Identification and partial characterization of a novel serpin from Eudiplozoon nipponicum (Monogenea, Polyopisthocotylea)

Background: Serpins are a superfamily of serine peptidase inhibitors that participate in the regulation of many physiological and cell peptidase-mediated processes in all organisms (e.g. in blood clotting, complement activation, fibrinolysis, inflammation, and programmed cell death). It was postulated that in the blood-feeding members of the monogenean family Diplozoidae, serpins could play an important role in the prevention of thrombus formation, activation of complement, inflammation in the host, and/or in the endogenous regulation of protein degradation. Results: In silico analysis showed that the DNA and primary protein structures of serpin from Eudiplozoon nipponicum (EnSerp1) are similar to other members of the serpin superfamily. The inhibitory potential of EnSerp1 on four physiologically-relevant serine peptidases (trypsin, factor Xa, kallikrein, and plasmin) was demonstrated and its presence in the worm’s excretory-secretory products (ESPs) was confirmed. Conclusion: EnSerp1 influences the activity of peptidases that play a role in blood coagulation, fibrinolysis, and complement activation. This inhibitory potential, together with the serpin’s presence in ESPs, suggests that it is likely involved in host-parasite interactions and could be one of the molecules involved in the control of feeding and prevention of inflammatory responses.

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Major acid endopeptidases of the blood-feeding monogenean Eudiplozoon nipponicum (Heteronchoinea: Diplozoidae)

Parasitology ◽

10.1017/s0031182015001808 ◽

2016 ◽

Vol 143 (4) ◽

pp. 494-506 ◽

Cited By ~ 10

Author(s):

LUCIE JEDLIČKOVÁ ◽

HANA DVOŘÁKOVÁ ◽

MARTIN KAŠNÝ ◽

JANA ILGOVÁ ◽

DAVID POTĚŠIL ◽

...

Keyword(s):

Soluble Protein ◽

Cathepsin D ◽

Cathepsin L ◽

Blood Feeding ◽

Host Immune System ◽

Significant Activity ◽

Molecular Tools ◽

Secretory Products ◽

Major Acid ◽

Eudiplozoon Nipponicum

SUMMARYIn parasitic flatworms, acid endopeptidases are involved in crucial processes, including digestion, invasion, interactions with the host immune system, etc. In haematophagous monogeneans, however, no solid information has been available about the occurrence of these enzymes. Here we aimed to identify major cysteine and aspartic endopeptidase activities in Eudiplozoon nipponicum, an invasive haematophagous parasite of common carp. Employing biochemical, proteomic and molecular tools, we found that cysteine peptidase activities prevailed in soluble protein extracts and excretory/secretory products (ESP) of E. nipponicum; the major part was cathepsin L-like in nature supplemented with cathepsin B-like activity. Significant activity of the aspartic cathepsin D also occurred in soluble protein extracts. The degradation of haemoglobin in the presence of ESP and worm protein extracts was completely inhibited by a combination of cysteine and aspartic peptidase inhibitors, and diminished by particular cathepsin L, B and D inhibitors. Mass spectrometry revealed several tryptic peptides in ESP matching to two translated sequences of cathepsin L genes, which were amplified from cDNA of E. nipponicum and bioinformatically annotated. The dominance of cysteine peptidases of cathepsin L type in E. nipponicum resembles the situation in, e.g. fasciolid trematodes.

