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

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

In situ formed depot of elastin-like polypeptide-hirudin fusion protein for long-acting antithrombotic therapy.

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.

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.

Hirudin ameliorates myocardial ischemia-reperfusion injury in a rat model of hemorrhagic shock and resuscitation: roles of NLRP3-signaling pathway.

Severe hemorrhage shock and resuscitation (HSR) has been reported to induce myocardial ischemia-reperfusion injury (MIRI), resulting in a poor prognosis. Hirudin, an effective thrombin inhibitor, can offer protection against MIRI. This study aimed to determine if hirudin administration ameliorates HSR-induced MIRI and the underlying mechanism. A rat model of HSR was established by bleeding rats to a mean arterial blood pressure of 30-35 mmHg for 45 min and then resuscitating them with all the shed blood through the left femoral vein. After HSR, 1 mg/kg of hirudin was administrated immediately. At 24 h after HSR, the cardiac injury was assessed using serum CK-MB, cTnT, hematoxylin-eosin (HE) staining, echocardiography, M1-polarized macrophages, and pyroptosis-associated factors, including cleaved caspase-1, Gasdermin D (GSDMD) N-terminal, IL-1ß, and IL-18 were measured by immunofluorescence and western blot assays. Nigericin, a unique agonist, was utilized to evaluate the responsibilities of NLRP3 signaling. Under the HSR condition, rats exhibited a significant increase in myocardial injury score, an elevation of serum cTnT, CK-MB levels, an aggrandization of M1-polarized macrophages, an upregulation of pyroptosis-associated factors, including cleaved caspase-1, GSDMD N-terminal, IL-1ß, and IL-18, but a significant decrease in left ventricular ejection fraction (EF%) and a reduction of left ventricular fractional shortening (FS%), while hirudin administration partially restored the changes. However, the NLRP3 agonist nigericin reversed the cardioprotective effects of hirudin. We determined the cardioprotective effects of hirudin against HSR-induced MIRI. The mechanism may involve the inhibition of NLRP3-induced pyroptosis.

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.

Effect of Sulfotyrosine and Negatively Charged Amino Acid of Leech-Derived Peptides on Binding and Inhibitory Activity Against Thrombin.

Hirudins, natural sulfo(glyco)proteins, are clinical anticoagulants that directly inhibit thrombin, a key coagulation factor. Their potent thrombin inhibition primarily results from antagonistic interactions with both the catalytic and non-catalytic sites of thrombin. Hirudins often feature sulfate moieties on Tyr residues in their anionic C-terminus region, enabling strong interactions with thrombin exosite-I and effectively blocking its engagement with fibrinogen. Although sulfotyrosines have been identified in various hirudin variants, the precise relationship between sulfotyrosine and the number of negatively charged amino acids within the anionic-rich C-terminus peptide domain for the binding of thrombin has remained elusive. By using Fmoc-SPPS, hirudin dodecapeptides homologous to the C-terminus of hirudin variants from various leech species were successfully synthesized, and the effect of sulfotyrosine and the number of negatively charged amino acids on hirudin-thrombin interactions was investigated. Our findings did not reveal any synergistic effect between an increasing number of sulfotyrosines or negatively charged amino acids and their inhibitory activity on thrombin or fibrinolysis in the assays, despite a higher binding level toward thrombin in the sulfated dodecapeptide Hnip_Hirudin was observed in SPR analysis.

Direct thrombin inhibitors fail to reverse the negative effects of heparin on lung growth and function after murine left pneumonectomy.

