Unravelling the Antifibrinolytic Mechanism of Action of the 1,2,3-Triazole Derivatives.

A new family of antifibrinolytic drugs has been recently discovered, combining a triazole moiety, an oxadiazolone, and a terminal amine. Two of the molecules of this family have shown activity that is greater than or similar to that of tranexamic acid (TXA), the current antifibrinolytic gold standard, which has been associated with several side effects and whose use is limited in patients with renal impairment. The aim of this work was to thoroughly examine the mechanism of action of the two ideal candidates of the 1,2,3-triazole family and compare them with TXA, to identify an antifibrinolytic alternative active at lower dosages. Specifically, the antifibrinolytic activity of the two compounds (1 and 5) and TXA was assessed in fibrinolytic isolated systems and in whole blood. Results revealed that despite having an activity pathway comparable to that of TXA, both compounds showed greater activity in blood. These differences could be attributed to a more stable ligand-target binding to the pocket of plasminogen for compounds 1 and 5, as suggested by molecular dynamic simulations. This work presents further evidence of the antifibrinolytic activity of the two best candidates of the 1,2,3-triazole family and paves the way for incorporating these molecules as new antifibrinolytic therapies.

Exploration of Novel Plasmin Inhibitor from ß-Lactoglobulin for Enhancing the Storage Stability of UHT Milk.

Plasmin-induced protein hydrolysis significantly compromises the stability of ultrahigh-temperature (UHT) milk. ß-Lactoglobulin (ß-Lg) was observed to inhibit plasmin activity, suggesting that there were active sites as plasmin inhibitors in ß-Lg. Herein, plasmin inhibitory peptides were explored from ß-Lg using experimental and computational techniques. The results revealed that increased denaturation of ß-Lg enhanced its affinity for plasmin, leading to a stronger inhibition of plasmin activity. Molecular dynamics simulations indicated that electrostatic and van der Waals forces were the primary binding forces in the ß-Lg/plasmin complex. Denatured ß-Lg increased hydrogen bonding and reduced the binding energy with plasmin. The sites of plasmin bound to ß-Lg were His624, Asp667, and Ser762. Four plasmin inhibitory peptides, QTMKGLDI, EKTKIPAV, TDYKKYLL, and CLVRTPEV, were identified from ß-Lg based on binding sites. These peptides effectively inhibited plasmin activity and enhanced the UHT milk stability. This study provided new insights into the development of novel plasmin inhibitors to improve the stability of UHT milk.

Prolonged joint cavity retention of tranexamic acid achieved by a solid-in-oil-in-gel system: A preliminary study.

Tranexamic acid (TXA) is an anti-fibrinolysis agent widely used in postoperative blood loss management. As a highly water-soluble drug, TXA is suffering from rapid clearance from the action site, therefore, large amount of drug is required when administered either by intravenously or topically. In this study, a TXA preparation with prolonged action site residence was designed using the nano-micro strategy. TXA nanoparticles were dispersed in oil by emulsification followed by lyophilization to give a solid-in-oil suspension, which was used as the oil phase for the preparation of TXA-loaded solid-in-oil-in-water (TXA@S/O/W) system. The particle size of TXA in oil was 207.4 ± 13.50 nm, and the particle size of TXA@S/O/W was 40.5 µm. The emulsion-in-gel system (TXA@S/O/G) was prepared by dispersing TXA@S/O/W in water solution of PLGA-b-PEG-b-PLGA (PPP). And its gelling temperature was determined to be 26.6 ℃ by a rheometer. Sustained drug release was achieved by TXA@S/O/G with 72.85 ± 7.52 % of TXA released at 120 h. Formulation retention at the joint cavity was studied by live imaging, and the fluorescent signals dropped gradually during one week. Drug escape from the injection site via drainage and absorption was investigated by a self-made device and plasma TXA concentration determination, respectively. TXA@S/O/G showed the least drug drainage during test, while more than 70 % of drug was drained in TXA@S/O/W group and TXA solution group. Besides, low yet steady plasma TXA concentration (less than 400 ng/mL) was found after injecting TXA@S/O/G into rat knees at a dosage of 2.5 mg/kg, which was much lower than those of TXA dissolved in PPP gel or TXA solution. In conclusion, sustained drug release as well as prolonged action site retention were simultaneously achieved by the designed TXA@S/O/G system. More importantly, due to the steady plasma concentration, this strategy could be further applied to other highly water-soluble drugs with needs on sustained plasma exposure.