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Excretory–secretory products of helminth parasites: effects on host immune responses

Parasitology ◽

10.1017/s0031182000086017 ◽

1988 ◽

Vol 96 (S1) ◽

pp. S123-S166 ◽

Cited By ~ 136

Author(s):

M. W. Lightowlers ◽

M. D. Rickard

Keyword(s):

Immune Responses ◽

Inflammatory Responses ◽

Helminth Parasites ◽

Host Immune Responses ◽

Parasitic Helminths ◽

Protective Antigens ◽

Helminth Infections ◽

Secretory Products ◽

Antigen Presenting ◽

Host Parasite

SUMMARYParasitic helminths excrete or secrete (ES) a variety of molecules into their mammalian hosts. The effects of these ES products on the host's immune responses are reviewed. Investigations into the source of antigenic or immunoregulatory ES products have identified the cuticular and tegumental surfaces of some nematodes and trematodes respectively as being important sources of ES products; other ES molecules are released through specialized excretory or secretory organs. It is proposed that the active shedding of surface antigens may serve as an important source of parasite antigens available to the immune system in a form in which they can be taken up and processed by antigen-presenting dendritic cells, macro-phages and certain B cells for presentation to T helper cells. The ES products of nematodes, trematodes and cestodes contribute to immune evasion strategies of the parasites through mechanisms including shedding of surface-bound ligands and cells, alteration of lymphocyte, macrophage and granulocyte functions and modulation of complement and other host inflammatory responses. Immunopathology may be induced by ES products as in the development of granulomas around entrapped schistosome eggs. In some host – parasite systems ES antigens may induce host-protective immune responses and this source of protective antigens has been utilized in the successful vaccination against helminth infections, particularly against infection with trichurid nematodes and the metacestode stage of cestode parasites. The use of ES antigens in immunodiagnosis of helminth infection is also briefly discussed.

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Oxidative Stress and Atherosclerosis: Its Relationship to Growth Factors, Thrombus Formation and Therapeutic Approaches

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615550 ◽

1999 ◽

Vol 82 (S 01) ◽

pp. 32-37 ◽

Cited By ~ 22

Author(s):

Karlheinz Peter ◽

Wolfgang Kübler ◽

Johannes Ruef ◽

Thomas K. Nordt ◽

Marschall S. Runge ◽

...

Keyword(s):

Oxidative Stress ◽

Growth Factors ◽

Vessel Wall ◽

Inflammatory Responses ◽

Plaque Rupture ◽

Thrombus Formation ◽

Vascular Lesion ◽

Coagulation System ◽

Therapeutic Approaches ◽

Causative Relationship

SummaryThe initiating event of atherogenesis is thought to be an injury to the vessel wall resulting in endothelial dysfunction. This is followed by key features of atherosclerotic plaque formation such as inflammatory responses, cell proliferation and remodeling of the vasculature, finally leading to vascular lesion formation, plaque rupture, thrombosis and tissue infarction. A causative relationship exists between these events and oxidative stress in the vessel wall. Besides leukocytes, vascular cells are a potent source of oxygen-derived free radicals. Oxidants exert mitogenic effects that are partially mediated through generation of growth factors. Mitogens, on the other hand, are potent stimulators of oxidant generation, indicating a putative self-perpetuating mechanism of atherogenesis. Oxidants influence the balance of the coagulation system towards platelet aggregation and thrombus formation. Therapeutic approaches by means of antioxidants are promising in both experimental and clinical designs. However, additional clinical trials are necessary to assess the role of antioxidants in cardiovascular disease.

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Heparin is more effective than apixaban in inhibiting in vitro contact activated coagulation

European Heart Journal ◽

10.1093/ehjci/ehaa946.3776 ◽

2020 ◽

Vol 41 (Supplement_2) ◽

Author(s):

M.M Engelen ◽

C Van Laer ◽

M Jacquemin ◽

C Vandenbriele ◽

K Peerlinck ◽

...

