Joannsin, a novel Kunitz-type FXa inhibitor from the venom of Prospirobolus joannsi

SummaryThe repugnatorial glands of millipedes release various defensive chemical secretions. Although varieties of such defensive secretions have been studied, none of them is protein or peptide. Herein, a novel factor Xa (FXa) inhibitor named joannsin was identified and characterised from repugnatorial glands of Prospirobolus joannsi. Joannsin is composed of 72 amino acid residues including six cysteines, which form three intra-molecular disulfide bridges. It is a member of Kunitz-type protease inhibitor family, members of which are also found in the secretory glands of other arthropods. Recombinant joannsin exhibited remarkable inhibitory activity against trypsin and FXa with a Ki of 182.7 ± 14.6 and 29.5 ± 4.7 nM, respectively. Joannsin showed strong anti-thrombosis functions in vitro and in vivo. Joannsin is the first peptide component in millipede repugnatorial glands to be identified and is a potential candidate and/or template for the development of anti-thrombotic agents. These results also indicated that there is Kunitz-type protease inhibitor toxin in millipede repugnatorial glands as in other arthropods secretory glands.

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PIVL, a snake venom Kunitz-type serine protease inhibitor, inhibits in vitro and in vivo angiogenesis

Microvascular Research ◽

10.1016/j.mvr.2014.08.006 ◽

2014 ◽

Vol 95 ◽

pp. 149-156 ◽

Cited By ~ 14

Author(s):

Maram Morjen ◽

Stéphane Honoré ◽

Amine Bazaa ◽

Zaineb Abdelkafi-Koubaa ◽

Ameneallah Ellafi ◽

...

Keyword(s):

Protease Inhibitor ◽

Serine Protease ◽

Snake Venom ◽

Serine Protease Inhibitor ◽

Kunitz Type

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Impact of Antithrombin Deficiency on Efficacies of DU-176b, a Novel Orally Active Direct Factor Xa Inhibitor, and Antithrombin Dependent Anticoagulants, Fondaparinux and Heparin.

Blood ◽

10.1182/blood.v106.11.1874.1874 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1874-1874 ◽

Cited By ~ 1

Author(s):

Toshio Fukuda ◽

Yuko Honda ◽

Chikako Matsumoto ◽

Nobutoshi Sugiyama ◽

Tadashi Matsushita ◽

...

Keyword(s):

Thrombus Formation ◽

Factor Xa ◽

In Vitro Study ◽

Antithrombotic Effects ◽

At Deficiency ◽

Fxa Inhibitor ◽

Orally Active ◽

Relative Potencies

Abstract Antithrombin (AT) is a major physiological inhibitor of coagulation factors, primarily inhibiting thrombin and factor Xa (FXa). Binding of heparin and its related pentasaccharides, fondaparinux, to AT dramatically accelerates inhibition of thrombin and FXa. Entire AT-dependency of heparins may result in decreased anticoagulant effects in patients with inherited or acquired AT deficiencies. Objectives: We have developed an orally active direct (i.e. AT-independent) FXa inhibitor, DU-176b. The objectives of this study were to examine the anticoagulant and antithrombotic effects of DU-176b, fondaparinux, and heparin in heterozygous AT deficient (AT+/−) mice (Refs 1, 2), and to determine the impact of AT deficiency on the efficacies of these anticoagulants. Methods: [In vitro study] Plasma obtained from wild type (AT+/+, C57BL/6J) and AT+/− mice were subjected to measurement of levels of AT antigen and activity. The anticoagulant effects on prothrombin time (PT) and activated partial thromboplastin time (APTT) was measured and the drug concentrations were calculated required to double the clotting time (CT2). [In vivo study] Male AT+/+ and AT+/− mice were fasted over night. Thrombosis was induced in the inferior vena cava by applying filter paper (1 x 5 mm) presoaked in 15% FeCl3 for 10 min. Thrombus was removed 60 min after FeCl3 treatment and its protein content was assessed by Bradford method. DU-176b was orally administered 60 min before, fondaparinux was given s.c. 30 min before, and heparin was injected into the jugular vein 3 min before thrombus induction. Relative potencies of antithrombotic effects in AT+/− mice to those in AT+/+ mice were analyzed by parallel line assay. Results: [In vitro study] Plasma levels of AT antigen and activity in AT+/− mice were deceased to 40% compared with AT+/+ plasma. PT-CT2 of DU-176b was 0.72 μM in AT+/+ plasma and 0.74 μM in AT+/− plasma, respectively, indicating that anticoagulant activity of the direct FXa inhibitor was not affected by heterozygous AT deficiency. APTT-CT2 of fondaparinux and heparin in AT+/+ plasma was 3.8 μM and 14 mU/mL, respectively, whereas APTT-CT2 in AT+/− plasma was 9.2 μM and 20 mU/mL, respectively. Therefore, anticoagulant activities of such AT-dependent inhibitors were attenuated in AT+/− plasma. [In vivo study] All three anticoagulants inhibited venous thrombus formation of AT+/+ mice in dose-dependent manners. In AT+/− mice, the antithrombotic effects of fondaparinux and heparin were less potent than those in AT+/+ mice. In contrast, DU-176b prevented thrombus formation equipotently in both mice. Relative potencies of DU-176b, fondaparinux and heparin were 0.84, 0.40, and 0.70, respectively. Conclusion: DU-176b exerts a comparable antithrombotic effect even in individuals with low plasma AT antigens and activities. Thus, DU-176b may be prioritized over AT-dependent agents for use at the fixed dose in patients with lower plasma AT concentrations.