Tranexamic acid loaded in a physically crosslinked trilaminate dressing for local hemorrhage control: Preparation, characterization, and in-vivo assessment using two different animal models.

This work aimed at formulating a trilaminate dressing loaded with tranexamic acid. It consisted of a layer of 3 % sodium hyaluronate to initiate hemostasis. It was followed by a mixed porous layer of 5 % polyvinyl alcohol and 2 % kappa-carrageenan. This layer acted as a drug reservoir that controlled its release. The third layer was 5 % ethyl cellulose backing layer for unidirectional release of tranexamic acid towards the wound. The 3 layers were physically crosslinked by hydrogen bonding as confirmed by Infrared spectroscopy. Swelling and release studies were performed, and results proposed that increasing number of layers decreased swelling properties and sustained release of tranexamic acid for 8 h. In vitro blood coagulation study was performed using human blood and showed that the dressing significantly decreased coagulation time by 70.5 % compared to the negative control. In vivo hemostatic activity was evaluated using tail amputation model in Wistar rats. Statistical analysis showed the dressing could stop bleeding in a punctured artery of the rat tail faster than the negative control by 59 %. Cranial bone defect model in New Zealand rabbits was performed to check for bone hemostasis and showed significant decrease in the hemostatic time by 80 % compared to the control.

Synthesis and Structural Characterization of Macrocyclic Plasmin Inhibitors.

Two series of macrocyclic plasmin inhibitors with a C-terminal benzylamine group were synthesized. The substitution of the N-terminal phenylsulfonyl group of a previously described inhibitor provided two analogues with sub-nanomolar inhibition constants. Both compounds possess a high selectivity against all other tested trypsin-like serine proteases. Furthermore, a new approach was used to selectively introduce asymmetric linker segments. Two of these compounds inhibit plasmin with Ki values close to 2 nM. For the first time, four crystal structures of these macrocyclic inhibitors could be determined in complex with a Ser195Ala microplasmin mutant. The macrocyclic core segment of the inhibitors binds to the open active site of plasmin without any steric hindrance. This binding mode is incompatible with other trypsin-like serine proteases containing a sterically demanding 99-hairpin loop. The crystal structures obtained experimentally explain the excellent selectivity of this inhibitor type as previously hypothesized.

The Importance of 6-Aminohexanoic Acid as a Hydrophobic, Flexible Structural Element.

6-aminohexanoic acid is an ω-amino acid with a hydrophobic, flexible structure. Although the ω-amino acid in question is mainly used clinically as an antifibrinolytic drug, other applications are also interesting and important. This synthetic lysine derivative, without an α-amino group, plays a significant role in chemical synthesis of modified peptides and in the polyamide synthetic fibers (nylon) industry. It is also often used as a linker in various biologically active structures. This review concentrates on the role of 6-aminohexanoic acid in the structure of various molecules.

The role of SERPIN citrullination in thrombosis.

Aberrant protein citrullination is associated with many pathologies; however, the specific effects of this modification remain unknown. We have previously demonstrated that serine protease inhibitors (SERPINs) are highly citrullinated in rheumatoid arthritis (RA) patients. These citrullinated SERPINs include antithrombin, antiplasmin, and t-PAI, which regulate the coagulation and fibrinolysis cascades. Notably, citrullination eliminates their inhibitory activity. Here, we demonstrate that citrullination of antithrombin and t-PAI impairs their binding to their cognate proteases. By contrast, citrullination converts antiplasmin into a substrate. We recapitulate the effects of SERPIN citrullination using in vitro plasma clotting and fibrinolysis assays. Moreover, we show that citrullinated antithrombin and antiplasmin are increased and decreased in a deep vein thrombosis (DVT) model, accounting for how SERPIN citrullination shifts the equilibrium toward thrombus formation. These data provide a direct link between increased citrullination and the risk of thrombosis in autoimmunity and indicate that aberrant SERPIN citrullination promotes pathological thrombus formation.

Novel IMB16-4 Compound Loaded into Silica Nanoparticles Exhibits Enhanced Oral Bioavailability and Increased Anti-Liver Fibrosis In Vitro.

