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.

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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.

Potent and selective inhibition of the structurally homologous proteases of coagulation poses challenges for drug development. Hematophagous organisms frequently accomplish this by fashioning peptide inhibitors combining exosite and active site binding motifs. Inspired by this biological strategy, we create several EXACT inhibitors targeting thrombin and factor Xa de novo by linking EXosite-binding aptamers with small molecule ACTive site inhibitors. The aptamer component within the EXACT inhibitor (1) synergizes with and enhances the potency of small-molecule active site inhibitors by many hundred-fold (2) can redirect an active site inhibitor\'s selectivity towards a different protease, and (3) enable efficient reversal of inhibition by an antidote that disrupts bivalent binding. One EXACT inhibitor, HD22-7A-DAB, demonstrates extraordinary anticoagulation activity, exhibiting great potential as a potent, rapid onset anticoagulant to support cardiovascular surgeries. Using this generalizable molecular engineering strategy, selective, potent, and rapidly reversible EXACT inhibitors can be created against many enzymes through simple oligonucleotide conjugation for numerous research and therapeutic applications.

OBJECTIVE: The activated partial thromboplastin time (aPTT) is the most frequently used monitoring assay for bivalirudin in children and young adults on mechanical circulatory support including ventricular assist devices (VADs) and extracorporeal membrane oxygenation (ECMO). However, intrinsic variability of the aPTT complicates management and risks bleeding or thrombotic complications. We evaluated the utility and reliability of a bivalirudin-calibrated dilute thrombin time (Bival dTT) assay for bivalirudin monitoring in this population.
METHODS: Retrospective analysis of clinical data (including aPTT, dilute thrombin time [dTT]) and results of residual plasma samples from VAD patients were assessed in two drug-calibrated experimental assays. One assay (Bival dTT) was validated for clinical use in VAD patients, and subsequently used by clinicians in ECMO patients. Pearson correlation and simple linear regression were used to determine R2 correlation coefficients between the different laboratory parameters using Statistical Package for Social Sciences (Armonk, NY).
METHODS: ICUs at Cincinnati Children\'s Hospital Medical Center.
METHODS: Children on VAD or ECMO support anticoagulated with bivalirudin.
METHODS: None.
RESULTS: One hundred fifteen plasma samples from 11 VAD patients were analyzed. Both drug-calibrated experimental assays (anti-IIa and Bival dTT) showed excellent correlation with each other ( R2 = 0.94) and with the dTT ( R2 = 0.87), but poor correlation with aPTT ( R2 = 0.1). Bival dTT was selected for validation in VAD patients. Subsequently, clinically ordered results (105) from 11 ECMO patients demonstrated excellent correlation between the Bival dTT and the standard dTT ( R2 = 0.86) but very poor correlation with aPTT ( R2 = 0.004).
CONCLUSIONS: APTT is unreliable and correlates poorly with bivalirudin\'s anticoagulant effect in ECMO and VAD patients. A drug-calibrated Bival dTT offers superior reliability and opportunity to standardize results across institutions. Additional studies are needed to determine an appropriate therapeutic range and correlation with clinical outcomes.

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.

OBJECTIVE: The efficacy and safety of bivalirudin when used concurrently with glycoprotein IIb/IIIa inhibitors (GPI) is uncertain. In this systematic review and meta-analysis, we aimed to evaluate the efficacy and safety of bivalirudin versus heparin in patients with ST-segment elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention (PCI) and to explore the impact of differential use (greater and balanced) of GPI.
METHODS: Online databases were queried from inception to March 2023 to identify eight randomized controlled trials (n = 22,483) for inclusion. The primary outcomes included all-cause mortality, major bleeding, major adverse cardiovascular events (MACE), and net adverse clinical events (NACE). Secondary efficacy endpoints included cardiac death, reinfarction, stent thrombosis (ST), and stroke. Data were pooled using a random-effects model to derive risk ratios (RRs) and 95% confidence intervals (CIs).
RESULTS: When compared to heparin, bivalirudin was associated with a significant reduction in all-cause mortality (RR 0.83; 95% CI 0.72-0.97; P = 0.02), major bleeding (RR 0.73; 95% CI 0.57-0.93; P = 0.01), cardiac death (RR 0.79; 95% CI 0.66-0.94; P = 0.01), and NACE (RR 0.80; 95% CI 0.72-0.89; P < 0.0001). However, while the bivalirudin arm showed an increased likelihood of ST in the greater GPI subgroup (RR 1.70; 95% CI 1.13-2.56; P = 0.01), it was associated with a decreased likelihood of ST in the balanced GPI subgroup (RR 0.40; 95% CI 0.24-0.65; P = 0.0003).
CONCLUSIONS: Overall, our findings suggest that bivalirudin may be a more efficacious intervention than heparin for reducing certain adverse events in patients with STEMI undergoing primary PCI.

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.