Recombinant Hirudin Research Articles

Recombinant hirudin and PAR-1 regulate macrophage polarisation status in diffuse large B-cell lymphoma

BackgroundDiffuse large B-cell lymphoma (DLBCL) is a malignant tumour. Although some standard therapies have been established to improve the cure rate, they remain ineffective for specific individuals. Therefore, it is meaningful to find more novel therapeutic approaches. Macrophage polarisation is extensively involved in the process of tumour development. Recombinant hirudin (rH) affects macrophages and has been researched frequently in clinical trials lately. Our article validated the regulatory role of rH in macrophage polarisation and the mechanism of PAR-1 by collecting clinical samples and subsequently establishing a cellular model to provide a scientifically supported perspective for discovering new therapeutic approaches.MethodWe assessed the expression of macrophage polarisation markers, cytokines and PAR-1 in clinical samples. We established a cell model by co-culture with THP-1 and OCI-Ly10 cell. We determined the degree of cell polarisation and expression of validation cytokines by flow cytometry, ELISA, and RT-qPCR to confirm the success of the cell model. Subsequently, different doses of rH were added to discover the function of rH on cell polarisation. We confirmed the mechanism of PAR-1 in macrophage polarisation by transfecting si-PAR-1 and pcDNA3.1-PAR-1.ResultsWe found higher expression of M2 macrophage markers (CD163 + CMAF+) and PAR-1 in 32 DLBCL samples. After inducing monocyte differentiation into M0 macrophages and co-culturing with OCI-Ly10 lymphoma cells, we found a trend of these expressions in the cell model consistent with the clinical samples. Subsequently, we discovered that rH promotes the polarisation of M1 macrophages but inhibits the polarisation of M2 macrophages. We also found that PAR-1 regulates macrophage polarisation, inhibiting cell proliferation, migration, invasion and angiogenic capacity.ConclusionrH inhibits macrophage polarisation towards the M2 type and PAR-1 regulates polarisation, proliferation, migration, invasion, and angiogenesis of DLBCL-associated macrophages.

Combined drug anti-deep vein thrombosis therapy based on platelet membrane biomimetic targeting nanotechnology

After orthopedic surgeries, such as hip replacement, many patients are prone to developing deep vein thrombosis (DVT), which in severe cases can lead to fatal pulmonary embolism or major bleeding. Clinical intervention with high-dose anticoagulant therapy inevitably carries the risk of bleeding. Therefore, a targeted drug delivery system that adjusts local DVT lesions and potentially reduces drug dosage and toxic side effects important. In this study, we developed a targeted drug delivery platelet-derived nanoplatform (AMSNP@PM-rH/A) for DVT treatment that can simultaneously deliver a direct thrombin inhibitor (DTI) Recombinant Hirudin (rH), and the Factor Xa inhibitor Apixaban (A) by utilizing Aminated mesoporous silica nanoparticles (AMSNP). This formulation exhibits improved biocompatibility and blood half-life and can effectively eliminate deep vein thrombosis lesions and achieve therapeutic effects at half the dosage. Furthermore, we employed various visualization techniques to capture the targeted accumulation and release of a platelet membrane (PM) coating in deep vein thrombosis and explored its potential targeting mechanism.

Recombinant hirudin suppresses angiogenesis of diffuselarge B-cell lymphoma through regulation of the PAR-1-VEGF.

Hirudin is one of the specific inhibitors of thrombin, which has been confirmed to have strong bioactivities, including inhibiting tumors. However, the function and mechanism of hirudin and protease-activated receptor 1 (PAR-1) in diffuse large B-cell lymphoma (DLBCL) have not been clear. Detecting the expression PAR-1 in DLBCL tissues and cells by RT-qPCR and IHC. Transfected sh-NC, sh-PAR-1, or pcDNA3.1-PAR-1 in DLBCL cells or processed DLBCL cells through added thrombin, Vorapaxar, Recombinant hirudin (RH), or Na2S2O4 and co-culture with EA.hy926. And built DLBCL mice observed tumor growth. Detecting the expression of related genes by RT-qPCR, Western blot, IHC, and immunofluorescence, measured the cellular hypoxia with Hypoxyprobe-1 Kit, and estimated the cell inflammatory factors, proliferation, migration, invasion, and apoptosis by ELISA, CCK-8, flow cytometry, wound-healing and Transwell. Co-immunoprecipitation and pull-down measurement were used to verify the relationship. PAR-1 was highly expressed in DLBCL tissues and cells, especially in SUDHL2. Na2S2O4 induced SUDHL2 hypoxia, and PAR-1 did not influence thrombin-activated hypoxia. PAR-1 could promote SUDHL2 proliferation, migration, and invasion, and it was unrelated to cellular hypoxia. PAR-1 promoted proliferation, migration, and angiogenesis of EA.hy926 or SUDHL2 through up-regulation vascular endothelial growth factor (VEGF). RH inhibited tumor growth, cell proliferation, and migration, promoted apoptosis of DLBCL, and inhibited angiogenesis by down-regulating PAR-1-VEGF. RH inhibits proliferation, migration, and angiogenesis of DLBCL cells by down-regulating PAR-1-VEGF.

