitory potencies in coagulation assays. RESULTS: We were successful in the identification and functional characterization of several putative hirudins in Whitmania pigra. Some, but not all, of these factors are indeed thrombin inhibitors. Whitmania pigra hence expresses both hirudins (factors that inhibit thrombin) and hirudin-like factors (that do not or only very weakly inhibit thrombin). Furthermore, we revealed the exon/intron structures of the corresponding genes. Coding sequences of some putative hirudins of Whitmania pigra were present also in transcriptome datasets of Hirudo nipponia, a hematophagous leech that is likewise used in TCM. CONCLUSIONS: Based on both structural and functional data we provide very strong evidence for the expression of hirudins in Whitmania pigra. This is the first description of hirudins in a non-hematophagous leech.

Enhanced anticoagulant activity of hirudin-i analogue co-expressed with arylsulfotransferase in periplasm of E. coli BL21(DE3).

Hirudin, a blood anticoagulant, is the most potent natural thrombin inhibitor of leech origin. Its application is limited because it is difficult to obtain abundant natural hirudin directly from the leech. Although some bioengineering methods can significantly increase the production of hirudin, the reduced efficacy of recombinant hirudin (rH) remains a critical shortcoming. The lack of sulfation of tyrosine 63 in rH is an important cause of its inadequate performance. This article is the first report of periplasmic co-expression of an rH-I analogue with arylsulfotransferase (ASST) in E. coli BL21(DE3). Co-expressed rH-I analogue with sulfate donor substrate (p-nitrophenyl sulfate potassium) showed anticoagulant (rabbit and goat serum) activity twice more than rH-I analogue expressed without ASST, indicating its potential periplasmic sulfation. Moreover, purified rH-I analogue showed above 4.5 times higher anticoagulant activity compared to therapeutic anti-thrombotic heparin (HE). At the same time, pH-dependent differential solubility was employed to purify rH analogues from fermentation broth, which is a simple, fast and inexpensive purification technology, and can potentially be used for larger scale purification. This will also greatly improve the application of rH in clinical treatment.

Draft genome of the European medicinal leech Hirudo medicinalis (Annelida, Clitellata, Hirudiniformes) with emphasis on anticoagulants.

The European medicinal leech has been used for medicinal purposes for millennia, and continues to be used today in modern hospital settings. Its utility is granted by the extremely potent anticoagulation factors that the leech secretes into the incision wound during feeding and, although a handful of studies have targeted certain anticoagulants, the full range of anticoagulation factors expressed by this species remains unknown. Here, we present the first draft genome of the European medicinal leech, Hirudo medicinalis, and estimate that we have sequenced between 79-94% of the full genome. Leveraging these data, we searched for anticoagulation factors across the genome of H. medicinalis. Following orthology determination through a series of BLAST searches, as well as phylogenetic analyses, we estimate that fully 15 different known anticoagulation factors are utilized by the species, and that 17 other proteins that have been linked to antihemostasis are also present in the genome. We underscore the utility of the draft genome for comparative studies of leeches and discuss our results in an evolutionary context.

The hirudin-like factors HLF3 and HLF4-hidden hirudins of European medicinal leeches.

The hirudin-like factors 3 (HLF3) and 4 (HLF4) belong to a new class of leech-derived factors and are present in specimens of the three European medicinal leeches, Hirudo medicinalis, Hirudo verbana, and Hirudo orientalis, respectively. Here we describe the functional analysis of natural and synthetic variants of HLF3 and HLF4. Whereas the natural variants display only very low or no detectable anti-coagulatory activities, modifications within the N-termini in combination with an exchange of the central globular domain have the potency to greatly enhance the inhibitory effects of respective HLF3 and HLF4 variants on blood coagulation. Our results support previous observations on the crucial importance of all parts (both the N- and C-termini as well as the central globular domains) of hirudin and HLF molecules for thrombin inhibition.

Hirudin or hirudin-like factor - that is the question: insights from the analyses of natural and synthetic HLF variants.