Neonates with congenital diaphragmatic hernia (CDH) frequently require cardiopulmonary bypass and systemic anticoagulation. We previously demonstrated that even subtherapeutic heparin impairs lung growth and function in a murine model of compensatory lung growth (CLG). The direct thrombin inhibitors (DTIs) bivalirudin and argatroban preserved growth in this model. Although DTIs are increasingly used for systemic anticoagulation clinically, patients with CDH may still receive heparin. In this experiment, lung endothelial cell proliferation was assessed following treatment with heparin-alone or mixed with increasing concentrations of bivalirudin or argatroban. The effects of subtherapeutic heparin with or without DTIs in the CLG model were also investigated. C57BL/6J mice underwent left pneumonectomy and subcutaneous implantation of osmotic pumps. Pumps were preloaded with normal saline, bivalirudin, or argatroban; treated animals received daily intraperitoneal low-dose heparin. In vitro, heparin-alone decreased endothelial cell proliferation and increased apoptosis. The effect of heparin on proliferation, but not apoptosis, was reversed by the addition of bivalirudin and argatroban. In vivo, low-dose heparin decreased lung volume compared with saline-treated controls. All three groups that received heparin demonstrated decreased lung function on pulmonary function testing and impaired exercise performance on treadmill tolerance testing. These findings correlated with decreases in alveolarization, vascularization, angiogenic signaling, and gene expression in the heparin-exposed groups. Together, these data suggest that bivalirudin and argatroban fail to reverse the inhibitory effects of subtherapeutic heparin on lung growth and function. Clinical studies on the impact of low-dose heparin with DTIs on CDH outcomes are warranted.NEW & NOTEWORTHY Infants with pulmonary hypoplasia frequently require cardiopulmonary bypass and systemic anticoagulation. We investigate the effects of simultaneous exposure to heparin and direct thrombin inhibitors (DTIs) on lung growth and pulmonary function in a murine model of compensatory lung growth (CGL). Our data suggest that DTIs fail to reverse the inhibitory effects of subtherapeutic heparin on lung growth and function. Clinical studies on the impact of heparin with DTIs on clinical outcomes are thus warranted.

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 → ß-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.

Chromogenic and Clot-Based Bivalirudin Assays for Monitoring Anticoagulation.

BACKGROUND: Direct thrombin inhibitors (DTIs) are usually monitored with the activated partial thromboplastin time (aPTT) or activated clotting time (ACT). Both are complex assays with multiple enzymatic steps, and performance may be influenced by physiologic and pathologic factors unrelated to the DTI. Simpler systems, such as clot-based dilute thrombin time (dTT) and chromogenic anti-factor IIa assays, have been developed for monitoring DTIs, but there is limited data on their performance in clinical settings. METHODS: Medical records of patients who received bivalirudin between March 2020 and April 2022 at a single institution were reviewed for demographic data and adverse outcomes. Plasma samples drawn for aPTT testing were analyzed with chromogenic anti-IIa and dTT bivalirudin assays. Results were compared to bivalirudin dosing. RESULTS: Results of aPTT assays from 32 patients were compared with the chromogenic (n = 136) and dTT (n = 120) bivalirudin assays. Correlations between the aPTT and the chromogenic and dTT assays were poor (Spearman coefficients 0.55 and 0.62, respectively). There was a stronger correlation when results of the chromogenic and dTT assays were compared to each other (Spearman coefficient 0.92). When assay results were compared to bivalirudin dose, there were stronger correlations with the chromogenic and dTT assays than with the aPTT (Spearman coefficients 0.51, 0.63 and 0.22, respectively). CONCLUSIONS: There was considerable variation between results of specific bivalirudin assays and the aPTT. While bivalirudin assay results correlated better with administered drug dose, suggesting improving reliability, more studies are needed to determine if there is correlation between testing and clinical outcomes.

Exploiting Chemical Protein Synthesis to Study the Role of Tyrosine Sulfation on Anticoagulants from Hematophagous Organisms.