Keyword(s):

Thrombin Generation ◽

Heart Valves ◽

Thrombus Formation ◽

Oral Anticoagulants ◽

Direct Oral Anticoagulants ◽

Factor Xa ◽

Coagulation Factor ◽

Mechanical Heart Valves ◽

Contact Activation

Abstract Introduction Contact of blood with artificial surfaces such as mechanical support devices, catheters, and mechanical heart valves activates the contact activation (CA) pathway of coagulation. Furthermore, recent animal data and clinical studies suggest a more important contribution of CA in pathological thrombus formation in other cardiovascular diseases. Direct oral anticoagulants (DOACs) are recommended as first-line treatment in most patients who require long-term anticoagulation. However, because DOACs directly inhibit a single downstream coagulation factor (thrombin (fXIIa) or factor Xa (fXa)), it has been suggested that their efficacy could be reduced in the presence of strong activation of the CA pathway as compared to anticoagulants that target multiple, more upstream located coagulation factors. Purpose To compare the efficacy of a DOAC (apixaban) and heparin to suppress thrombin generation in the presence of strong CA pathway activation. Methods Pooled platelet-poor plasma was spiked with either apixaban (dissolved in DMSO and PBS) or unfractionated heparin to achieve therapeutic plasma levels. SynthASil, a commercially available mixture of phospholipids and silica, was used to stimulate the CA pathway in two different dilutions (1–80 and 5–80). Downstream coagulation was accessed by Thrombin Generation Test using Thrombinoscope by Stago and associated Thrombin Calibrator (activity 640 nM). The endogenous thrombin potential (area under the thrombin generation curve; ETP), peak thrombin generation (PTG), time to peak (ttPeak) and time to start (ttStart) were accessed. Results With decreasing concentrations of apixaban, stimulation with the lower dose SynthASil reveals an increasing ETP and PTG. As expected, ttPeak and ttStart decreased. Even supratherapeutic levels of apixaban (i.e. 1120 ng/mL) could not inhibit thrombin from being generated, in striking contrast with UFH where no thrombin was formed. Using a five times higher dose of SynthASil showed comparable ETP for all concentrations of apixaban, allocated around the control value. PTG, however, slightly increased with decreasing concentrations of apixaban. ttPeak and ttStart slightly decreased. Except for the subtherapeutic UFH concentration of 0,114 IU/mL, no thrombin was generated with UFH. Conclusion UFH is more effective in inhibiting downstream thrombin generation compared to apixaban as a response to activation of the CA pathway in vitro. These findings could help explain why direct inhibitors were not able to show non-inferiority in patients with mechanical heart valves and support the development of specific CA pathway inhibitors for patients with conditions that activate the CA pathway. Thrombin generation curves Funding Acknowledgement Type of funding source: None

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Recognition Pattern of the Fasciola hepatica Excretome/Secretome during the Course of an Experimental Infection in Sheep by 2D Immunoproteomics

Pathogens ◽

10.3390/pathogens10060725 ◽

2021 ◽

Vol 10 (6) ◽

pp. 725

Author(s):

David Becerro-Recio ◽

Javier González-Miguel ◽

Alberto Ucero ◽

Javier Sotillo ◽

Álvaro Martínez-Moreno ◽

...

Keyword(s):

Experimental Infection ◽

Fasciola Hepatica ◽

Cathepsin L ◽

Helminth Parasites ◽

Glutathione S Transferase ◽

Serum Samples ◽

Secretory Products ◽

Immunogenic Proteins ◽

Host Parasite ◽

First Time

Excretory/secretory products released by helminth parasites have been widely studied for their diagnostic utility, immunomodulatory properties, as well as for their use as vaccines. Due to their location at the host/parasite interface, the characterization of parasite secretions is important to unravel the molecular interactions governing the relationships between helminth parasites and their hosts. In this study, the excretory/secretory products from adult worms of the trematode Fasciola hepatica (FhES) were employed in a combination of two-dimensional electrophoresis, immunoblot and mass spectrometry, to analyze the immune response elicited in sheep during the course of an experimental infection. Ten different immunogenic proteins from FhES recognized by serum samples from infected sheep at 4, 8, and/or 12 weeks post-infection were identified. Among these, different isoforms of cathepsin L and B, peroxiredoxin, calmodulin, or glutathione S-transferase were recognized from the beginning to the end of the experimental infection, suggesting their potential role as immunomodulatory antigens. Furthermore, four FhES proteins (C2H2-type domain-containing protein, ferritin, superoxide dismutase, and globin-3) were identified for the first time as non-immunogenic proteins. These results may help to further understand host/parasite relationships in fasciolosis, and to identify potential diagnostic molecules and drug target candidates of F. hepatica.

