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.

UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: R-hirudin attenuated pulmonary hypertension and thrombosis in APE rats, suggesting its potential as a novel treatment strategy for APE.

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.

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.

Hirudin, an acidic polypeptide secreted by the salivary glands of Hirudo medicinalis (also known as \"Shuizhi\" in traditional Chinese medicine), is the strongest natural specific inhibitor of thrombin found so far. Hirudin has been demonstrated to possess potent anti-thrombotic effect in previous studies. Recently, increasing researches have focused on the anti-thrombotic activity of the derivatives of hirudin, mainly because these derivatives have stronger antithrombotic activity and lower bleeding risk. Additionally, various bioactivities of hirudin have been reported as well, including wound repair effect, anti-fibrosis effect, effect on diabetic complications, anti-tumor effect, anti-hyperuricemia effect, effect on cerebral hemorrhage, and others. Therefore, by collecting and summarizing publications from the recent two decades, the pharmacological activities, pharmacokinetics, novel preparations and derivatives, as well as toxicity of hirudin were systematically reviewed in this paper. In addition, the clinical application, the underlying mechanisms of pharmacological effects, the dose-effect relationship, and the development potential in new drug research of hirudin were discussed on the purpose of providing new ideas for application of hirudin in treating related diseases.

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.

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 5 kDa 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 20 kDa 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.

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.

OBJECTIVE: To investigate the antithrombotic effects of recombinant hirudin and its mechanism.
METHODS: 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.
RESULTS: 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.
CONCLUSIONS: 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.

In this article, the surface of intraocular len material PMMA was first aminated for activation on which some polar groups generated such as C-N, COO(-), -OH, NH3(+), etc. Then the anticoagulant drugs recombinant hirudin (rH) was grafted with amido bonds to look forward to resist the adsorption of nonspecific protein or cells in tear, even the cataract. The detailed analysis and discussion about the grafting quantity, molography, wettability, electric charges, chemical structure, and the dynamic adsorption of protein Fn on the material surface were carried on by the technology of ultraviolet photometric, contact angle, solid Zeta potential, X-ray photoelectron spectroscopy, and quartz crystal microbalance. The surface with a certain amount of rH modification existed more hydrophilic due to the amphiphilic structure than before, on which the protein adsorption was the most unstable. The results indicated that the rH modification improved the resistance of PMMA to nonspecific adsorption of protein Fn to achieve the expectative effect.