Tyrosine sulfation is a post-translational modification (PTM) that modulates function by mediating key protein-protein interactions. One of the early proteins shown to possess this PTM was hirudin, produced in the salivary glands of the medicinal leech Hirudo medicinalis, whereby tyrosine sulfation led to a ∼10-fold improvement in α-thrombin inhibitory activity. Outside of this pioneering discovery, the involvement of tyrosine sulfation in modulating the activity of salivary proteins from other hematophagous organisms was unknown. We hypothesized that the intrinsic instability of the tyrosine sulfate functionality, particularly under the acidic conditions used to isolate and analyze peptides and proteins, has led to poor detection during the isolation and/or expression of these molecules.Herein, we summarize our efforts to interrogate the functional role of tyrosine sulfation in the thrombin inhibitory and anticoagulant activity of salivary peptides and proteins from a range of different blood feeding organisms, including leeches, ticks, mosquitoes, and flies. Specifically, we have harnessed synthetic chemistry to efficiently generate homogeneously sulfated peptides and proteins for detailed structure-function studies both in vitro and in vivo.Our studies began with the leech protein hirudin P6 (from Hirudinaria manillensis), which is both sulfated on tyrosine and O-glycosylated at a nearby threonine residue. Synthetically, this was achieved through solid-phase peptide synthesis (SPPS) with a late-stage on-resin sulfation, followed by native chemical ligation and a folding step to generate six differentially modified variants of hirudin P6 to assess the functional interplay between O-glycosylation and tyrosine sulfation. A one-pot, kinetically controlled ligation of three peptide fragments was used to assemble homogeneously sulfoforms of madanin-1 and chimadanin from the tick Haemaphysalis longicornis. Dual tyrosine sulfation at two distinct sites was shown to increase the thrombin inhibitory activity by up to 3 orders of magnitude through a novel interaction with exosite II of thrombin. The diselenide-selenoester ligation developed by our lab provided us with a means to rapidly assemble a library of different sulfated tick anticoagulant proteins: the andersonins, hyalomins, madanin-like proteins, and hemeathrins, thus enabling the generation of key structure-activity data on this family of proteins. We have also confirmed the presence of tyrosine sulfation in the anticoagulant proteins of Anopheles mosquitoes (anophelins) and the Tsetse fly (TTI) via insect expression and mass spectrometric analysis. These molecules were subsequently synthesized and assessed for thrombin inhibitory and anticoagulant activity. Activity was significantly improved by the addition of tyrosine sulfate modifications and led to molecules with potent antithrombotic activity in an in vivo murine thrombosis model.The Account concludes with our most recent work on the design of trivalent hybrids that tandemly occupy the active site and both exosites (I and II) of α-thrombin, with a TTI-anophelin hybrid (Ki = 20 fM against α-thrombin) being one of the most potent protease inhibitors and anticoagulants ever generated. Taken together, this Account highlights the importance of the tyrosine sulfate post-translational modification within salivary proteins from blood feeding organisms for enhancing anticoagulant activity. This work lays the foundation for exploiting native or engineered variants as therapeutic leads for thrombotic disorders in the future.

Targeting endothelial coagulation signaling ameliorates liver obstructive cholestasis and dysfunctional angiogenesis.

Cholestatic fibrogenesis is a pathobiological process in which cumulative injury to the bile ducts coincides with progressive liver fibrosis. The pathobiologic mechanisms underlying fibrogenesis and disease progression remain poorly understood. Currently, there is no effective treatment for liver fibrosis. In this study, we discovered that components of the coagulation cascade were associated with the advanced progression of obstructive cholestasis, and anticoagulant therapy could improve liver cholestasis-induced fibrosis. In a mouse model of common bile duct ligation (BDL), which mimics cholestatic liver injury, RNA sequencing analysis revealed an increased expression of coagulation factors in endothelial cells. Pharmacological targeting of the coagulation signaling by hirudin, an anticoagulatory antagonist of thrombin, ameliorated obstructive cholestasis, and attenuated liver fibrosis symptoms. Hirudin attenuated fibrosis-associated angiogenesis, endothelial-to-mesenchymal transition (EndMT), and tissue hypoxia and reduced liver inflammation after BDL. Furthermore, hirudin suppressed YAP (Yes-associated protein) signaling and its downstream effectors in vascular endothelial cells, which are considered with profibrotic characteristics. In conclusion, we demonstrated that pharmacological targeting of coagulation signaling by hirudin has the potential to alleviate liver obstructive cholestasis and fibrosis.

Hirudin inhibits glioma growth through mTOR-regulated autophagy.