Recombinant hirudin attenuates pulmonary hypertension and thrombosis in acute pulmonary embolism rat model.

Acute pulmonary embolism (APE) is classified as a subset of diseases that are characterized by lung obstruction due to various types of emboli. Current clinical APE treatment using anticoagulants is frequently accompanied by high risk of bleeding complications. Recombinant hirudin (R-hirudin) has been found to have antithrombotic properties. However, the specific impact of R-hirudin on APE remains unknown. Sprague-Dawley (SD) rats were randomly assigned to five groups, with thrombi injections to establish APE models. Control and APE group rats were subcutaneously injected with equal amounts of dimethyl sulfoxide (DMSO). The APE+R-hirudin low-dose, middle-dose, and high-dose groups received subcutaneous injections of hirudin at doses of 0.25 mg/kg, 0.5 mg/kg, and 1.0 mg/kg, respectively. Each group was subdivided into time points of 2 h, 6 h, 1 d, and 4 d, with five animals per point. Subsequently, all rats were euthanized, and serum and lung tissues were collected. Following the assessment of right ventricular pressure (RVP) and mean pulmonary artery pressure (mPAP), blood gas analysis, enzyme-linked immunosorbnent assay (ELISA), pulmonary artery vascular testing, hematoxylin-eosin (HE) staining, Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining, immunohistochemistry, and Western blot experiments were conducted. R-hirudin treatment caused a significant reduction of mPAP, RVP, and Malondialdehyde (MDA) content, as well as H2O2 and myeloperoxidase (MPO) activity, while increasing pressure of oxygen (PaO2) and Superoxide Dismutase (SOD) activity. R-hirudin also decreased wall area ratio and wall thickness to diameter ratio in APE rat pulmonary arteries. Serum levels of endothelin-1 (ET-1) and thromboxaneB2 (TXB2) decreased, while prostaglandin (6-K-PGF1α) and NO levels increased. Moreover, R-hirudin ameliorated histopathological injuries and reduced apoptotic cells and Matrix metalloproteinase-9 (MMP9), vascular cell adhesion molecule-1 (VCAM-1), p-Extracellular signal-regulated kinase (ERK)1/2/ERK1/2, and p-P65/P65 expression in lung tissues. R-hirudin attenuated pulmonary hypertension and thrombosis in APE rats, suggesting its potential as a novel treatment strategy for APE.

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.

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 → β-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.

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.

Aptamer-thrombin loaded magnetic microspheres for bio-specific extraction and precise detection of hirudin

Hirudin, that is naturally occurring in leeches (Hirudo medicinalis) and known as the most potent natural inhibitor of thrombin, exerts double-edged effects in clinic application. It can be used as a therapeutic ingredient for cardiovascular disease, while it can be regarded as a toxic polypeptide with bleeding risk. Effective detection of hirudin in biological samples contributes greatly to reasonable therapy. In this study, we proposed a smart adsorbent based on affinity magnetic microspheres, where thrombin was immobilized for capturing hirudin in the animal serum. Aptamer was introduced as a ligand for linking the magnetic agarose microspheres and thrombin, thereby avoiding loss of biological activity of the enzyme to hirudin. Taken recombinant hirudin variant 2-Lys47 (rHV2) as a model, we established a rapid and bio-specific extraction method coupled with liquid chromatography and quadrupole-time-of-flight mass spectrometry (LC-QTOF/MS) for determination of hirudin in the serum. Owing to this strategy, a low limit of detection (LOD) of rHV2 (0.5 nM), a good linearity with correlation coefficient of 0.9975, an acceptable precision with relative standard deviation (RSD) below 3.6% (n = 6) and acceptable recoveries ranging from 85.7% to 90.2% were achieved. Moreover, the functionalized magnetic composite could be reused for at least nine cycles. Our work combined the merits of affinity separation and advanced instrument analysis for hirudin, providing a new vision to precise determination of hirudin in medical and pharmaceutical fields.