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Kunitz type protease inhibitor EgKI-1 from the canine tapeworm Echinococcus granulosus as a promising anti-cancer therapeutic

10.1101/357590 ◽

2018 ◽

Author(s):

Shiwanthi L Ranasinghe ◽

Glen M Boyle ◽

Katja Fischer ◽

Jeremy Potriquet ◽

Jason P Mulvenna ◽

...

Keyword(s):

Cell Growth ◽

Protease Inhibitor ◽

Hydatid Cyst ◽

Echinococcus Granulosus ◽

Cancer Cell ◽

Dependent Manner ◽

Anti Cancer ◽

Kunitz Type ◽

Kunitz Type Protease Inhibitor

AbstractEgKI-1, a member of the Kunitz type protease inhibitor family, is highly expressed by the oncosphere of the canine tapeworm Echinococcus granulosus, the stage that is infectious to humans and ungulates, giving rise to a hydatid cyst localized to the liver and other organs. Larval protoscoleces, which develop within the hydatid cyst, have been shown to possess anti-cancer properties, although the precise molecules involved have not been identified. We show that recombinant EgKI-1 inhibits the growth and migration of a range of human cancers including breast, melanoma and cervical cancer cell lines in a dose-dependent manner in vitro without affecting normal cell growth. Furthermore, EgKI-1 treatment arrested the cancer cell growth by disrupting the cell cycle and induced apoptosis of cancer cells in vitro. An in vivo model of triple negative breast cancer (MDA-MB-231) in BALB/c nude mice showed significant tumor growth reduction in EgKI-1-treated mice compared with controls. These findings indicate that EgKI-1 shows promise for future development as an anti-cancer therapeutic.

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In Vitro Characteristics, Anticoagulant Effects and In Vivo Antithrombotic Efficacy of a Novel, Potent and Orally Active Direct Factor Xa Inhibitor, DU-176b.

Blood ◽

10.1182/blood.v104.11.1862.1862 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1862-1862 ◽

Cited By ~ 3

Author(s):

Yoshiyuki Morishima ◽

Taketoshi Furugohri ◽

Koji Isobe ◽

Yuko Honda ◽

Chikako Matsumoto ◽

...