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Biochemical and Pharmacological Properties of SANORG 34006, a Potent and Long-Acting Synthetic Pentasaccharide

Blood ◽

10.1182/blood.v91.11.4197 ◽

1998 ◽

Vol 91 (11) ◽

pp. 4197-4205 ◽

Cited By ~ 151

Author(s):

J.M. Herbert ◽

J.P. Hérault ◽

A. Bernat ◽

R.G.M. van Amsterdam ◽

J.C. Lormeau ◽

...

Keyword(s):

Thrombin Generation ◽

Ex Vivo ◽

Thrombus Formation ◽

Factor Xa ◽

Therapeutic Index ◽

Inhibitory Effect ◽

Experimental Models ◽

Treatment And Prevention ◽

Long Acting

Abstract SANORG 34006 is a new sulfated pentasaccharide obtained by chemical synthesis. It is an analog of the “synthetic pentasaccharide” (SR 90107/ ORG 31540) which represents the antithrombin (AT) binding site of heparin. SANORG 34006 showed a higher affinity to human AT than SR 90107/ORG 31540 (kd = 1.4 ± 0.3 v 48 ± 11 nmol/L), and it is a potent and selective catalyst of the inhibitory effect of AT on factor Xa (1,240 ± 15 anti–factor Xa U/mg v850 ± 27 anti-factor Xa U/mg for SR 90107/ORG 31540). In vitro, SANORG 34006 inhibited thrombin generation occurring via both the extrinsic and intrinsic pathway. After intravenous (IV) or subcutaneous (SC) administration to rabbits, SANORG 34006 displayed a long-lasting anti–factor Xa activity and inhibition of thrombin generation (TG) ex vivo. SANORG 34006 was slowly eliminated after IV or SC administration to rats, rabbits, and baboons, showed exceptionally long half-lives (between 9.2 hours in rats and 61.9 hours in baboons), and revealed an SC bioavailability near 100%. SANORG 34006 displayed antithrombotic activity by virtue of its potentiation of the anti–factor Xa activity of AT. It strongly inhibited thrombus formation in experimental models of thromboplastin/stasis-induced venous thrombosis in rats (IV) and rabbits (SC) (ED50values = 40.0 ± 3.4 and 105.0 ± 9.4 nmol/kg, respectively). The duration of its antithrombotic effects closely paralleled the ex vivo anti–factor Xa activity. SANORG 34006 enhanced rt-PA–induced thrombolysis and inhibited accretion of125I-fibrinogen onto a preformed thrombus in the rabbit jugular vein suggesting that concomitant use of SANORG 34006 during rt-PA therapy might be helpful in facilitating thrombolysis and preventing fibrin accretion onto the thrombus under lysis. Contrary to standard heparin, SANORG 34006 did not enhance bleeding in a rabbit ear incision model at a dose that equals 10 times the antithrombotic ED50 in this species and, therefore, exhibited a favorable therapeutic index. We suggest that SANORG 34006 is a promising compound in the treatment and prevention of various thrombotic diseases.

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RPR120844, a Novel, Specific Inhibitor of Coagulation Factor Xa Inhibits Venous Thrombosis in the Rabbit

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614434 ◽

1999 ◽

Vol 81 (01) ◽

pp. 157-160 ◽

Cited By ~ 14

Author(s):

Ross Bentley ◽

Suzanne Morgan ◽

Karen Brown ◽

Valeria Chu ◽

Richard Ewing ◽

...