The hirudin-like factor 1 (HLF1) of Hirudo medicinalis belongs to a new class of leech-derived factors. In previous investigations, HLF1 did not exhibit anticoagulatory activities. Here, we describe the analysis of natural and synthetic variants of HLF1 and HLF-Hyb, a yet uncharacterized member of the HLF family. Modifications within the N terminus of HLF1 have a strong impact on its activity. Some variants of HLF1 exhibit thrombin-inhibiting activity comparable to hirudins, whereas others have reduced or no activity. The analyses of HLF-Hyb variants revealed a strong impact of the central globular domain on activity. Our results indicate a comparable mode of action of hirudins and thrombin-inhibiting HLF variants. Finally, we propose and discuss criteria for classifying hirudins and HLFs.

Transcriptomic Analysis of the Influence of Methanol Assimilation on the Gene Expression in the Recombinant Pichia pastoris Producing Hirudin Variant 3.

Hirudin and its variants, as strong inhibitors against thrombin, are present in the saliva of leeches and are recognized as potent anticoagulants. However, their yield is far from the clinical requirement up to now. In this study, the production of hirudin variant 3 (HV3) was successfully realized by cultivating the recombinant Pichia pastoris GS115/pPIC9K-hv3 under the regulation of the promoter of AOX1 encoding alcohol oxidase (AOX). The antithrombin activity in the fermentation broth reached the maximum value of 5000 ATU/mL. To explore an effective strategy for improving HV3 production in the future, we investigated the influence of methanol assimilation on the general gene expression in this recombinant by transcriptomic study. The results showed that methanol was partially oxidized into CO2, and the rest was converted into glycerone-P which subsequently entered into central carbon metabolism, energy metabolism, and amino acid biosynthesis. However, the later metabolic processes were almost all down-regulated. Therefore, we propose that the up-regulated central carbon metabolism, energy, and amino acid metabolism should be beneficial for methanol assimilation, which would accordingly improve the production of HV3.

Hirudin and Decorsins of the North American Medicinal Leech Macrobdella decora: Gene Structure Reveals Homology to Hirudins and Hirudin-like Factors of Eurasian Medicinal Leeches.

Blood-sucking leeches, some of which are referred to as medicinal leeches, have caught attention not only because of their medical purposes, but also as study organisms to conduct research within fields as diverse as neurobiology, osmoregulation, ecology, and phylogeny. Of particular interest is the question whether hemophagy in leeches is of single origin or evolved independently several times. A key component in the saliva of hematophagous leeches is hirudin, a strong natural inhibitor of thrombin and hence the blood coagulation cascade. Multiple isoforms of hirudin have been described within and among several leech species and genera, often based on sequence data only. The identification of hirudin-like factors (HLFs) illustrated the necessity to underpin such predictions by functional tests. We overexpressed and purified the hirudin of the North American medicinal leech, Macrobdella decora, and proved its thrombin-inhibiting activity. In addition, analysis of the gene structure of both hirudin and some of the decorsins of M. decora clearly indicated conserved exon and intron positions when compared to genes of hirudins and HLFs of Eurasian medicinal leeches. Our data provide evidence for the incorporation of decorsins into the hirudin superfamily and support the concept of a single origin of blood feeding in jawed leeches.

Therapeutic effects of the rhSOD2-Hirudin fusion protein on bleomycin-induced pulmonary fibrosis in mice.

