Recombinant Hirudin Research Articles

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.

Chemical modification of recombinant hirudin with palmitic acid in mixed aqueous-organic solutions

Abstract In this study, chemical modification of recombinant hirudin variant-2 (HV2) with palmitic acid (PAL) was proposed as an alternative approach to circumvent the limitations of PEGylation. To facilitate a sufficient contact of the hydrophilic HV2 to the hydrophobic PAL, thereby improving the reaction specificity to achieve the desired mono-PAL-HV2 with high retained bioactivity, the reaction was developed in mixed aqueous-organic solutions. Compared with HV2 conjugation with PAL-benzotriazole (PAL-BTA) in mixed aqueous-NMP solution, the conjugation of HV2 with PAL-N-hydroxysuccinimide (PAL-NHS) in mixed aqueous-DMSO solution could improve the site-specific conjugation of one PAL molecule to a particular lysine residue. Furthermore, the reaction mixture of the latter was further purified by preparative liquid chromatography. Three mono-PAL-HV2 isomers were obtained and retained 36%, 4% and 89% of the in vitro anticoagulant activity of unmodified HV2, respectively. One of the mono-PAL-HV2 isomers, namely, mono-PAL-HV2-3, was isolated with the highest selectivity and exhibited the highest in vitro anticoagulant bioactivity. Modification site analysis of mono-PAL-HV2-3 revealed that a single PAL molecule was conjugated at Lys27 of HV2. This study presented a successful PAL modification in which site-specific reaction was improved to achieve the desired mono-PAL-HV2 with highly retained bioactivity in mixed aqueous-organic solutions.

Combined optimization of N-terminal site-specific PEGylation of recombinant hirudin using response surface methodology and kinetic analysis.

In this study, a combined optimization method was developed to optimize the N-terminal site-specific PEGylation of recombinant hirudin variant-2 (HV2) with different molecular weight mPEG-propionaldehyde (mPEG-ALD), which is a multifactor-influencing process. The HV2-PEGylation with 5kDa mPEG-ALD was first chosen to screen significant factors and determine the locally optimized conditions for maximizing the yield of mono-PEGylated product using combined statistical methods, including the Plackett-Burman design, steepest ascent path analysis, and central composition design for the response surface methodology (RSM). Under the locally optimized conditions, PEGylation kinetics of HV2 with 5, 10, and 20kDa mPEG-ALD were further investigated. The molar ratio of polyethylene glycol to HV2 and reaction time (the two most significant factors influencing the PEGylation efficiency) were globally optimized in a wide range using kinetic analysis. The data predicted by the combined optimization method using RSM and kinetic analysis were in good agreement with the corresponding experiment data. PEGylation site analysis revealed that almost 100% of the obtained mono-PEGylated-HV2 was modified at the N-terminus of HV2. This study demonstrated that the developed method is a useful tool for the optimization of the N-terminal site-specific PEGylation process to obtain a homogeneous mono-PEGylated protein with desirable yield.