Glioma is the most common primary malignant brain tumour, and survival is poor. Hirudin has anticancer pharmacological effects through suppression of glioma cell progression, but the molecular target and mechanism are poorly understood. In this study, we observed that hirudin dose- and time-dependently inhibited glioma invasion, migration and proliferation. Mechanistically, hirudin activated LC3-II but not Caspase-3 to induce the autophagic death of glioma cells by decreasing the phosphorylation of mTOR and its downstream substrates ULK1, P70S6K and 4EBP1. Furthermore, hirudin inhibited glioma growth and induced changes in autophagy in cell-derived xenograft (CDX) nude mice, with a decrease in mTOR activity and activation of LC3-II. Collectively, our results highlight a new anticancer mechanism of hirudin in which hirudin-induced inhibition of glioma progression through autophagy activation is likely achieved by inhibition of the mTOR signalling pathway, thus providing a molecular basis for hirudin as a potential and effective clinical drug for glioma therapy.

Hirudin versus citrate as an anticoagulant for ROTEM platelet whole blood impedance aggregometry in thrombocytopenic patients.

Citrate is widely used as an anticoagulant for platelet function tests (PFTs). Due to an intrinsic inhibitory effect of citrate on platelet function, hirudin is used as an alternative. However, studies comparing the effect of these anticoagulants on rotational thromboelastometry (ROTEM) platelet whole blood impedance aggregometry in thrombocytopenic patients are scant. Cross-sectional study was done in 105 patients who entered the critical phase of Dengue hemorrhagic fever with plasma leakage and severe thrombocytopenia (<100 × 109/L). Samples were collected on two consecutive days and considered as a combined data set for analysis, out of which 200 have been included in the data analysis. Platelet count was used from routine full blood count. ROTEM platelet used TRAPTEM assay, which was performed with 3.2% sodium citrate and 525 ATU/ml hirudin anticoagulated blood. Means of all the TRAPTEM parameters were significantly higher in hirudin, compared to citrate samples (p < .05). Significantly higher overall platelet aggregation was observed in hirudinized samples with a significant mean difference (p < .05) compared to citrate in each quartile of platelet count. Higher platelet aggregation was observed with hirudin compared to citrate in ROTEM platelet whole blood impedance aggregometry in thrombocytopenic patients elaborating the importance of using hirudin anticoagulation in PFTs, particularly in patients with severe thrombocytopenia.

Citrate is the most commonly used anticoagulant for coagulation studies including rotational thromboelastometry (ROTEM).Hirudin is an alternative option to be used as an anticoagulant for PFTs because of the inhibitory effect of citrate on platelet function.One study (Nissen et al. (2020)) reported higher precision and platelet aggregation with hirudinized blood of healthy individuals, over citrate using ROTEM platelet.However, none of the studies were performed in patients in actual clinical context.We evaluated the potential benefit of using hirudin anticoagulated blood over citrate in thrombocytopenic patients due to Dengue hemorrhagic fever using ROTEM platelet.We observed higher platelet aggregation with hirudin compared to citrate suggesting the importance of using hirudin anticoagulation in PFTs, particularly in patients with severe thrombocytopenia.

Assays to Monitor Bivalirudin.

Bivalirudin (Angiomax, Angiox) is a parenteral direct thrombin inhibitor (DTI) that is used for patients with heparin-induced thrombocytopenia (HIT), where heparin cannot be used due to the risk of thrombosis. Bivalirudin is also licensed for use in cardiology procedures (e.g., percutaneous transluminal coronary angioplasty; PTCA). Bivalirudin is a synthetic analogue of hirudin found in the saliva of the medicinal leech and has a relatively short half-life of ~25 min. Several assays can be used to monitor bivalirudin; these include the activated partial thromboplastin time (APTT), activated clotting time (ACT), ecarin clotting time (ECT), an ecarin-based chromogenic assay, thrombin time (TT), the dilute TT, and the prothrombinase-induced clotting time (PiCT). Drug concentrations can also be measured using liquid chromatography tandem mass spectrometry (LC/MS) and clotting or chromogenic-based assays with specific drug calibrators and controls.

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.

Intracerebral hirudin injection alleviates cognitive impairment and oxidative stress and promotes hippocampal neurogenesis in rats subjected to cerebral ischemia.

