BACKGROUND: Diffuse 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.
METHODS: We 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.
RESULTS: We 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.
CONCLUSIONS: rH inhibits macrophage polarisation towards the M2 type and PAR-1 regulates polarisation, proliferation, migration, invasion, and angiogenesis of DLBCL-associated macrophages.

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.

The use of medicinal leeches in clinical therapy has been employed for a long time, as it was originally recognized for exerting antithrombin effects. These effects were due to the ability of the leech to continuously suck blood while attached to human skin. According to Chinese Pharmacopoei, leeches used in traditional Chinese medicine mainly consist of Whitmania pigra Whitman, Hirudo nipponia Whitman, and Whitmania acranulata, but the latter two species are relatively scarce. The main constituents of leeches are protein and peptide macromolecules. They can be categorized into two categories based on their pharmacological effects. One group consists of active ingredients that directly target the coagulation system, such as hirudin, heparin, and histamine, which are widely known. The other group comprises protease inhibitor components like Decorsin and Hementin. Among these, hirudin secreted by the salivary glands of the leech is the most potent thrombin inhibitor and served as the sole remedy for preventing blood clotting until the discovery of heparin. Additionally, leeches play a significant role in various traditional Chinese medicine formulations. In recent decades, medicinal leeches have been applied in fields including anti-inflammatory treatment, cardiovascular disease management, antitumor treatment, and many other medical conditions. In this review, we present a comprehensive overview of the historical journey and medicinal applications of leeches in various medical conditions, emphasizing their pharmaceutical significance within traditional Chinese medicine. This review offers valuable insights for exploring additional therapeutic opportunities involving the use of leeches in various diseases and elucidating their underlying mechanisms for future research.

Vascular transplantation is a common treatment for Cardiovascular disease (CVD). However, the mismatch of mechanical, structural, or microenvironmental properties of materials limits the clinical application. Therefore, the functional construction of artificial vessels or other blood contact materials remains an urgent challenge. In this paper, the composite nanofibers of polycaprolactone (PCL) with dopamine and polyethylenimine (PEI) coating are first prepared, which are further self-assembled by anticoagulant hirudin (rH) and antimicrobial peptide (AMP) of HHC36 through layer-by-layer (LBL) method. The results of FTIR and XPS analysis show that hirudin and AMP are successfully loaded on PEI-PDA/PCL nanofibers and the hydrophilicity is improved. They also show good mechanical properties that the ultimate tensile strength and elongation at break are better than natural blood vessels. The antibacterial results show that the antibacterial effect is still 93% against E. coli on the fifth day because of the stable and continuous release of HHC36 and rH. The performance of anticoagulant activity also exhibited the same results, which APTT is even 9.7s longer in the experimental group than the control group on the fifth day. The novel materials would be effectively solve the formation of thrombosis around artificial blood vessel grafts and the treatment of inflammation.

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.

BACKGROUND: 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?
OBJECTIVE: 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.
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.
CONCLUSIONS: 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.

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.

OBJECTIVE: Hirudin, a potent anticoagulant, is used in traditional Chinese medicine (TCM) to treat thrombotic conditions and prevent postoperative thrombosis. Coagulation-related vascular complications are a common cause of perforator flaps failure. This study explores hirudin\'s potential to enhance flap growth by mitigating coagulation-related issues.
METHODS: Patients were divided into GroupⅠ(hirudin group) and GroupⅡ(control). Laboratory tests covered red blood cell count (RBC), hematocrit (HCT), platelet count (PLT), monocyte count (MONO), prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB), and D-Dimer. Clinical parameters, including capillary refill time (CRT), flap swelling, and survival status, were evaluated. Animal experiments used Sprague-Dawley (SD) rats to establish random skin flaps. The experimental side received hirudin injection, while the control side received saline. Flaps were photographed to calculate survival rate, and CD31 immunohistochemical (IHC) analysis was performed to calculate microvessel density (MVD).
RESULTS: The study, with 29 patients, found significant CRT differences between groups on postoperative days 2 and 6 (p = 0.027; p = 0.019), favoring GroupⅠ. Swelling severity varied significantly over time; GroupⅡhad more pronounced swelling. GroupⅠshowed superior flap growth with fewer complications, statistically significant (p = 0.033). Specific lab indicators (MONO, PT, and FIB) were significant at certain times. In animal experiments, the experimental side consistently had higher flap survival and slightly increased CD31 expression at various times, with higher MVD on days 2 and 6.
CONCLUSIONS: Hirudin enhances flap survival through diverse mechanisms, supporting its role as a complementary approach in perforator flap surgeries.

Chronic kidney disease (CKD) is a common public health concern. The global burden of CKD is increasing due to the high morbidity and mortality associated with it, indicating the shortcomings of therapeutic drugs at present. Renal fibrosis is the common pathology of CKD, which is characterized by glomerulosclerosis, renal tubular atrophy, and renal interstitial fibrosis. Natural hirudin is an active ingredient extracted from Hirudo medicinalis, which has been found to be the strongest natural specific inhibitor of thrombin. Evidence based on pharmacological data has shown that hirudin has important protective effects in CKD against diabetic nephrology, nephrotic syndrome, and renal interstitial fibrosis. The mechanisms of hirudin in treating CKD are mainly related to inhibiting the inflammatory response, preventing apoptosis of intrinsic renal cells, and inhibiting the interactions between thrombin and protease-activated receptors. In this review, we summarize the function and beneficial properties of hirudin for the treatment of CKD, and its underlying mechanisms.

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.