Idiopathic pulmonary fibrosis (IPF) is a disease with a poor prognosis and high mortality, posing a major threat to human health. Increased levels of inflammatory cytokines, reactive oxygen species and coagulation cascade have been extensively reported in IPF. We previously fused Hirudin and human manganese superoxide dismutase (hSOD2) to generate a dual-feature fusion protein, denoted as rhSOD2-Hirudin fusion protein. In this study, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and Hydroxyproline (HYP) assays were used to investigate the effects of rhSOD2-Hirudin protein on thrombin-induced fibroblast proliferation and collagen accumulation in vitro. Subsequently, the mice model of pulmonary fibrosis induced by bleomycin was used for evaluating the anti-inflammatory and anti-fibrotic effects of rhSOD2-Hirudin protein in vivo. Results showed that rhSOD2-Hirudin protein could inhibit the proliferation of fibroblasts and reduce the HYP production in vitro by inhibiting the activity of thrombin. In vivo experiments showed that lung inflammation and fibrosis were significantly decreased in rhSOD2-Hirudin protein-treated mice. Furthermore, rhSOD2-Hirudin protein treatment reduced profibrotic protein and gene expression while reducing the number of inflammatory cells in the lung. In conclusion, rhSOD2-Hirudin protein can effectively attenuate pulmonary fibrosis in vitro and in vivo, mainly by inhibiting the activity of thrombin meanwhile increasing SOD2 levels prevent cells from being damaged by reactive oxygen species, thereby mitigating IPF progression. This study provided important information on the feasibility and efficacy of rhSOD2-Hirudin protein as a novel therapeutic agent for IPF.

The effects of overexpression of cytoplasmic chaperones on secretory production of hirudin-PA in E. coli.

The secretory production of heterologous proteins in E. coli has revolutionized biotechnology. Efficient periplasmic production of foreign proteins in E. coli often requires a signal peptide to direct proteins to the periplasm. However, the presence of attached signal peptide does not guarantee periplasmic expression of target proteins. Overproduction of auxiliary proteins, such as chaperones can be a useful approach to enhance protein export. In the current study, three chaperone plasmid sets, including GroEL-GroES (GroELS), Dnak-Dnaj-GrpE (DnaKJE), and trigger factor (TF), were coexpressed in E. coli BL21 (DE3) in a pairwise manner with two pET22-b vectors carrying the recombinant hirudin-PA (Hir) gene and different signal sequences alkaline phosphatase (PhoA) and l-asparaginase II (l-ASP). Overexpression of cytoplasmic combinations of molecular chaperones containing GroELS and DnaKJE with PhoAHir increased the secretory production of PhoAHir by 2.6fold (p < 0.05) and 3.5fold (p < 0.01) compared with their controls, respectively. By contrast, secretory production of PhoAHir significantly reduced in the presence of overexpressed TF (p = 0.02). Further, periplasmic expression of l-ASP was significantly increased only in the presence of DnaKJE (p = 0.04). These findings suggest that using molecular chaperones can be helpful for improving periplasmic expression of Hir. However, tagged signal peptides may affect the physicochemical properties and secondary and tertiary structures of mature Hir, which may alter their interactions with chaperones. Hence, using overexpressed chaperones has various effects on secretory production of PhoAHir and l-ASPHir.

Novel Strategy of Gene Delivery System Based on Dendrimer Loaded Recombinant Hirudine Plasmid for Thrombus Targeting Therapy.

This study proposed a new nonviral gene delivery system for thrombus targeting therapy based on PEGlyation polyamides dendrimer (PAMAM) modified with RGDyC to condense the pDNA with recombinant hirudine (rHV) gene (RGDyC-rHV-EGFP). The RGDyC-mPEG-PAMAM was synthesized and characterized by 1H NMR, PAMAM/pDNA was characterized by particle size, zeta potential, cellular uptake, and gel retraction assay. The transfection was carried out between lipofectamine 2000 and PAMAM/pDNA on HUVEC cells at various N/P ratios. The antithrombotic effect in vivo was evaluated by venous thrombosis model on Wistar rats. It showed that the drug delivery system of RGDyC modified PAMMA, which entrapped pDNA could significantly improve the transfection efficiency. It was about 7.56-times higher than that of lipofectamine 2000. In addition, the expression level of hirudine fusion protein was the highest at N/P ratio of 0.5. The results of antithrombotic effect showed that the weight of thrombus was reduced in RGDyC modified group; compared with heparin group, there was no significant difference ( P > 0.05). Overall, we take the advantage of the unique advantages of hirudine, combining the genetic engineering, nanocarriers, and targeting technology, to achieve the targeted enrichment and activation the hirudine fusion protein in the thrombus site, to improve the concentration of drugs in the thrombus site, finally increasing the curative effect and reduce the risk of bleeding. The strategy of gene delivery system holds unique properties as a gene delivery system and has great promises in thrombus targeting therapy.