BACKGROUND: Liver fibrosis, as a common and refractory disease, is challenging to treat due to the lack of effective agents worldwide. Recently, we have developed a novel compound, N-(3,4,5-trichlorophenyl)-2(3-nitrobenzenesulfonamide) benzamide (IMB16-4), which is expected to have good potential effects against liver fibrosis. However, IMB16-4 is water-insoluble and has very low bioavailability. METHODS: Mesoporous silica nanoparticles (MSNs) were selected as drug carriers for the purpose of increasing the dissolution of IMB16-4, as well as improving its oral bioavailability and inhibiting liver fibrosis. The physical states of IMB16-4 and IMB16-4-MSNs were investigated using nitrogen adsorption, thermogravimetric analysis (TGA), HPLC, UV-Vis, X-ray diffraction (XRD) and differential scanning calorimetry (DSC). RESULTS: The results show that MSNs enhanced the dissolution rate of IMB16-4 significantly. IMB16-4-MSNs reduced cytotoxicity at high concentrations of IMB16-4 on human hepatic stellate cells LX-2 cells and improved oral bioavailability up to 530% compared with raw IMB16-4 on Sprague-Dawley (SD) rats. In addition, IMB16-4-MSNs repressed hepatic fibrogenesis by decreasing the expression of hepatic fibrogenic markers, including α-smooth muscle actin (α-SMA), transforming growth factor-beta (TGF-ß1) and matrix metalloproteinase-2 (MMP2) in LX-2 cells. CONCLUSIONS: These results provided powerful information on the use of IMB16-4-MSNs for the treatment of liver fibrosis in the future.

Ifenprodil Stereoisomers: Synthesis, Absolute Configuration, and Correlation with Biological Activity.

Ifenprodil (1) is a potent GluN2B-selective N-methyl-d-aspartate (NMDA) receptor antagonist that is used as a cerebral vasodilator and has been examined in clinical trials for the treatment of drug addiction, idiopathic pulmonary fibrosis, and COVID-19. To correlate biological data with configuration, all four ifenprodil stereoisomers were prepared by diastereoselective reduction and subsequent separation of enantiomers by chiral HPLC. The absolute configuration of ifenprodil stereoisomers was determined by X-ray crystal structure analysis of (1R,2S)-1a and (1S,2S)-1d. GluN2B affinity, ion channel inhibitory activity, and selectivity over α, σ, and 5-HT receptors were evaluated. (1R,2R)-Ifenprodil ((1R,2R)-1c) showed the highest affinity toward GluN2B-NMDA receptors (Ki = 5.8 nM) and high inhibition of ion flux in two-electrode voltage clamp experiments (IC50 = 223 nM). Whereas the configuration did not influence considerably the GluN2B-NMDA receptor binding, (1R)-configuration is crucial for elevated inhibitory activity. (1R,2R)-Configured ifenprodil (1R,2R)-1c exhibited high selectivity for GluN2B-NMDA receptors over adrenergic, serotonergic, and σ1 receptors.

Nasal Powder Formulation of Tranexamic Acid and Hyaluronic Acid for the Treatment of Epistaxis.

PURPOSE: The aim of this study was to develop a nasal powder formulation of the antifibrinolytic drug, tranexamic acid (TXA), in combination with the wound-healing agent hyaluronic acid (HA) for the local treatment of epistaxis (nose bleeding). METHODS: Formulations of TXA alone and with different concentrations of HA were freeze-dried and characterised according to their physicochemical properties. Aerosol performance was assessed to ensure nasal deposition with minimal lung deposition. Nasal epithelial cells were used to assess cytotoxicity, transport across the nasal epithelium, antioxidant, wound-healing and anti-inflammatory properties of all formulations. RESULTS: Formulations containing TXA and HA were produced and found to be mostly deposited in the nasal cavity (more than 90%). Formulation of TXA + 0.3%HA showed wound reduction of 29.3% when assessed in ALI culture. At this concentration, formulations also reduced ROS production in RPMI 2650, and IL-8 production in primary nasal epithelial cells. Furthermore, for formulations containing HA, the higher viscosity may lead to larger residence time in the nasal cavity. CONCLUSIONS: Combination of TXA with HA shows promising results for the treatment of nasal epistaxis.

Field-based rational design of p300 histone acetyltransferase inhibitor and systematic evaluation as an anti-fibrotic agent.