Antithrombotic effects of recombinant hirudin in mice and its mechanism

To investigate the antithrombotic effects of recombinant hirudin and its mechanism. Sixty male Kunming mice were randomly divided into 6 group (n=10):control group, model group, aspirin (25 mg/kg) group, recombinant hirudinlow, middle and high dose (0.05, 0.1, 0.2 mg/kg) groups.Except mice in control group, 2.5 mg/kg carrageenan was injected intraperitoneallyto mice in the other groups to produce thrombosis on the mice tail. The mice in aspirin group were administrated intraperitoneally 25 mg/kg aspirin, the mice in recombinant hirudinlow, middle and high dose groups were administrated intraperitoneally 0.05, 0.1, 0.2 mg/kg combinanthirudin, the mice in control group and model group were administrated intraperitoneallynormal saline at the same volume respectively at 24 h, 0.5 h before injecting carrageenan and 24 h after injecting carrageenan. The black tail length of mice and the incidence of black tail were observed at 48h after injection of carrageenan; prothrombin time (PT), activated partial thromboplastin time (APTT), tissue plasminogen activator (t-PA), type-1 plasminogen activator inhibitor (PAI-1), 6-keto-PGF1α, and thromboxane B2 (TXB2) level in mice plasma were determined. As compared with control group, the mice in model group presented tail thrombosis; PT level in plasma was significantly shortened (P<0.01), PAI-1 and TXB2levels in plasma were significantly increased (P<0.01), while the t-PA and 6-keto-PGF1α levels in plasma in model group were significantly decreased (P<0.01). As compared with model group, the thrombus length in the tail was significantly shortened (P<0.05, P<0.01), PT level was obviously prolonged (P<0.01), and the plasma levels of PAI-1 and TXB2 were significantly decreased (P<0.01), while the plasma levels of t-PA and 6-keto-PGF1α were significantly increased (P<0.01)in the mice of recombinant hirudin low dose, middle dose, high dose groups and aspirin group. As compared with aspirin group, the thrombus length in the tail was significantly increased (P<0.05), PT level was obviously shortened (P<0.01), and the plasma levels of PAI-1 and TXB2 were significantly increased (P<0.01)in the mice of recombinant hirudin low dose group; the plasma level of 6-keto-PGF1α was significantly decreased (P<0.01, P<0.05) in the mice of recombinant hirudin low dose and middle dose groups; the plasma levels of PAI-1 and TXB2 were significantly increased (P<0.01, P<0.05)in the mice of recombinant hirudin middle dose group. The recombinant hirudin can fight against thrombosis, its antithrombotic mechanisms may be related to its influence on the exogenous coagulation system and the promotion of fibrinolysis function.

Randomized, double‐blind, placebo‐controlled, interventional phase IV investigation to assess the efficacy and safety of r‐hirudin gel (1120I.U) in patients with hematomas

Unlabelled Box BackgroundHirudin is the most potent direct thrombin inhibitor, and recombinant forms are routinely used in anticoagulation therapy. Recombinant hirudin gels are commercially available for the treatment of hematomas and associated symptoms. ObjectivesTo assess the efficacy and safety of a topically administered recombinant hirudin gel in patients with hematomas. Patients/MethodsThis double‐blind, placebo‐controlled, phase IV investigation recruited patients presenting with at least one hematoma. Subjects were randomly assigned (1:1) recombinant hirudin gel (1120 IU/100 g) or a placebo, administered 2‐3 times daily for 16 days. Changes in hematoma size, flare, and the proportion of patients achieving complete resolution of hematomas and associated edemas were investigated. ResultsBy study end, a greater proportion of subjects in the treatment group achieved a complete resolution of hematomas versus placebo (98.0% vs 71.9%; P < .001) and edemas (99% vs 50%; P < .001). Patients in the recombinant hirudin group exhibited a marginally larger, yet significant, reduction in mean hematoma size versus placebo (99.9% vs 96.6%; P < .001) and flare (93.6% vs 78.6%; P < .001). Median time to hematoma resolution for the recombinant hirudin and placebo administered cohorts was 8 and 16 days, respectively (P < .001). No adverse events were reported for the recombinant hirudin cohort. ConclusionsTopical recombinant hirudin is an effective, safe, and well tolerated intervention for the symptomatic treatment of hematomas. This trial was registered at www.clinicaltrials.gov as NCT01960569.

Development, validation, and clinical pharmacokinetic application of ultra-performance liquid chromatography/tandem mass spectrometry method for simultaneously determining a novel recombinant hirudin derivative (Neorudin) and its active metabolite in human serum.