Keyword(s):

Oral Administration ◽

Factor Xa ◽

Coagulation Cascade ◽

Venous Stasis ◽

Factor Xa Inhibitor ◽

Fxa Inhibitor ◽

Antithrombotic Efficacy ◽

Orally Active

Abstract Factor Xa (FXa) is a key serine protease in the coagulation cascade and is a promising target enzyme for developing a new antithrombotic agent. Our first clinical candidate for a small molecular direct FXa inhibitor DX-9065a potently inhibits FXa (Ki = 41 nM) and exerts antithrombotic effects in animal models. However, due to its poor bioavailability (10% in monkeys) the compound is used only as an injectable formulation in clinical studies. Here we report in vitro characteristics of serine proteases inhibition, anticoagulant effects and in vivo antithrombotic efficacy of DU-176b, a novel, potent and orally active direct FXa inhibitor. DU-176b competitively inhibited human FXa with a Ki value of 0.561 nM, indicating 70-fold increase in FXa inhibitory activity compared with DX-9065a. DU-176b demonstrated 10,000-fold selectivity relative to inhibition of thrombin (Ki = 6.00 μM), and had no effects on the enzymatic activities of factor VIIa, t-PA, plasmin, trypsin and chymotrypsin. In human plasma, DU-176b prolonged prothrombin time (PT) and activated partial thromboplastin time (APTT) in a concentration-dependent manner. Its concentrations for doubling these clotting times were 0.256 and 0.508 μM, respectively. After oral administration of DU-176b to rats, significant anti-Xa activity was observed in plasma over 4 h. The oral bioavailability of DU-176b (approximately 50%) was significantly higher than that of DX-9065a (10%) in monkeys. The antithrombotic efficacy of DU-176b was examined by oral administration to rats 30 minutes prior to thrombogenic stimuli. In a venous stasis thrombosis model, DU-176b (0.5 – 12.5 mg/kg, p.o.) dose-dependently inhibited thrombus formation, prolonged PT, and revealed plasma anti-Xa activity. DU-176b also exerted significant anticoagulant effect in a rat model of tissue factor-induced disseminated intravascular coagulation at doses of 0.1 – 2.5 mg/kg, p.o. These results demonstrate that DU-176b is a potent and selective factor Xa inhibitor that possesses antithrombotic effect after oral administration. DU-176b has the potential to be clinically useful for prophylaxis and treatment of several thrombotic diseases.

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Members of the Kunitz-type protease inhibitor gene family of potato inhibit soluble tuber invertase in vitro

Potato Research ◽

10.1007/bf02736112 ◽

2002 ◽

Vol 45 (2-4) ◽

pp. 163-176 ◽

Cited By ~ 26

Author(s):

Heike Glaczinski ◽

A. Heibges ◽

F. Salamini ◽

Christiane Gebhardt

Keyword(s):

Protease Inhibitor ◽

Gene Family ◽

Kunitz Type ◽

Kunitz Type Protease Inhibitor ◽

Inhibitor Gene

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Kunitz type protease inhibitor from the canine tapeworm as a potential therapeutic for melanoma

Scientific Reports ◽

10.1038/s41598-019-52609-4 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Shiwanthi L. Ranasinghe ◽

Vanessa Rivera ◽

Glen M. Boyle ◽

Donald P. McManus

Keyword(s):

Tumor Microenvironment ◽

Protease Inhibitor ◽

Survivin Expression ◽

Surrounding Tissue ◽

Axillary Lymph Nodes ◽

Axillary Lymph ◽

Anti Cancer ◽

Kunitz Type ◽

Kunitz Type Protease Inhibitor

Abstract Modulating the tumor microenvironment to promote an effective immune response is critical in managing any type of tumor. Melanoma is an aggressive skin cancer and the incidence rate is increasing worldwide. Potent protease inhibitors have recently been extensively researched as potential therapeutic agents against various cancers. EgKI-1 is a potent Kunitz type protease inhibitor identified from the canine tapeworm Echinococcus granulosus that has shown anti-cancer activities in vivo. In this study we show that EgKI-1 significantly reduced the growth of melanoma in the B16-F0 mouse model and was not toxic to normal surrounding tissue. Moreover, EgKI-1 treatment significantly reduced survivin expression levels and increased the CD8+ T cell population in draining axillary lymph nodes. Therefore, EgKI-1 potentially reduces tumor growth by inducing apoptosis and modulating the tumor microenvironment, and has potential for development as an intra-lesional treatment for melanoma.