(E)-3-(3-(4-((3-Carbamoylbenzyl)oxy)-3-iodo-5-methoxyphenyl) acryloyl)benzamide (A6) was found to be a potent p300 inhibitor (IC50 = 870 nM) showing a similar binding mode to that of acetyl-CoA, a p300 substrate, and effective anti-fibrotic activity in both TGF-ß1-stimulated lung fibroblast cells and bleomycin-induced in vivo lung fibrosis mice.

A pirfenidone loaded spray dressing based on lyotropic liquid crystals for deep partial thickness burn treatment: healing promotion and scar prophylaxis.

A deep partial thickness (DPT) burn injury refers to burn damage involving the epidermis and major dermis, whose prognosis depends greatly on wound management. Lack of effective management can lead to an elongated healing process and aggravated scar formation, which can severely disturb patients, both physically and mentally. A dressing with good water absorption and moderate mechanical properties is crucial for healing promotion, and the prevention of scar formation is highly desirable. In this project, a hyaluronic acid combined lyotropic liquid crystal based spray dressing (HLCSD) loaded with the anti-fibrotic drug pirfenidone (PFD) has been designed. HLCSD is expected to achieve the goals of both wound healing promotion and scar prophylaxis. Its water absorption capacity, mechanical properties, drug release behavior and phase transition are fully evaluated. HLCSD possesses low viscosity for spray administration and high levels of water absorption for exudate absorption. An in situ gel composed of self-assembled lattice nanostructures provides excellent mechanical protection to promote the healing process and steady PFD release to exert a scar prophylaxis effect. The benefit of HLCSD on the wound healing rate is verified in vivo. In the DPT burn wound model we established, HLCSD also exhibits excellent healing promotion effects, and PFD-loaded HLCSD shows scar prophylaxis effects and displays an ideal prognosis, with skin as smooth as healthy skin. The healing promotion of HLCSD is considered to be related to the alleviation of inflammation, with an obviously shortened inflammation phase, with contributions from water management, mechanical protection and anti-inflammation by HLCSD. The scar prophylaxis of PFD-loaded HLCSD is proven to be related to the regulation of collagen synthesis and degradation, involving key cytokines like TGF-ß and MMP-1. Taken together, the PFD-loaded HLCSD with healing promotion and scar prophylaxis offers significant promise as a spray dressing for DPT burn injuries.

The antifibrotic effect and mechanism of a novel tyrosine kinase inhibitor, ZSP1603, in preclinical models of pulmonary fibrosis.

OBJECTIVE: This study aimed to investigate the efficacy and molecular mechanisms of ZSP1603 as a novel anti-fibrotic compound. MATERIALS AND METHODS: The unilateral left pulmonary fibrosis model was established in the Sprague Dawley (SD) rats. The bilateral pulmonary fibrosis model was established in the C57BL/6J mice. The therapeutic treatment regimen began after the induction of pulmonary fibrosis. The preventive treatment regimen began on the first day of bleomycin administration. Animals were randomly divided into the sham, model, Nintedanib, and ZSP1603 treatment groups. Haematoxylin and eosin (H&E) and Masson's trichrome staining were performed to evaluate pulmonary injury, inflammation, and fibrosis. Cell Counting Kit-8 (CCK-8) assay and Western blot were used to investigate the effects and mechanisms of ZSP1603 on the proliferation of primary human pulmonary fibroblasts (pHPFs). The messenger ribonucleic acid (mRNA) expression of transforming growth factor (TGF)-ß1, tissue inhibitor of metalloproteinase 1 (TIMP-1), and collagen 1A1 (COL1A1) in pHPFs was detected by quantitative Real Time-Polymerase Chain Reaction (PCR). RESULTS: ZSP1603 inhibited the proliferation of pHPFs in vitro by blocking the platelet-derived growth factor receptor-ß (PDGF-Rß) and extracellular signal-regulated kinase (ERK) signalling pathway. ZSP1603 also inhibited the differentiation of pHPFs by reducing the expression of TGF-ß1, TIMP-1, and COL1A1. ZSP1603 significantly attenuated pulmonary injury, inflammation, and fibrosis in vivo in four independent animal studies of pulmonary fibrosis. CONCLUSIONS: ZSP1603 is an effective anti-fibrotic compound with clear mechanisms.

Fibrinolysis Inhibitors: Potential Drugs for the Treatment and Prevention of Bleeding.