Recombinant Neorudin (EPR-hirudin, EH), a novel, low-bleeding anticoagulant fusion protein, has been developed as an inactive prodrug that is converted to an active metabolite, hirudin variant 2-Lys47 (HV2), at the thrombus site and is undergoing Phase I clinical trials in China. The goal of our present research was to establish a novel ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) method for simultaneously quantifying EH and HV2 in human serum. Furthermore, the method was used in clinical pharmacokinetic study after validation. The stock and dilute working solutions were dissolved in methanol/water (1/1, v/v) to avoid their adsorption. The internal standard (IS) used, had a similar structure to that of EH. The serum sample pretreatment involved protein precipitation with methanol. The volume ratio of the precipitating solvent to the serum sample was 3:1 (300μL methanol: 100μL serum sample). The chromatographic separation was performed using a 300Å C18 column using a multi-step gradient with a mobile phase consisting of acetonitrile:water containing 0.1% formic acid. The detection was carried out using an ESI source in the positive multiple reaction monitoring (MRM) mode. The within and between run precision were in the range of 3.5%-10.3% for EH and 3.3%-8.8% for HV2, and the accuracy of both EH and HV2 was between -4.6% and 2.1%. The extraction recoveries and matrix effect at three quality control (QC) levels for EH and HV2 were satisfactory. The stabilities of EH and HV2 during the storage, preparation, and analysis were confirmed, and the carryover also proved to be acceptable. This technique was efficiently used in Phase I clinical pharmacokinetic trials of EH following intravenous administration of 0.2mg/kg to healthy volunteers.

Effects of linear and branched polyethylene glycol on PEGylation of recombinant hirudin: Reaction kinetics and in vitro and in vivo bioactivities

Abstract To improve the therapy efficacy of recombinant hirudin variant-2 (HV2), its PEGylation was investigated using linear mPEG-succinimidyl carbonate (mPEG-SC) and branched mPEG2-N-hydroxysuccinimide (mPEG2-NHS). The reaction mixtures of PEGylation were analyzed by RP-HPLC and the mono-PEG-HV2 products were purified by anion exchange chromatography (IEC). Effects of linear and branched PEG on the hydrolysis kinetics of the PEG reagent, the PEGylation kinetics of HV2 and the in vitro and in vivo bioactivity of mono-PEG-HV2 were investigated. The RP-HPLC and IEC analyses showed that linear and branched PEG-HV2 with identical molecular weight had different chromatographic behaviors. The reaction kinetics showed that branched mPEG2-NHS displayed higher hydrolysis rate but lower PEGylation rates than linear mPEG-SC. Consequently, HV2 conjugated with mPEG2-NHS required a greater molar ratio of PEG to HV2 than that of mPEG-SC to achieve the identically desired yield of mono-PEG-HV2. The in vitro and in vivo bioactivities of mono-PEG-HV2 showed that branched PEG-HV2 had higher therapeutic efficacy than linear PEG-HV2 with identical molecular weight. The in vivo bioactivity of mono-B-PEG40k-HV2 (mono-PEG-HV2 derived from 40 kDa branched mPEG2-NHS) had a markedly longer duration in rabbits than did unmodified HV2, which showed its potential to be developed as a candidate antithrombotic drug.

Production and purification of constructed recombinant hirudin in BL21(DE3) strain

Background/Objective: Hirudin, an extract from the leech, has powerful antithrombin activity affecting the blood coagulation pathway, it is the most potent natural inhibitor of thrombin, it binds thrombin with high affinity, so, the aim of this study was to build hirudin gene by overlapping extension PCR then cloning and expression in BL21(DE3) strain.Methods: Hirudin gene constructed with four modified primers then the final product amplified by two primers named as A, B by using overlapping extension PCR, for gene expression, BL21(DE3) strain was used under the control of T7 promoter in pET-16b vector and for hirudin production, LB broth medium was used as fermentation medium. Hirudin protein purified at first by (Immobilized metal affinity chromatography) IMAC, then this protein was dialysis and treated with factor Xa to eliminate His-tag. Then hirudin purified by DEAE sepharose and SP sepharose column, the concentration of protein determined by ELISA, furthermore the activity evaluated by thrombin titration and activated partial thromboplastin time (APPT) test.Results: Hirudin gene constructed in two round PCR first round produced two products (product1 117 bp while product 2,114 bp) the second-round PCR gave the final product 213 bp. The resulted band from gel electrophoresis for constructed vector pET-16b-HirudinS was (5,901 bp). The analysis on 15% SDS-PAGE for the SP sepharose column illustrated the hirudinS protein band with size about ~10.8. Concentration of produced hirudin within its solution reached to 1.75 ng, thrombin titration method showed that the hirudin protein required 300 µl from thrombin to clot, also, APPT test showed that hirudin elongated clotting time to 7min in comparison with 6min for aspirin and the statistical analysis results for APPT test illustrated that there was no significant difference between hirudin and aspirin.Conclusion: This study approved that overlapping extension PCR is a good strategy for building hirudin gene and it’s successfully expressed in BL21(DE3) strain.