Functionalization of PCL-based nanofibers loaded with hirudin as blood contact materials

Blood-contacting materials with good mechanical property, excellent anticoagulant function and promoting effect on endothelialization are in great demand for clinical application such as vascular grafts in treating cardiovascular diseases. In this study, electrospinning nanofiber scaffolds of polycaprolactone (PCL) were functionalized by oxidative self-polymerization of dopamine (PDA) on the surface followed by the modification of anticoagulant recombinant hirudin (rH) molecules. The morphology, structure, mechanical property, degradation behavior, cellular compatibility and blood compatibility of the multifunctional PCL/PDA/rH nanofiber scaffolds were evaluated. The diameter of the nanofibers was between 270–1030 nm. The ultimate tensile strength of the scaffolds was around 4 MPa and the elastic modulus increased with the amount of rH. The degradation tests in vitro indicated that the nanofiber scaffolds began to crack on the 7th day, but still maintained the nanoscale architecture within a month. The cumulative release of rH from the nanofiber scaffold was up to 95.9 % at 30th day. The functionalized scaffolds promoted the adhesion and proliferation of endothelial cells, while resisting platelet adhesion and enhancing anticoagulation effects. The hemolysis ratios of all scaffolds were <2 %. The nanofiber scaffolds are promising candidates for vascular tissue engineering.

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.

Enhanced production of recombinant proteins in Corynebacterium glutamicum using a molecular chaperone.

Protein synthesis in Corynebacterium glutamicum is critical for applications in biotechnology and medicine. However, the use of C. glutamicum for protein production is limited by its low expression and aggregation. To overcome these limitations, a molecular chaperone plasmid system was developed in this study to improve the efficiency of recombinant protein synthesis in C. glutamicum. The effect of molecular chaperones on target protein synthesis (Single-chain variable fragment, Scfv) under three different promoter strengths was tested. In addition, the plasmid containing the molecular chaperone and target protein was verified for growth stability and plasmid stability. This expression model was further validated using two recombinant proteins, human interferon-beta (Hifn) and hirudin variant III (Rhv3). Finally, the Rhv3 protein was purified, and analysis of Rhv3 activity confirmed that the use of a molecular chaperone led to an improvement in test protein synthesis. Thus, the use of molecular chaperones is believed to will improve recombinant proteins synthesis in C. glutamicum.

LC-MS/MS method for determination of r-RGD-hirudin in human plasma and its application in pharmacokinetic study

ObjectiveThis study aimed to develop a simple, sensitive, and selective Liquid chromatography with a Mass spectroscopic method for simultaneous quantification of a recombinant bifunctional hirudin (r-RGD-Hirudin, Bifunctional Hirudin, BFH) in human plasma and verify its effectiveness. MethodsThe analytes and the internal standards from human plasma were extracted using the solid-phase extraction technique. The reconstituted samples were chromatographed on Waters C18 column (BEH 50 × 2.1 mm, 1.7 μm) using a mixture of 0.1% formic acid/acetonitrile (85%/15%, v/v) with gradient elution as the initial mobile phase at a flow rate of 0.3 mL/min. ResultsThe effectiveness of the proposed method was verified over the concentration range of 10–2000 ng/mL for r-RGD-Hirudin. A linear calibration curve was obtained. The precision and accuracy of BFH in the intra- and inter-day runs fell within the range of ±15% at LQC, GMQC, MQC and HQC concentrations. The extraction recoveries and matrix effect at two quality control (QC) levels for BFH were confirmed to conform to the relevant requirement. ConclusionThe proposed method was successfully adapted to examine the pharmacokinetics of BFH in 40 Chinese healthy volunteers, respectively.

Surface functionalization of anticoagulation and anti-nonspecific adsorption with recombinant hirudin modification.