Hyperfibrinolytic situations can lead to life-threatening bleeding, especially during cardiac surgery. The approved antifibrinolytic agents such as tranexamic acid, ε-aminocaproic acid, 4-aminomethylbenzoic acid, and aprotinin were developed in the 1960s without the structural insight of their respective targets. Crystal structures of the main antifibrinolytic targets, the lysine binding sites on plasminogen's kringle domains, and plasmin's serine protease domain greatly contributed to the structure-based drug design of novel inhibitor classes. Two series of ligands targeting the lysine binding sites have been recently described, which are more potent than the most-widely used antifibrinolytic agent, tranexamic acid. Furthermore, four types of promising active site inhibitors of plasmin have been developed: tranexamic acid conjugates targeting the S1 pocket and primed sites, substrate-analogue linear homopiperidylalanine-containing 4-amidinobenzylamide derivatives, macrocyclic inhibitors addressing nonprimed binding regions, and bicyclic 14-mer SFTI-1 analogues blocking both, primed and nonprimed binding sites of plasmin. Furthermore, several allosteric plasmin inhibitors based on heparin mimetics have been developed.

In vitro screening for compounds from Hypericum longistylum with anti-pulmonary fibrosis activity.

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with a poor prognosis and limited therapies, and transforming growth factor-ß1 (TGF-ß1) plays a central role in the pathogenesis of IPF. Here, we aimed to investigate the chemical constituents and biological activities of Hypericum longistylum and detect whether the isolated compounds inhibit the TGF-ß1/Smad3 signaling pathway to identify candidate compounds for the treatment of pulmonary fibrosis. Fifteen compounds (1-15) were isolated from H. longistylum and their structures were elucidated on the basis of spectroscopic analyses. An in vitro MTT assay was used to test the effect of these fifteen compounds on fibroblast cytotoxicity and vitality. Furthermore, their bioactivities were screened using a TGF-ß1/Smad3 pathway luciferase reporter in vitro. MTT screening found that compounds 1-15 had no deleterious effects on normal mouse lung fibroblasts and no significant inhibition of vitality. Luciferase assay showed that compounds 14 and 15 could significantly inhibit the TGF-ß1/Smad3 pathway with the inhibition rates of 67.92% and 93.10%, respectively. Both compounds can be used as lead compounds for structural modification and optimization to obtain more drug candidates for the treatment of pulmonary fibrosis.

Binding characteristics of polyphenols as milk plasmin inhibitors.

BACKGROUND: The potential use of polyphenols to improve the functional characteristics of dairy products has gained much attention. However, the effects of the polyphenols on naturally occurring enzymes in milk have not been studied extensively. Excess plasmin activity in dairy products might result in several quality defects. The objective of this study was to assess the ability of polyphenols to inhibit plasmin in milk using a molecular and kinetic approach. RESULTS: Epicatechin gallate (ECG), epigallocatechin gallate (EGCG), quercetin (QUER), and myricetin (MYR) caused a significant decrease in plasmin activity by 60, 86, 65, and 90%, respectively. The inhibition rates were alleviated in the presence of milk proteins. EGCG, QUER, and MYR, exhibited noncompetitive inhibition against plasmin, whereas ECG caused a mixed-type inhibition. A decrease in the random structure of plasmin upon the complex formation with ECG, EGCG, QUER, and MYR was found. The other phenolics that were evaluated did not cause any significant changes in plasmin conformation. The observed inhibitory phenolic-plasmin interactions were dominated by H-bonds and electrostatic attractions. Green tea extract (GTE) rich in catechins also inhibited plasmin activity in the milk. CONCLUSION: Significant changes in the secondary structure of plasmin upon binding of ECG, EGCG, QUER, and MYR led to diminished plasmin activity both in the absence and presence of milk proteins. These flavonoids with promising plasmin inhibitory potential could be used in new dairy formulations leading to controlled undesired consequences of plasmin activity. © 2019 Society of Chemical Industry.

Increased fibrinolysis-induced bradykinin formation in hereditary angioedema confirmed using stored plasma and biotechnological inhibitors.