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Ancylostoma caninum Anticoagulant Peptide Blocks Metastasis In Vivo and Inhibits Factor Xa Binding to Melanoma Cells In Vitro

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615117 ◽

1998 ◽

Vol 79 (05) ◽

pp. 1041-1047 ◽

Cited By ~ 20

Author(s):

Kathleen M. Donnelly ◽

Michael E. Bromberg ◽

Aaron Milstone ◽

Jennifer Madison McNiff ◽

Gordon Terwilliger ◽

...

Keyword(s):

Active Site ◽

Thrombin Generation ◽

Factor Xa ◽

Coagulation Factor ◽

Melanoma Cells ◽

Factor V ◽

Ancylostoma Caninum ◽

Metastatic Activity

SummaryWe evaluated the in vivo anti-metastatic activity of recombinant Ancylostoma caninum Anticoagulant Peptide (rAcAP), a potent (Ki = 265 pM) and specific active site inhibitor of human coagulation factor Xa originally isolated from bloodfeeding hookworms. Subcutaneous injection of SCID mice with rAcAP (0.01-0.2 mg/mouse) prior to tail vein injection of LOX human melanoma cells resulted in a dose dependent reduction in pulmonary metastases. In order to elucidate potential mechanisms of rAcAP’s anti-metastatic activity, experiments were carried out to identify specific interactions between factor Xa and LOX. Binding of biotinylated factor Xa to LOX monolayers was both specific and saturable (Kd = 15 nM). Competition experiments using antibodies to previously identified factor Xa binding proteins, including factor V/Va, effector cell protease receptor-1, and tissue factor pathway inhibitor failed to implicate any of these molecules as significant binding sites for Factor Xa. Functional prothrombinase activity was also supported by LOX, with a half maximal rate of thrombin generation detected at a factor Xa concentration of 2.4 nM. Additional competition experiments using an excess of either rAcAP or active site blocked factor Xa (EGR-Xa) revealed that most of the total factor Xa binding to LOX is mediated via interaction with the enzyme’s active site, predicting that the vast majority of cell-associated factor Xa does not participate directly in thrombin generation. In addition to establishing two distinct mechanisms of factor Xa binding to melanoma, these data raise the possibility that rAcAP’s antimetastatic effect in vivo might involve novel non-coagulant pathways, perhaps via inhibition of active-site mediated interactions between factor Xa and tumor cells.

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The Anticoagulant Properties of a Modified Form of Protein S

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647048 ◽

1988 ◽

Vol 60 (02) ◽

pp. 298-304 ◽

Cited By ~ 17

Author(s):

C A Mitchell ◽

S M Kelemen ◽

H H Salem

Keyword(s):

Monoclonal Antibody ◽

Anticoagulant Activity ◽

Activated Protein C ◽

Factor Xa ◽

Protein S ◽

Human Neutrophil Elastase ◽

Factor X ◽

Sds Page

SummaryProtein S (PS) is a vitamin K-dependent anticoagulant that acts as a cofactor to activated protein C (APC). To date PS has not been shown to possess anticoagulant activity in the absence of APC.In this study, we have developed monoclonal antibody to protein S and used to purify the protein to homogeneity from plasma. Affinity purified protein S (PSM), although identical to the conventionally purified protein as judged by SDS-PAGE, had significant anticoagulant activity in the absence of APC when measured in a factor Xa recalcification time. Using SDS-PAGE we have demonstrated that prothrombin cleavage by factor X awas inhibited in the presence of PSM. Kinetic analysis of the reaction revealed that PSM competitively inhibited factor X amediated cleavage of prothrombin. PS preincubated with the monoclonal antibody, acquired similar anticoagulant properties. These results suggest that the interaction of the monoclonal antibody with PS results in an alteration in the protein exposing sites that mediate the observed anticoagulant effect. Support that the protein was altered was derived from the observation that PSM was eight fold more sensitive to cleavage by thrombin and human neutrophil elastase than conventionally purified protein S.These observations suggest that PS can be modified in vitro to a protein with APC-independent anticoagulant activity and raise the possibility that a similar alteration could occur in vivo through the binding protein S to a cellular or plasma protein.