Keyword(s):

Jugular Vein ◽

Drug Effect ◽

Ex Vivo ◽

Thrombus Formation ◽

Specific Inhibitor ◽

Factor Xa ◽

Coagulation Factor ◽

Antithrombotic Activity ◽

Fxa Inhibitor

SummaryThe in vivo antithrombotic activity of RPR120844, a novel synthetic coagulation factor Xa (fXa) inhibitor (Ki = 7 nM), was assessed by its ability to inhibit thrombus formation in a damaged segment of the rabbit jugular vein. Intravenous dose-response studies were performed and thrombus mass (TM), activated partial thromboplastin time (APTT), prothrombin time (PT), inhibition of ex vivo fXa activity and plasma drug levels (PDL) were determined. TM, measured at the end of a 50 min infusion, was significantly reduced (p <0.05 vs saline-treated animals) by RPR120844 at 30 and 100 μg/kg/min. At doses of 10, 30 and 100 μg/kg/min, APTT was prolonged by 2.1, 4.2 and 6.1-fold, and PT was prolonged by 1.4, 2.2 and 3.5-fold, respectively. PDL were determined by measuring anti-fXa activity using an amidolytic assay. Peak PDL were 0.8 ± 0.3, 1.5 ± 0.9 and 2.4 ± 0.6 μM, respectively. The drug effect was reversible with APTT, PT and PDL returning toward pretreatment values 30 min after termination of treatment. The results suggest that RPR120844, or similar compounds, may provide an efficacious, yet easily reversible, means of inhibiting thrombus formation.

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Abstract 153: IL-10 Accelerates Re-Endothelialization and Inhibits Post-injury Intimal Hyperplasia following Carotid Artery Denudation by Attenuating TNF-alpha-induced Endothelial Cell Dysfunction

Circulation Research ◽

10.1161/res.115.suppl_1.153 ◽

2014 ◽

Vol 115 (suppl_1) ◽

Author(s):

Suresh K Verma ◽

Prasanna Krishnamurthy ◽

Alexander R Mackie ◽

Erin E Vaughan ◽

Mohsin Khan ◽

...

Keyword(s):

Endothelial Cell ◽

Inflammatory Responses ◽

Thrombus Formation ◽

Specific Effect ◽

Mononuclear Phagocytes ◽

Tnf Alpha ◽

Post Injury ◽

Knock Out ◽

Cytokines And Chemokines

The association of inflammation with atherosclerosis and restenosis is now fairly well established. Restenosis, a persistent complication of percutaneous vascular interventions, is thought to be a complex response to injury, which includes early thrombus formation, neointimal growth and acute inflammation. Mononuclear phagocytes are likely participants in the host response to vascular injury, via the secretion of cytokines and chemokines, including TNF-alpha (TNF). Others and we have previously shown that IL-10 inhibits TNF and other inflammatory mediators produced in response to cardiovascular injuries. The specific effect of IL-10 on endothelial cell (EC) biology is not well elucidated. Here we report that in a mouse model of carotid denudation, IL-10 knock-out mice (IL10KO) displayed significantly delayed ReEndothelialization and enhanced neointimal growth compared to their WT counterparts. Exogenous treatment of recombinant IL-10 dramatically blunted the inflammatory cell infiltration and neointimal thickening while significantly accelerating the recovery of the injured endothelium both WT and IL10KO mice. In vitro, IL10 co-treatment reversed TNF-mediated growth arrest, EC cell cycle inhibition, EC-monocyte adhesion and EC apoptosis. At signaling level, IL-10 reduced TNF-induced activation of JNK MAP kinase while simultaneously activating PI3K/Akt pathway. Because IL-10 function and signaling are important components for control of inflammatory responses, these results may provide insights necessary to develop strategies for modulating vascular repair and other accelerated arteriopathies, including transplant vasculopathy and vein graft hyperplasia.