Endothelial cell-specific anticoagulation reduces inflammation in a mouse model of acute lung injury.

Tissue factor (TF)-dependent coagulation contributes to lung inflammation and the pathogenesis of acute lung injury (ALI). In this study, we explored the roles of targeted endothelial anticoagulation in ALI using two strains of transgenic mice expressing either a membrane-tethered human tissue factor pathway inhibitor (hTFPI) or hirudin fusion protein on CD31+ cells, including vascular endothelial cells (ECs). ALI was induced by intratracheal injection of LPS, and after 24 h the expression of TF and protease-activated receptors (PARs) on EC in lungs were assessed, alongside the extent of inflammation and injury. The expression of TF and PARs on the EC in lungs was upregulated after ALI. In the two strains of transgenic mice, expression of either of hTFPI or hirudin by EC was associated with significant reduction of inflammation, as assessed by the extent of leukocyte infiltration or the levels of proinflammatory cytokines, and promoted survival after LPS-induced ALI. The beneficial outcomes were associated with inhibition of the expression of chemokine CCL2 in lung tissues. The protection observed in the CD31-TFPI-transgenic strain was abolished by injection of an anti-hTFPI antibody, but not by prior engraftment of the transgenic strains with WT bone marrow, confirming that the changes observed were a specific transgenic expression of anticoagulants by EC. These results demonstrate that the inflammation in ALI is TF and thrombin dependent, and that expression of anticoagulants by EC significantly inhibits the development of ALI via repression of leukocyte infiltration, most likely via inhibition of chemokine gradients. These data enhance our understanding of the pathology of ALI and suggest a novel therapeutic strategy for treatment.

Hirudin variants production by genetic engineered microbial factory.

Hirudin was discovered as an active anticoagulant in leech extracts almost 60 years ago. Since their initial discovery, hirudin and its variants have been produced with various anti-thrombotic, cancer cell inhibition, diabetic cataract treatment and anti-fatigue activities. Some hirudin variants have been approved for clinical use and released into the marketplace. Recent progress has seen made in relation to hirudin variants expressed in several well-established microbial hosts, including Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris and others, with high levels of activity and yield. This review summarizes the current progress on hirudin production using microbial producers, and considers the outlook for future development.

SAK-HV Triggered a Short-period Lipid-lowering Biotherapy Based on the Energy Model of Liver Proliferation via a Novel Pathway.

The accumulations of excess lipids within liver and serum are defined as non-alcoholic fatty liver disease (NAFLD) and hyperlipemia respectively. Both of them are components of metabolic syndrome that greatly threaten human health. Here, a recombinant fusion protein (SAK-HV) effectively treated NAFLD and hyperlipemia in high-fat-fed ApoE-/- mice, quails and rats within just 14 days. Its triglyceride and cholesterol-lowering effects were significantly better than that of atorvastatin during the observation period. We explored the lipid-lowering mechanism of SAK-HV by the hepatic transcriptome analysis and serials of experiments both in vivo and in vitro. Unexpectedly, SAK-HV triggered a moderate energy and material-consuming liver proliferation to dramatically decrease the lipids from both serum and liver. We provided the first evidence that PGC-1α mediated the hepatic synthesis of female hormones during liver proliferation, and proposed the complement system-induced PGC-1α-estrogen axis via the novel STAT3-C/EBPß-PGC-1α pathway in liver as a new energy model for liver proliferation. In this model, PGC-1α ignited and fueled hepatocyte activation as an "igniter"; PGC-1α-induced estrogen augmented the energy supply of PGC-1α as an "ignition amplifier", then triggered the hepatocyte state transition from activation to proliferation as a "starter", causing triglyceride and cholesterol-lowering effects via PPARα-mediated fatty acid oxidation and LDLr-mediated cholesterol uptake, respectively. Collectively, the SAK-HV-triggered distinctive lipid-lowering strategy based on the new energy model of liver proliferation has potential as a novel short-period biotherapy against NAFLD and hyperlipemia.