Aptameric hirudins as selective and reversible EXosite-ACTive site (EXACT) inhibitors.

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.

Recombinant hirudin suppresses angiogenesis of diffuse large 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.

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.

Exploring a Hirudin variant from nonhematophagous leeches: Unraveling full-length sequence, alternative splicing, function, and potential as a novel anticoagulant polypeptide.

ETHNOPHARMACOLOGICAL RELEVANCE: 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? AIM OF THE STUDY: 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. MATERIALS AND 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. CONCLUSION: 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.

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.

Mechanism of Hirudin-Mediated Inhibition of Proliferation in Ovarian Cancer Cells.

To investigate the inhibitory effect of hirudin on the cell proliferation of human ovarian cancer A2780 cells by preventing thrombin and its underlying molecular mechanism. Cell Counting Kit-8 (CCK-8) method was used to detect the effect of different concentrations of hirudin and thrombin on the cell proliferation of A2780 cells. PAR-1 wild-type overexpression plasmid was constructed utilizing enzyme digestion identification, and it was transferred to A2780 cells. Sequencing and Western blot were used to detect the changes in PAR-1 protein expression. Western blot detection of PKCα protein phosphorylation in A2780 cells was performed. We also implemented quantitative PCR to detect the mRNA expression levels of epithelial-mesenchymal transition (EMT)-related genes, CDH2, Snail, and Vimentin, in A2780 cells. 1 µg/ml hirudin treatment maximally inhibited the promotion of A2780 cell proliferation by thrombin. Hirudin inhibited the binding of thrombin to the N-terminus of PAR-1, hindered PKCα protein phosphorylation in A2780 cells, and downregulated the mRNA expression levels of CDH2, Snail, and Vimentin. In conclusion, hirudin inhibits the cell proliferation of ovarian cancer A2780 cells, and the underlying mechanism may be through downregulating the transcription level of EMT genes, CDH2, Snail, and Vimentin. This study indicates that hirudin may have a therapeutic potential as an anti-cancer agent for ovarian cancer.

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.

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.

Effect of bivalirudin on coagulation in neonatal (cord) and adult human blood in vitro.

INTRODUCTION: Bivalirudin is recommended as an alternative to heparin in cardiac surgery with cardiopulmonary bypass. Although it has been used in infants and children for this indication, there is a paucity of data on the pharmacologic effects of bivalirudin in neonates. Given the immaturity of the hemostatic system in neonates, we hypothesized that coagulation responses to bivalirudin in this population would be different than in adults. METHODS: Blood samples were drawn from placenta-cord units and from healthy adult donors. The study was carried out in two steps. First, bivalirudin was added to cord and adult blood samples at concentrations of 0, 5, 10, 15, and 20 µg/mL. Activated clotting time and thromboelastographic variables were recorded. Next, we used a Chandler loop system to assess the efficacy of bivalirudin in a simple model of cardiopulmonary bypass. The loops were primed with cord or adult blood and were run until thrombus was detected. Plasma bivalirudin concentrations were measured at 1, 15, 30, 45, 60, and 75 min after initiating rotation of the loops using liquid chromatography/mass spectrometry. RESULTS: Bivalirudin elicited a dose-dependent prolongation inhibition of coagulation in both cord and adult blood samples with greater potency in cord blood in comparison to adult blood (activated clotting time: 627 ± 50 vs. 452 ± 22 s at 15 µg/mL bivalirudin, p < .0001). This relative potency was also demonstrated in the Chandler loop system, but interestingly, cord blood appeared to inactivate bivalirudin more rapidly than adult blood with earlier clotting in loops containing cord blood. CONCLUSIONS: This study demonstrates that bivalirudin has greater potency in cord blood in vitro than in adult blood. Plasma degradation appears to proceed more rapidly in cord blood than in adults. Both of these findings should be considered when planning dosing regimens in neonatal patients.

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.

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.

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.

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 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.