In situ PEGylation of recombinant hirudin on an anion exchange chromatography column

Abstract In this study, an integrated process was developed for successive solid-phase PEGylation of recombinant hirudin variant-2 (HV2) and separation of PEGylated HV2 species on an anion exchange chromatography column (so-called in situ PEGylation). The effects of different PEG sizes, ion exchange resins and reaction conditions on in situ PEGylation were investigated. The results showed that in situ PEGylation efficiently integrates the reaction, separation and purification into a single-unit operation using the same column. In situ PEGylation could improve the selectivity of PEGylation reactions by significantly reducing the formation of multi-PEG-HV2. The pore sizes and internal surface structures of different resins had a significant impact on the yield of mono-PEG-HV2. In contrast to liquid-phase PEGylation, the yield of mono-PEG-HV2 decreased as PEG size increased during the in situ PEGylation process, indicating that in situ PEGylation is a pore diffusion-controlled process. The in vitro and in vivo anticoagulant activities of mono-PEG-HV2 derived from in situ PEGylation were higher than those from liquid-phase PEGylation, indicating that in situ PEGylation could enhance the bioactivity retention of mono-PEG-HV2. The results of this study demonstrated that in situ PEGylation can be used as an effective approach for the development of PEGylated protein drugs.

In silico designing of a new cysteine analogue of hirudin variant 3 for site specific PEGylation.

Hirudin is an anticoagulant agent of the salivary glands of the medicinal leech. Recombinant hirudin (r-Hir) displays certain drawbacks including bleeding and immunogenicity. To solve these problems, cysteine-specific PEGylation has been proposed as a successful technique. However, proper selection of the appropriate cysteine residue for substitution is a critical step. This study has, for the first time, used a computational approach aimed at identifying a single potential PEGylation site for replacement by cysteine residue in the hirudin variant 3 (HV3). Homology modeling (HM) was performed using MODELLER. All non-cysteine residues of the HV3 were replaced with the cysteine. The best model was selected based on the results of discrete optimized protein energy score, PROCHECK software, and Verify3D. The receptor binding was investigated using protein-protein docking by ClusPro web tool which was then visualized using LigPlot+ software and PyMOL. Finally, multiple sequence alignment (MSA) using ClustalW software and disulfide bond prediction were performed. According to the results of HM and docking, Q33C, which was located on the surface of the protein, was the best site for PEGylation. Furthermore, MSA showed that Q33 was not a conserved residue and LigPlot+ software showed that it is not involved in the hirudin-thrombin binding pocket. Moreover, prediction softwares established that it is not involved in disulfide bond formation. In this study, for the first time, the utility of the in silico approach for creating a cysteine analogue of HV3 was introduced. Our study demonstrated that the substitution of Q33 by cysteine probably has no effect on the biological activity of the HV3. However, experimental analyses are required to confirm the results.