Surface functionalization to improve the blood compatibility is pivotal for the application of biomaterials. In this article, the surface of silicon was first functionalized with chemical groups, such as amino, quinone and phenol groups by the self-polymerization of dopamine, which were used to immobilize anticoagulant drugs hirudin. The detailed analysis and discussion about the grafting groups, morphology, wettability, the dynamic adsorption of proteins, the cytological property and the blood compatibility on the surfaces were carried on by the technology of contact angle, X-ray photoelectron spectroscopy, quartz crystal microbalance, endothelial cells culture and anticoagulant blood test in vivo. The surface with hirudin modification exhibited hydrophilic property and significantly inhibited the nonspecific adsorption of albumin, while it was more approachable to fibronectin. In vitro study displayed that the surface loaded with hirudin could promote the proliferation of endothelial cells. The evaluation of anticoagulant showed good anti-adhesion effect on platelets and the hemolysis rate decreased significantly to less than 0.4%. Activated partial thromboplastin time (APTT) of the silicon wafer loaded with hirudin can exceed 38s, and the APTT prolongs as the hirudin concentration rises. This study suggested that such simple but effective surface functionalization technique, combining excellent anticoagulant activity together with reendothelialization potential due to the preferable fibronectin adsorption, provide great practical significance to the application of cardiovascular materials.

Design and fabrication of r-hirudin loaded dissolving microneedle patch for minimally invasive and long-term treatment of thromboembolic disease

Cardiovascular disease is the leading cause of global mortality, with anticoagulant therapy being the main prevention and treatment strategy. Recombinant hirudin (r-hirudin) is a direct thrombin inhibitor that can potentially prevent thrombosis via subcutaneous (SC) and intravenous (IV) administration, but there is a risk of haemorrhage via SC and IV. Thus, microneedle (MN) provides painless and sanitary alternatives to syringes and oral administration. However, the current technological process for the micro mould is complicated and expensive. The micro mould obtained via three-dimensional (3D) printing is expected to save time and cost, as well as provide a diverse range of MNs. Therefore, we explored a method for MNs array model production based on 3D printing and translate it to micro mould that can be used for fabrication of dissolving MNs patch. The results show that r-hirudin-loaded and hyaluronic acid (HA)-based MNs can achieve transdermal drug delivery and exhibit significant potential in the prevention of thromboembolic disease without bleeding in animal models. These results indicate that based on 3D printing technology, MNs combined with r-hirudin are expected to achieve diverse customizable MNs and thus realize personalized transdermal anticoagulant delivery for minimally invasive and long-term treatment of thrombotic disease.

Recombinant Hirudin Protects the Vasculature and Myocardium in Atherosclerotic Rats by Inhibiting the p38 MAPK/NF-kB Signaling Pathway

Recombinant hirudin (r-hirudin) has a good anticoagulant effect and has a certain inhibitory effect on atherosclerosis (AS), however, its intrinsic mechanism of inhibiting AS is still unclear. In this study, we investigated the mechanism underlying the vascular and myocardial protective effects of r-hirudin in AS rats through animal experiments. A rat AS model was established by high-fat diet feeding combined with common carotid artery balloon injury. The model rats were given low, medium, or high doses of r-hirudin (0.05, 0.1, or 0.2 mg/kg/ day), simvastatin tablets (1 mg/kg/day) and p38 mitogen-activated protein kinase (MAPK) pathway inhibitors (SB203580, 100 mg/kg/day) by gavage for 8 weeks. The results showed that in AS rats, r-hirudin significantly alleviated pathological changes in the common carotid artery and myocardial tissue; decreased serum total cholesterol (TC), triglyceride (TG) and low-density lipoprotein-cholesterol (LDL-C) levels; increased highdensity lipoprotein-cholesterol (HDL-C) levels; decreased serum oxidized low-density lipoprotein (ox-LDL), tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1β, and endothelin (ET)-1 levels; increased nitric oxide (NO) levels; and decreased p38 MAPK, nuclear factor-kappa B (NF-κB), caspase-9, caspase-3 mRNA and protein expression. This study showed that r-hirudin may protect blood vessels and the myocardium in AS rats by adjusting blood lipid levels and inhibiting the p38 MAPK/NF-κB signaling pathway to exert anti-inflammatory and anti-apoptotic effects and protect the vascular endothelium.