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Complement C5 inhibition protects against hemolytic anemia and acute kidney injury in anthrax peptidoglycan-induced sepsis in baboons

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2104347118 ◽

2021 ◽

Vol 118 (37) ◽

pp. e2104347118

Author(s):

Ravi Shankar Keshari ◽

Narcis Ioan Popescu ◽

Robert Silasi ◽

Girija Regmi ◽

Cristina Lupu ◽

...

Keyword(s):

Acute Kidney Injury ◽

Hemolytic Anemia ◽

Complement Activation ◽

Inflammatory Responses ◽

Atypical Hemolytic Uremic Syndrome ◽

Kidney Injury ◽

Multiple Organ ◽

Immune Recognition ◽

Gram Positive ◽

Terminal Complement

Late-stage anthrax infections are characterized by dysregulated immune responses and hematogenous spread of Bacillus anthracis, leading to extreme bacteremia, sepsis, multiple organ failure, and, ultimately, death. Despite the bacterium being nonhemolytic, some fulminant anthrax patients develop a secondary atypical hemolytic uremic syndrome (aHUS) through unknown mechanisms. We recapitulated the pathology in baboons challenged with cell wall peptidoglycan (PGN), a polymeric, pathogen-associated molecular pattern responsible for the hemostatic dysregulation in anthrax sepsis. Similar to aHUS anthrax patients, PGN induces an initial hematocrit elevation followed by progressive hemolytic anemia and associated renal failure. Etiologically, PGN induces erythrolysis through direct excessive activation of all three complement pathways. Blunting terminal complement activation with a C5 neutralizing peptide prevented the progressive deposition of membrane attack complexes on red blood cells (RBC) and subsequent intravascular hemolysis, heme cytotoxicity, and acute kidney injury. Importantly, C5 neutralization did not prevent immune recognition of PGN and shifted the systemic inflammatory responses, consistent with improved survival in sepsis. Whereas PGN-induced hemostatic dysregulation was unchanged, C5 inhibition augmented fibrinolysis and improved the thromboischemic resolution. Overall, our study identifies PGN-driven complement activation as the pathologic mechanism underlying hemolytic anemia in anthrax and likely other gram-positive infections in which PGN is abundantly represented. Neutralization of terminal complement reactions reduces the hemolytic uremic pathology induced by PGN and could alleviate heme cytotoxicity and its associated kidney failure in gram-positive infections.

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Molecular Docking, Computational, and Antithrombotic Studies of Novel 1,3,4-Oxadiazole Derivatives

International Journal of Molecular Sciences ◽

10.3390/ijms19113606 ◽

2018 ◽

Vol 19 (11) ◽

pp. 3606 ◽

Cited By ~ 3

Author(s):

Majda Batool ◽

Affifa Tajammal ◽

Firdous Farhat ◽

Francis Verpoort ◽

Zafar Khattak ◽

...

Keyword(s):

Factor Xa ◽

Clot Lysis ◽

Clotting Time ◽

Reference Drug ◽

Docking Score ◽

High Affinity ◽

Oxadiazole Derivatives ◽

Inhibitory Potential

A new series of 1,3,4-oxadiazoles derivatives was synthesized, characterized, and evaluated for their in vitro and in vivo anti-thrombotic activity. Compounds (3a–3i) exhibited significant clot lysis with respect to reference drug streptokinase (30,000 IU), and enhanced clotting time (CT) values (130–342 s) than heparin (110 s). High affinity towards 1NFY with greater docking score was observed for the compounds (3a, 3i, 3e, 3d, and 3h) than the control ligand RPR200095. In addition, impressive inhibitory potential against factor Xa (F-Xa) was observed with higher docking scores (5612–6270) with Atomic Contact Energy (ACE) values (−189.68 to −352.28 kcal/mol) than the control ligand RPR200095 (Docking score 5192; ACE −197.81 kcal/mol). In vitro, in vivo, and in silico results proposed that these newly synthesized compounds might be used as anticoagulant agents.

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