Effectiveness and Tolerability of Anticoagulants for Thromboprophylaxis after Major Joint Surgery: a Network Meta-Analysis

Background/Aims: Venous thromboembolism (VTE) is the most common complication after major joint surgery. VTE can easily develop into pulmonary embolism (PE), leading to cardiopulmonary dysfunction or sudden death. We aimed to comprehensively analyse the thromboprophylactic drugs that are used to prevent thrombosis and reduce bleeding risk. Methods: We searched the PubMed, EMBASE, and Cochrane databases for randomized controlled trials that evaluated the use of thromboprophylaxis after major joint surgery. The major outcomes were the numbers of all-cause VTE and bleeding events, and the secondary outcomes were major VTE and major bleeding/clinically relevant non-major bleeding events. A random-effects network meta-analysis was used to assess the effectiveness and tolerability of each anticoagulant after major joint surgery. Results: We included 104 trials that assessed 110,643 patients in our meta-analysis. The cluster ranking of major outcomes indicated that FXI-ASO, ardeparin, aspirin, and apixaban were ideal for preventing all-cause VTE and avoiding all bleeding events. Nadroparin, recombinant hirudin, and rivaroxaban effectively inhibited VTE but were associated with a high risk of bleeding. For secondary outcomes, we found that betrixaban, dalteparin, warfarin, and eribaxaban were ideal for preventing major VTE and reducing major bleeding, while rivaroxaban effectively inhibited major VTE but was associated with a high risk of major/clinically relevant non-major bleeding. A sensitivity analysis showed that the effect of apixaban was more robust for major outcomes, while aspirin was more robust for preventing all-cause bleeding events. In secondary outcomes, the effect of warfarin was more robust, while apixaban was still considered an ideal treatment to inhibit major VTE and bleeding events. Conclusion: Our study indicates that FXI-ASO, ardeparin, aspirin, and apixaban are ideal for preventing all-cause VTE and reducing all bleeding events, among which apixaban is the most reliable. Betrixaban, dalteparin, warfarin, and eribaxaban are ideal for preventing major VTE and reducing major/clinically relevant non-major bleeding events, among which warfarin is the most reliable.

Biological performance of functionalized biomedical polymers for potential applications as intraocular lens.

To study the biological performance of surface-modified biomedical polymer materials, a model of the functional mechanism of nonspecific adsorption resistance was constructed. Cell behavior on the surface and in vivo transplantation features of intraocular lens (IOL) materials, such as hydrophobic acrylic ester and polymethyl methacrylate (PMMA), were investigated. The results of cell adhesion and proliferation studies showed that the addition of hirudin can significantly resist epithelial cell adhesion, better than the pure amination process, and thereby inhibit excessive proliferation on the surface. Experiments on the eyes of rabbits indicated that the IOL surfaces with hirudin modification reduced the incidence of cell aggregation and inflammation. Combined with a study of protein-resistant layer construction with recombinant hirudin on the material surface, the mechanism of surface functionalization was determined. The biological performance indicated that nonspecific adsorption is greatly decreased due to the existence of amphiphilic ions or hydration layers, which lead to stability and long-term resistance to nonspecific adsorption. These results offer a theoretical basis for the use of traditional biomedical polymer materials in long-term clinical applications. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1961-1967, 2016.

Evaluating the Effect of Protein A Signal Peptide on Extracellular Expression of Recombinant Hirudin in E.Coli

Background and Objective: Hirudin is an anticoagulant polypeptide secreted from the salivary glands of leeches. Recombinant hirudin is a strong anticoagulant agent in arterial and venous thrombosis. The aim of this study was to evaluate the effect of inserting protein A signal peptide sequence of pEZZ18 plasmid on expression and secretion of the recombinant hirudin in E.coli. Methods: the synthetic hirudin gene was amplified by PCR using specific primers. First, the gene was purified and cloned into PTG19-T cloning vector, and then it was subcloned into pEZZ18 expression vector by SalI / SacI enzymatic digestion and finally transformed into E.coli JM107. After the expression of recombinant hirudin protein, different cellular fractions were isolated and analyzed on SDS-PAGE and further confirmed by Western blotting. Results: PCR product (522 bp) was first subcloned into the T-Vector (replicating vector) and then successfully subcloned into the pEZZ18 (expression vector). Cloning and subclonig were confirmed by enzymatic digestion and Colony PCR. After the expression and isolation of fractions, the presence of hirudin (about 29 kDa) in different cell fractions due to the effects of signal peptide was observed in SDS-PAGE and finally confirmed by Western blotting. Conclusion: The gene of anticoagulant hirudin protein (desirudin) was cloned into the pEZZ18 vector containing Protein A signal peptide sequence and later transformed into E.coli JM107. The recombinant hirudin protein expression in the extracellular space was approved.