Cerebral ischemia starts with cerebral blood flow interruption that causes severely limited oxygen and glucose supply, eliciting a cascade of pathological events, such as excitotoxicity, oxidative stress, calcium dysregulation, and inflammatory response, which could ultimately result in neuronal death. Hirudin has beneficial effects in ischemic stroke and possesses antioxidant and anti-inflammatory properties. Therefore, we investigated the biological functions of hirudin and its related mechanisms in cerebral ischemia. The ischemia-like conditions were induced by transient middle cerebral artery occlusion (MCAO). To investigate hirudin roles, intracerebroventricular injection of 10 U hirudin was given to the rats. Cognitive and motor functions were examined by beam walking and Morris water maze tests. 2,3,5-triphenyl tetrazolium chloride-stained brain sections were used to measure infarct volume. Oxidative stress was determined by assessment of oxidative stress markers. The proliferated cells were labeled by BrdU and Nestin double staining. Western blotting was performed to measure protein levels. Hirudin administration improved cognitive and motor deficits post-ischemia. Hirudin reduced brain infarction and neurological damage in MCAO-subjected rats. Hirudin alleviated oxidative stress and enhanced neurogenesis in ischemic rats. Hirudin facilitated the promotion of phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and serine-threonine kinase. In sum, hirudin alleviates cognitive deficits by attenuating oxidative stress and promoting hippocampal neurogenesis through the regulation of ERK1/2 and serine-threonine kinase in MCAO-subjected rats.

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.

Hirudin ameliorates diabetic nephropathy by inhibiting Gsdmd-mediated pyroptosis.

Our group previously reported that hirudin ameliorated diabetic nephropathy (DN) in streptozotocin (STZ)-injected rats, but the mechanism remained largely unknown. Therefore, we further explored its possible mechanism. We subcutaneously injected 5 U hirudin into STZ-induced WT mice or Gasdermin D (Gsdmd)-/- (KO) mice daily for 12 weeks, respectively, and evaluated their kidney injury. Next, glomerular endothelial cells (GECs), renal tubular epithelial cells (RTECs), and bone-marrow-derived macrophages (BMDMs) were isolated from WT mice and treated with hirudin in the presence of high glucose/lipopolysaccharides and ATP to measure the release of interleukin-18 and interleukin-1ß. Kidney injury induced by STZ injection was significantly ameliorated by hirudin through inhibiting Gsdmd-mediated pyroptosis in the mice, not Caspase 1-mediated apoptosis. Meanwhile, hirudin also suppressed pyroptosis in primary GECs, RTECs, and BMDMs in vitro. Moreover, the deletion of Gsdmd reduced pyroptosis and kidney injury both in vivo and in vitro. We also found that hirudin regulated the expression of Gsdmd by inhibiting interferon regulatory factor 2 (Irf2). Hirudin ameliorated Gsdmd-mediated pyroptosis by inhibiting irf2, leading to the improvement of kidney injury. Therefore, hirudin might serve as a potential therapeutic strategy to treat DN.

Monitoring Direct Thrombin Inhibitors With Calibrated Diluted Thrombin Time vs Activated Partial Thromboplastin Time in Pediatric Patients.

OBJECTIVES: Activated partial thromboplastin time (aPTT) is the primary test used to monitor intravenous (IV) direct thrombin inhibitors (DTIs) but has many limitations. The plasma diluted thrombin time (dTT) has shown better correlation with DTI levels than aPTT. This study compared dose-response curves for dTT and aPTT in pediatric patients receiving argatroban and bivalirudin. METHODS: A retrospective review of pediatric patients treated with argatroban (n = 45) or bivalirudin (n = 14) monitored with dTT and aPTT. RESULTS: The dTT assay was calibrated to report DTI concentrations in µg/mL for argatroban and bivalirudin with good analytic sensitivity and specificity. The dTT was fivefold more likely to show a stable dose-response slope than the aPTT (P < .0002; odds ratio, 4.9). For patients in whom both dTT and aPTT showed a significant correlation between dose and assay results, dTT had a higher average correlation factor compared with aPTT (P = .007). Argatroban dose-response slopes showed more inter- and intrapatient variation than bivalirudin (dose-response slope coefficient of variation, 132% vs 52%). CONCLUSIONS: The dTT assay was more likely to show a stable dose response and have a stronger correlation with DTI dose than aPTT. Argatroban shows more variation in dose response than bivalirudin.