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Comparison of the Anticoagulant and Antithrombotic Effects of Synthetic Thrombin and Factor Xa Inhibitors

Thrombosis and Haemostasis ◽

10.1055/s-0038-1645198 ◽

1990 ◽

Vol 63 (02) ◽

pp. 220-223 ◽

Cited By ~ 19

Author(s):

J Hauptmann ◽

B Kaiser ◽

G Nowak ◽

J Stürzebecher ◽

F Markwardt

Keyword(s):

Factor Xa ◽

Thrombin Inhibitors ◽

Factor Xa Inhibitors ◽

Venous Stasis ◽

Clotting Time ◽

Thrombosis Model ◽

Activity Factor ◽

Antithrombotic Effects

SummaryThe anticoagulant effect of selected synthetic inhibitors of thrombin and factor Xa was studied in vitro in commonly used clotting assays. The concentrations of the compounds doubling the clotting time in the various assays were mainly dependent on their thrombin inhibitory activity. Factor Xa inhibitors were somewhat more effective in prolonging the prothrombin time compared to the activated partial thromboplastin time, whereas the opposite was true of thrombin inhibitors.In vivo, in a venous stasis thrombosis model and a thromboplastin-induced microthrombosis model in rats the thrombin inhibitors were effective antithrombotically whereas factor Xa inhibitors of numerically similar IQ value for the respective enzyme were not effective at equimolar dosageThe results are discussed in the light of the different prelequisiles and conditions for inhibition of thrombin and factor Xa in the course of blood clotting.

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Effect of Depolymerized Holothurian Glycosaminoglycan (DHG) on Tissue Factor Pathway Inhibitor: In Vitro and In Vivo Studies

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657643 ◽

1997 ◽

Vol 78 (02) ◽

pp. 864-870 ◽

Cited By ~ 9

Author(s):

Hideki Nagase ◽

Kei-ichi Enjyoji ◽

Yu-ichi Kamikubo ◽

Keiko T Kitazato ◽

Kenji Kitazato ◽

...

Keyword(s):

Tissue Factor ◽

Tissue Factor Pathway Inhibitor ◽

Factor Xa ◽

Free Form ◽

Initial Reaction ◽

Extrinsic Pathway ◽

Factor Xa Inhibition ◽

Tissue Factor Pathway

SummaryDepolymerized holothurian glycosaminoglycan (DHG) is a glycosaminoglycan extracted from the sea cucumber Stichopus japonicusSelenka. In previous studies, we demonstrated that DHG has antithrombotic and anticoagulant activities that are distinguishable from those of heparin and dermatan sulfate. In the present study, we examined the effect of DHG on the tissue factor pathway inhibitor (TFPI), which inhibits the initial reaction of the tissue factor (TF)-mediated coagulation pathway. We first examined the effect of DHG on factor Xa inhibition by TFPI and the inhibition of TF-factor Vila by TFPI-factor Xa in in vitro experiments using human purified proteins. DHG increased the rate of factor Xa inhibition by TFPI, which was abolished either with a synthetic C-terminal peptide or with a synthetic K3 domain peptide of TFPI. In contrast, DHG reduced the rate of TF-factor Vila inhibition by TFPI-factor Xa. Therefore, the effect of DHG on in vitroactivity of TFPI appears to be contradictory. We then examined the effect of DHG on TFPI in cynomolgus monkeys and compared it with that of unfractionated heparin. DHG induced an increase in the circulating level of free-form TFPI in plasma about 20-fold when administered i.v. at 1 mg/kg. The prothrombin time (PT) in monkey plasma after DHG administration was longer than that estimated from the plasma concentrations of DHG. Therefore, free-form TFPI released by DHG seems to play an additive role in the anticoagulant mechanisms of DHG through the extrinsic pathway in vivo. From the results shown in the present work and in previous studies, we conclude that DHG shows anticoagulant activity at various stages of coagulation reactions, i.e., by inhibiting the initial reaction of the extrinsic pathway, by inhibiting the intrinsic Xase, and by inhibiting thrombin.

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