Test of hirudin activity by tracking the binding of hirudin to thrombin in the presence of BS3 cross-linking.

Hirudin has a great potential in inhibiting thrombin, and its antithrombin activity has direct bearing on its clinical application. Using bovine alpha-thrombin and recombinant hirudin of Poecilobdella javanica purified from Phichia pastoris as materials, this study introduced a novel method to testing antithrombin activity of hirudin visually and dynamically by tracking the binding of hirudin to thrombin. After incubating the mixture of thrombin and hirudin at 37 °C for 5 min, the binding of hirudin to thrombin was cross-linked by bis[sulfosuccinimidyl] suberate for 30 min and visualized by SDS-polyacrylamide gel electrophoresis. With the aid of image analysis on the basis of INRA-Noésis E1D analysis software, antithrombin activity of hirudin was calculated through intensity variations of protein bands of either thrombin-hirudin compound, unbound thrombin, or unbound hirudin. In this regard, activity of the given hirudin was tested to be 5625 ATU/mg based on a single reaction, and 5675.3 ATU/mg based on a series of reactions in a stepwise manner, close to the result of 6000 ATU/mg concluded by titration method. The superiorities of the method include good accuracy (the minimum testable concentration of hirudin is 1.5 μg/ml) and little sample consumption (sample consumption of hirudin is generally 1-11.5 μl using the apparatus of Mini Protean 3 Cell). Easy operation, low input, and equipment requirement also grant it as an effective way.

PEGylation of recombinant hirudin in mixed aqueous–organic solutions

Serious loss of polyethylene glycol (PEG) reagent is confronted during PEGylation of proteins in mildly acidic aqueous solution. In this study, a novel approach of PEGylation of recombinant hirudin variant-2 (HV2) with monomethoxy-PEG-succinimidyl carbonate (mPEG-SC) was developed in six mixed aqueous–organic solutions. The PEGylation efficiency in each mixed solutions was greater than that in pure aqueous solution. Structure analysis of HV2, hydrolysis kinetics of mPEG-SC, and PEGylation kinetics of HV2 in different solutions revealed that solvent effects occurred in mixed aqueous–organic solutions. According to the dominant solvent effect of PEGylation acceleration and mPEG-SC hydrolysis inhibition, the selected mixed aqueous–organic solutions could be divided into different types (PEGylation-driven, mPEG-SC hydrolysis-driven, both PEGylation and mPEG-SC hydrolysis-driven). In aqueous-DMSO solutions, the desired yield (approximately 50%) of mono-PEG-HV2 was achieved under the optimized reaction conditions. The optimal usage of PEG reagent could decrease 3–5 fold compared with that of PEGylation in pure aqueous solution. The mono-PEG-HV2 could restore its structure and bioactivity after being purified into aqueous solution. This study demonstrated that PEGylation of proteins in mixed aqueous–organic solutions could be used as an alternative method to PEGylation in pure aqueous solution.

Recombinant hirudin suppresses the viability, adhesion, migration and invasion of Hep-2 human laryngeal cancer cells.