Microneedle patch-assisted transdermal administration of recombinant hirudin for the treatment of thrombotic diseases

The painless microneedle patch (MNP), widely explored for transdermal drug delivery of macromolecules, can overcome the limitations of traditional administrations of protein and polypeptide anticoagulant drugs. We constructed a recombinant hirudin-loaded microneedle patch, suitable for patients with thrombotic diseases requiring long-term preventive medication. The recombinant hirudin-loaded dissoluble microneedle patch (RHDMNP) was created using a mold casting technique and polyvinylpyrrolidone and polyvinyl alcohol were used as the matrix material with a 1:1.2 ratio. We prepared bilayer RHDMNPs with pyramidal appearance and 0.37 N/needle strength. The intradermal dissolution height of the microneedle reached approximately 78.67% of the total height, and 68.12% of the drug was delivered into the skin. The 12-hour cumulative permeation of the MNP was 21.69 ± 3.90 μg/cm2. The MNP showed a Tmax of 1.5 h, Cmax of 144 ± 71 ng/mL, and area under curve (AUC) of 495 ± 66 ng/mL·min compared to Tmax of 0.5 h, Cmax of 249 ± 89 ng/mL, and AUC of 944 ± 65 ng/mL·min for the subcutaneous injection group. Both in vivo and in vitro experiments showed that the RHDMNP exerted effective anticoagulant effects, prevented the incidence of acute pulmonary embolism, and revealed the potential for venous thrombosis prevention.

Investigating the Roles of Platelet PAR4 in Hemostasis, Thrombosis and Viral Infection Using a Newly Generated PAR4 Floxed Mouse

Introduction: Protease-activated receptor 4 (PAR4) is expressed by a wide variety of cells, including megakaryocytes/platelets, immune cells, cardiomyocytes and lung epithelial cells, and activated by multiple ligands including thrombin and cathepsin G. Importantly, PAR4 is the only functional thrombin receptor on murine platelets. A global deficiency of PAR4 is associated with impaired hemostasis and protection from thrombosis which are attributed to loss of platelet PAR4, but this has not been specifically demonstrated in mice. Additionally, global PAR4 deficiency increases mortality after influenza A virus infection, but the cell type/s responsible for the enhanced mortality have not been determined. Here, we describe the generation of PAR4 floxed (PAR4 fl/fl) mice that can be used to delete PAR4 in a cell type-specific manner, and examine the effect of megakaryocyte/platelet-specific deletion of PAR4 on hemostasis, thrombosis and viral infection using PAR4 fl/fl;PF4Cre + mice.Methods: PAR4global knockout (PAR4 -/-), MK/platelet-specific knockout (PAR4 fl/fl;PF4Cre +) and appropriate littermate control mice were used for experiments. Platelet function was determined by light transmission aggregometry and flow cytometry. Hemostasis was assessed in the saphenous vein laser injury model. Platelet plug formation was visualized by intravital microscopy following saphenous vein laser ablation (~50 μm diameter injury), followed by 2 subsequent ablations to reinjure the same site unless on-going bleeding was occurring. Mice were treated with ibrutinib (12.5 mg/kg) to inhibit GPVI signaling, or dabigatran etexilate (chow containing 10 mg/g) or recombinant hirudin (50 mg/kg) to inhibit thrombin activity. Thrombosis was assessed in the carotid artery FeCl 3 model. The carotid artery was exposed and 8% FeCl 3 applied for 3 mins. Blood flow was observed for 30 mins and occlusion was defined as no blood flow for 2 mins. To study susceptibility to viral infection, mice were challenged intranasally with a mouse-adapted H1N1 influenza A virus (H1N1 IAV PR8; 0.02 hemagglutination assay units), which induces mortality in 20% of WT mice. Mortality was defined as body weight loss greater than 25%, which required euthanasia.Results: As expected, PAR4 fl/fl;PF4Cre + platelets were unresponsive to thrombin or PAR4-specific stimulation, while the response to other agonists was retained. In the saphenous vein laser injury hemostasis model, PAR4 fl/fl;PF4Cre + mice were able to rapidly form a hemostatic platelet plug, but the majority of plugs (7/8) were unstable and re-opened after several minutes, leading to severely prolonged total bleeding times. We observed similar findings in global PAR4 -/- mice with 8/12 plugs re-opening. To investigate the mechanism mediating initial platelet plug formation, we inhibited GPVI signaling in PAR4 fl/fl;PF4Cre + mice using the Btk inhibitor ibrutinib. Ibrutinib administration shortened time to plug re-opening in PAR4 fl/fl;PF4Cre + mice but plugs were still able to form, which is likely mediated by GPIbα/VWF. We observed a similar phenotype to PAR4 fl/fl;PF4Cre + mice in mice treated with the direct thrombin inhibitor hirudin, suggesting thrombin is the primary activator of PAR4 during hemostatic plug formation. In the FeCl 3-induced carotid artery thrombosis model, both PAR4 fl/fl;PF4Cre + and PAR4 -/- mice were significantly protected compared to controls. Finally, when challenged with the mouse-adapted H1N1 IAV PR8, PAR4 fl/fl;PF4Cre + mice demonstrated similar body weight loss and survival as littermate controls.Conclusions: Our results in mice demonstrate that 1) platelet PAR4 is not required for initial hemostatic plug formation but is necessary for maintaining hemostatic plug stability, 2) loss of platelet PAR4 protects from arterial thrombosis, and 3) platelet PAR4 does not alter the course of H1N1 IAV infection, at least at the virus dose used in this study. In summary, we generated a novel mouse line carrying a floxed PAR4 allele which can be used to investigate cell-specific roles of PAR4 in disease. DisclosuresNo relevant conflicts of interest to declare.