OBJECTIVE: We recently investigated the pathways of immunoreactive bradykinin (iBK) formation in fresh blood of normal volunteers and of patients with hereditary angioedema due to C1-esterase inhibitor deficiency (HAE-1/-2). Herein, we adapted the techniques to small volumes (200 µl) of previously frozen citrated plasma and further analyzed the mechanisms of iBK formation with additional biotechnological inhibitors. RESULTS: Measurable iBK formation was observed under stimulation with tissue kallikrein (KLK-1, 10 nM), the particulate material Kontact-APTT (concentration reduced to 2% v/v) or recombinant tissue plasminogen activator (tPA, 169 nM), with little background in unstimulated plasma incubated for up to 2 h. Plasma samples from HAE-1/-2 patients responded earlier to tPA than those from controls, as previously reported with whole blood. Lanadelumab inhibited iBK formation induced by Kontact-APTT and tPA. A highly specific plasmin inhibitor, DX-1000, abolished tPA-induced iBK formation in plasma but had no effect against Kontact-APTT, confirming the role of fibrinolysis in tPA-induced kinin formation. The anti-lanadelumab neutralizing antibody M293-D02 reversed the inhibitory effects of lanadelumab. Frozen plasma is a suitable material for measuring iBK formation kinetics, with possible applications such as investigating the effect of rare disease states on the kallikrein-kinin system and monitoring the effect of HAE prophylactic treatments.

Topical tranexamic acid inhibits fibrinolysis more effectively when formulated with self-propelling particles.

BACKGROUND: Endogenous fibrinolytic activation contributes to coagulopathy and mortality after trauma. Administering tranexamic acid (TXA), an antifibrinolytic agent, is one strategy to reduce bleeding; however, it must be given soon after injury to be effective and minimize adverse effects. Administering TXA topically to a wound site would decrease the time to treatment and could enable both local and systemic delivery if a suitable formulation existed to deliver the drug deep into wounds adequately. OBJECTIVES: To determine whether self-propelling particles could increase the efficacy of TXA. METHODS: Using previously developed self-propelling particles, which consist of calcium carbonate and generate CO2 gas, TXA was formulated to disperse in blood and wounds. The antifibrinolytic properties were assessed in vitro and in a murine tail bleeding assay. Self-propelled TXA was also tested in a swine model of junctional hemorrhage consisting of femoral arteriotomy without compression. RESULTS: Self-propelled TXA was more effective than non-propelled formulations in stabilizing clots from lysis in vitro and reducing blood loss in mice. It was well tolerated when administered subcutaneously in mice up to 300 to 1000 mg/kg. When it was incorporated in gauze, four of six pigs treated after a femoral arteriotomy and without compression survived, and systemic concentrations of TXA reached approximately 6 mg/L within the first hour. CONCLUSIONS: A formulation of TXA that disperses the drug in blood and wounds was effective in several models. It may have several advantages, including supporting local clot stabilization, reducing blood loss from wounds, and providing systemic delivery of TXA. This approach could both improve and simplify prehospital trauma care for penetrating injury.

A novel Kunitz protein with proposed dual function from Eudiplozoon nipponicum (Monogenea) impairs haemostasis and action of complement in vitro.

Serine peptidases are involved in many physiological processes including digestion, haemostasis and complement cascade. Parasites regulate activities of host serine peptidases to their own benefit, employing various inhibitors, many of which belong to the Kunitz-type protein family. In this study, we confirmed the presence of potential anticoagulants in protein extracts of the haematophagous monogenean Eudiplozoon nipponicum which parasitizes the common carp. We then focused on a Kunitz protein (EnKT1) discovered in the E. nipponicum transcriptome, which structurally resembles textilinin-1, an antihemorrhagic snake venom factor from Pseudonaja textilis. The protein was recombinantly expressed, purified and biochemically characterised. The recombinant EnKT1 did inhibit in vitro activity of Factor Xa of the coagulation cascade, but exhibited a higher activity against plasmin and plasma kallikrein, which participate in fibrinolysis, production of kinins, and complement activation. Anti-coagulation properties of EnKT1 based on the inhibition of Factor Xa were confirmed by thromboelastography, but no effect on fibrinolysis was observed. Moreover, we discovered that EnKT1 significantly impairs the function of fish complement, possibly by inhibiting plasmin or Factor Xa which can act as a C3 and C5 convertase. We localised Enkt1 transcripts and protein within haematin digestive cells of the parasite by RNA in situ hybridisation and immunohistochemistry, respectively. Based on these results, we suggest that the secretory Kunitz protein of E. nipponicum has a dual function. In particular, it impairs both haemostasis and complement activation in vitro, and thus might facilitate digestion of a host's blood and protect a parasite's gastrodermis from damage by the complement. This study presents, to our knowledge, the first characterisation of a Kunitz protein from monogeneans and the first example of a parasite Kunitz inhibitor that impairs the function of the complement.