Recombinant hirudin (rH) is a highly potent and specific inhibitor of thrombin, and has been shown to inhibit the growth and metastasis of several types of cancers in experimental tumor models. The objective of this study was to evaluate the antitumor effects and explore the underlying mechanisms of rH in Hep-2 human laryngeal carcinoma (LC) cells. Hep-2 cells were treated with various concentrations of rH for 24 h. The cell viability was evaluated by a water-soluble tetrazolium salt (WST) assay. The adhesion ability of the cells was evaluated by cell adhesion to fibronectin. Cell migration and invasion were measured with the Boyden chamber assay. Cell apoptosis was detected by Hoechst 33324 fluorescence staining. A chicken chorioallantoic membrane (CAM) assay was used to assess the effects of rH on angiogenesis in vivo. Western blotting was used to detect the expression levels of vascular endothelial growth factor receptor (VEGF-R), focal adhesion kinase (FAK), Bcl-2-associated agonist of cell death (Bad) and B-cell CLL/lymphoma 2 (Bcl-2) proteins. rH significantly inhibited the cell viability and induced apoptosis in LC Hep-2 cells in a dose-dependent manner, as compared with phosphate-buffered saline (PBS) as control. These results were accompanied by a decrease in the anti-apoptotic protein Bcl-2 and an increase in the pro-apoptotic protein Bad. Moreover, rH dose-dependently inhibited the adhesion, migration and invasion of the Hep-2 cells, compared to the vehicle PBS. In addition, rH robustly suppressed angiogenesis in the CAM assay. Importantly, the expression of adhesion and angiogenesis-associated proteins FAK and VEGF-R was significantly downregulated by rH in a dose-dependent manner. The present findings demonstrate that rH exerts antitumor effects in Hep-2 human laryngeal cancer cells via multiple mechanisms and suggests that targeting thrombin by rH is a potential strategy for the treatment of LC.

Improving the bioactivity of rHirudin with boronophenylalanine site-specific modification.

To improve the bioactivity of recombinant (r)Hirudin, the orthogonal pair MjBTyrRS/tRNATyr cua (made up of the boronophenylalanine, tRNA and tRNA synthetase), was selected to incorporate boronophenylalanine site‑specifically into rHirudin at the 63 sites in an Escherichia coli system in response to the TAG codon. Following fusion with the gIII signal peptide and a hexahistidine tag, the modified protein was secreted into Luria‑Bertani culture medium and purified by nickel-nitrilotriacetic acid affinity chromatography following a gel filtration column. In a 200 ml flask, the yield of boronophenylalanine‑modified hirudin was 10 mg l‑1 and that of rHirudin was 19 mg l‑1. The authenticity of the purified proteins was verified using matrix-assisted laser desorption ionization time of flight mass spectroscopy and antithrombin activity assays. The results revealed that the antithrombin activity of the boronophenylalanine‑modified hirudin to human thrombin was more enhanced than that of rHirudin. The modified hirudin demonstrated stronger proliferation inhibiting ability on fibroblast L929 cells compared with that of rHirudin.

A comparative study between fondaparinux, a low-molecular-weight heparin, and recombinant hirudin in thromboembolic prophylaxis after major abdominal surgery in the surgical intensive care unit

Objective Evaluation of the safety and the efficacy of different anticoagulants in thromboembolic prophylaxis after major abdominal surgeries. Background Thromboembolic events are serious complications after major abdominal surgeries, and there are different modalities to prevent them. Materials and methods Sixty patients who underwent major abdominal surgery were assigned in a randomized, double-blinded manner and classified into three groups: group A ( n = 20) was started on enoxaparin at a daily dose of 40 mg. Group B ( n = 20) was given recombinant hirudin 15 mg twice daily. Group C ( n = 20) was given fondaparinux 2.5 mg subcutaneously daily. The duration of treatment was 5-12 days. The efficacy of the three drugs was compared by the occurrence of DVT (assessed by Doppler ultrasound) or fatal and nonfatal pulmonary embolism (by computed tomography). The safety was assessed by postoperative bleeding in terms of the number of transfused whole blood units, plasma expanders, or packed red blood cells and fatal bleeding. Results Results showed that enoxparin, fondaparinux, and recombinant hirudin were equally effective in the prevention of thromboembolism. The least risk of postoperative bleeding was noticed in patients receiving enoxparin in comparison with patients receiving fondaparinux or recombinant hirudin. Conclusion Enoxaparin, hirudin, and fondaparinux are equally effective in protection against thromboembolic events in patients undergoing major abdominal surgeries; however, enoxaparin is superior to the others regarding safety.