Wolffia arrhiza as a promising producer of recombinant hirudin.

The production of recombinant proteins in transgenic plants is becoming an increasingly serious alternative to classical biopharming methods as knowledge about this process grows. Wolffia arrhiza, an aquatic plant unique in its anatomy, is a promising expression system that can grow in submerged culture in bioreactors. In our study 8550 explants were subjected to Agrobacterium-mediated transformation, and 41 independent hygromycin-resistant Wolffia lines were obtained, with the transformation efficiency of 0.48%. 40 of them contained the hirudin-1 gene (codon-optimized for expression in plants) and were independent lines of nuclear-transformed Wolffia, the transgenic insertion has been confirmed by PCR and Southern blot analysis. We have analyzed the accumulation of the target protein and its expression has been proven in three transgenic lines. The maximum accumulation of recombinant hirudin was 0.02% of the total soluble protein, which corresponds to 775.5 ± 111.9ngg-1 of fresh weight of the plant. The results will be used in research on the development of an expression system based on Wolffia plants for the production of hirudin and other recombinant pharmaceutical proteins.

Cell-free synthesis of the hirudin variant 1 of the blood-sucking leech Hirudo medicinalis

Synthesis and purification of peptide drugs for medical applications is a challenging task. The leech-derived factor hirudin is in clinical use as an alternative to heparin in anticoagulatory therapies. So far, recombinant hirudin is mainly produced in bacterial or yeast expression systems. We describe the successful development and application of an alternative protocol for the synthesis of active hirudin based on a cell-free protein synthesis approach. Three different cell lysates were compared, and the effects of two different signal peptide sequences on the synthesis of mature hirudin were determined. The combination of K562 cell lysates and the endogenous wild-type signal peptide sequence was most effective. Cell-free synthesized hirudin showed a considerably higher anti-thrombin activity compared to recombinant hirudin produced in bacterial cells.

Construction of a 3A system from BioBrick parts for expression of recombinant hirudin variants III in Corynebacterium glutamicum.

Standardized parts can be efficiently assembled into novel biological systems using the three antibiotic (3A) system, ensuring the reusability of components and repeatability of experiments. In this study, we created the 3A expression system for easy construction of gene expression cassettes in Corynebacterium glutamicum (C. glutamicum), which was applied to screen combinations of promoters and signal peptides for improved secreted rhv3 production. We first obtained three strong promoters P2252, Podhi, and PyweA from all of promoters, which drive the highest expression of green fluorescent protein (egfp). The three promoters were then assembled with different signal peptides to generate a series of constructs using the 3A expression system developed in this study, from which the highest activity of rhv3 reached 3187.5 ATU/L of PyweA-CspA-rhv3. Further increased production of rhv3 achieved large-scale fermentation using 5-L jar bioreactor, with the highest rhv3 accumulation 1.21 g/L obtained after 40 h of cultivation, which is higher than 0.95 g/L reported in E. coli. To the best of our knowledge, this is the first report of rhv3 secretory expression in C. glutamicum, which could be applied for the production of other recombinant proteins with significant applications.Key points• We have exploited a 3A system for the genetic manipulation in C. glutamicum.• We constructed element libraries for assembling standard sequence in C. glutamicum.• The secreted expression of rhv3 was realized by 3A system in C. glutamicum.