Different Classes of Anticoagulant Drugs in Clinical Use. Is there a Class Effect?

Hippocrates first proposed ‘Blood thinners’ in ancient Greek medicine. Historically, extracts from many plants, bloodletting, leech bleeding, acid fruits and clear wines were considered as anticoagulants. Although, Haycraft in 1884 identified the anticoagulant in the saliva of European medicinal leech, later named hirudin, the extract was found to be too toxic. Following Jay McLean’s discovery of an anticoagulant in 1916, and naming of this anticoagulant as ‘heparin’ by Howel and Holt in 1918 and Howel’s purification of this compound in 1925, it was only in 1930s that the effective anticoagulant treatment started. Soon after the discovery of the first oral anticoagulant, dicoumarin in 1935, the purified injectable preparations of heparin were used. Later, other types of antithrombotic and anticoagulant treatment, including antiplatelet drugs, snake venoms, direct and indirect thrombin inhibitors, activated protein C, direct Factor Xa inhibitors and recombinant hirudins have since been introduced [1]. Warfarin, a conventionally used oral anticoagulant, despite its limitations of drugfood, drug-drug interactions, need for frequent PT/INR monitoring, adverse drug reactions, bleeding, warfarin-induced skin necrosis, frequent need for dosage adjustments because of changes in the dietary patterns, effects due to VKORC polymorphism, has stood the test of time and still continues to be used in clinical practice. Recently the New Oral Anticoagulant drugs, direct Factor Xa such as Rivaroxaban, Apixaban and Edoxaban and direct thrombin inhibitors such as Dabigatran were approved by the USFDA. There are several limitations in the use of NOACs such as the lack of an effective test to monitor their anticoagulant activities, the lack of an effective antidote, and dose adjustments in patients with renal impairment [2]. Thus, a bewildering wide array of anticoagulant drugs are now available for use and it is interesting to see whether or not these drugs representing different classes of anticoagulant drugs, especially direct thrombin and direct Factor Xa inhibitors have any class effects in terms of differences in their clinical outcomes.

Role of the extracellular signal-regulated kinase 1/2 signaling pathway in the process of thrombin-promoting airway remodeling in ovalbumin-allergic rats

Context: Although it is recognized that thrombin plays a key role in airway remodeling during chronic asthma. In a previous study, we have proved that thrombin promotes airway remodeling via PAR-1 in OVA-allergic rats, but little is known about intracellular signaling pathway involved in the event.Objective: In this study, we intend to explore the impact of pERK1/2 signaling pathway on the process of thrombin-induced airway remodeling in OVA-allergic rats.Materials and methods: A rat model of chronic asthma was set up by systemic sensitization and repeated challenge to OVA. The doses of thrombin, recombinant hirudin, PAR-1 inhibitor ER-112780-06, and pERK1/2 inhibitor PD98059 varied for different groups. The expression of pERK1/2 was analyzed by western blot and RT-PCR. Secretion of TGF-β1 and IL-6 was detected by ELISA.Results: The expression of pERK1/2 was higher in the airway of asthmatic rats than those of normal rats, and was significantly increased by thrombin treatment but decreased by thrombin-inhibitor treatment. Airway remodeling was enhanced by thrombin but weakened by pERK1/2 inhibitor. Expression of growth factors and IL-6 in asthmatic rats was significantly increased by thrombin treatment and decreased by thrombin-inhibitor treatment and pERK1/2 inhibitor treatment.Conclusion: These results suggest that ERK1/2 signaling pathway may play an important role in the process of thrombin-promoting airway remodeling in OVA-allergic rats, and pERK1/2 inhibitor effectively inhibits the process.