Lufaxin, a Novel Factor Xa Inhibitor From the Salivary Gland of the Sand Fly
            Lutzomyia longipalpis
            Blocks Protease-Activated Receptor 2 Activation and Inhibits Inflammation and Thrombosis In Vivo

AbstractThe protozoan parasite Leishmania possesses a single flagellum, which is remodelled during the parasite’s life cycle from a long motile flagellum in promastigote forms in the sand fly to a short immotile flagellum in amastigotes residing in mammalian phagocytes. This study examined the protein composition and in vivo function of the promastigote flagellum. Protein mass spectrometry and label free protein enrichment testing of isolated flagella and deflagellated cell bodies defined a flagellar proteome for L. mexicana promastigote forms (available via ProteomeXchange with identifier PXD011057). This information was used to generate a CRISPR-Cas9 knockout library of 100 mutants to screen for flagellar defects. This first large-scale knockout screen in a Leishmania sp. identified 56 mutants with altered swimming speed (52 reduced and 4 increased) and defined distinct mutant categories (faster swimmers, slower swimmers, slow uncoordinated swimmers and paralysed cells, including aflagellate promastigotes and cells with curled flagella and disruptions of the paraflagellar rod). Each mutant was tagged with a unique 17-nt barcode, providing a simple barcode sequencing (bar-seq) method for measuring the relative fitness of L. mexicana mutants in vivo. In mixed infections of the permissive sand fly vector Lutzomyia longipalpis, paralysed promastigotes and uncoordinated swimmers were severely diminished in the fly after defecation of the bloodmeal. Subsequent examination of flies infected with a single mutant lacking the central pair protein PF16 showed that these paralysed promastigotes did not reach anterior regions of the fly alimentary tract. These data show that L. mexicana need directional motility for successful colonisation of sand flies.Author SummaryLeishmania are protozoan parasites, transmitted between mammals by the bite of phlebotomine sand flies. Promastigote forms in the sand fly have a long flagellum, which is motile and used for anchoring the parasites to prevent clearance with the digested blood meal remnants. To dissect flagellar functions and their importance in life cycle progression, we generated here a comprehensive list of >300 flagellar proteins and produced a CRISPR-Cas9 gene knockout library of 100 mutant Leishmania. We studied their behaviour in vitro before examining their fate in the sand fly Lutzomyia longipalpis. Measuring mutant swimming speeds showed that about half behaved differently compared to the wild type: a few swam faster, many slower and some were completely paralysed. We also found a group of uncoordinated swimmers. To test whether flagellar motility is required for parasite migration from the fly midgut to the foregut from where they reach the next host, we infected sand flies with a mixed mutant population. Each mutant carried a unique tag and tracking these tags up to nine days after infection showed that paralysed and uncoordinated Leishmania were rapidly lost from flies. These data indicate that directional swimming is important for successful colonisation of sand flies.

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A specific factor Xa inhibitor, DX-9065a, prevents experimental DIC in primates by the inhibition of factor Xa in vivo

The Japanese Journal of Pharmacology ◽

10.1016/s0021-5198(19)40826-3 ◽

1998 ◽

Vol 76 ◽

pp. 179

Author(s):

Toshio Fukuda ◽

Yoshiyuki Morishima ◽

Tsuyoshi Hara ◽

Satoshi Kunitada

Keyword(s):

Factor Xa ◽

Specific Factor ◽

Factor Xa Inhibitor

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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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Leishmania amazonensis: Sensitivity of Different Promastigote Morphotypes to Salivary Gland Homogenates of the Sand Fly Lutzomyia longipalpis

Experimental Parasitology ◽

10.1006/expr.1995.1021 ◽

1995 ◽

Vol 80 (2) ◽

pp. 167-175 ◽

Cited By ~ 11

Author(s):

R. Charlab ◽

R.B. Tesh ◽

E.D. Rowton ◽

J.M.C. Ribeiro

Keyword(s):

Salivary Gland ◽

Leishmania Amazonensis ◽

Sand Fly ◽

Lutzomyia Longipalpis

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Effect of Factor Xa Inhibitors on the Platelet-derived Microparticles Procoagulant Activity In Vitro> and In Vivo in Rats

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614085 ◽

2000 ◽

Vol 84 (10) ◽

pp. 668-674 ◽

Cited By ~ 5

Author(s):

J. P. Hérault ◽

B. Perrin ◽

C. Jongbloet ◽

A. M. Pflieger ◽

A. Bernat ◽

...

Keyword(s):

Factor Xa ◽

Procoagulant Activity ◽

Factor Xa Inhibitors ◽

Gel Chromatography ◽

Factor Xa Inhibitor ◽

Dependent Manner ◽

Platelet Surface ◽

Prothrombinase Activity

SummaryThe aim of this study was to investigate the effect of factor Xa inhibitors on the prothrombinase activity of platelet-derived microparticles in vitro and in vivo. The factor Xa inhibitors studied were DX9065A (a direct factor Xa inhibitor) and Sanorg34006 (an antithrombin (AT)-mediated factor Xa inhibitor). Microparticles formed from the platelet surface following activation were isolated by size exclusion gel chromatography. After purification, their presence was detected by their procoagulant activity and by flow cytometry. Our results show that factor Xa and/or factor Va were present at the surface of the platelet-derived microparticles. Prothrombinase formed on the microparticles was inhibited by factor Xa inhibitors at IC50 values of 0.45 ± 0.05 and 0.045 ± 0.005 µM for DX9065A and AT-Sanorg34006 respectively. In an experiment aimed at determining the kinetics of microparticles formation we demonstrated that thrombin traces were sufficient to induce the formation of a significant quantity of microparticles. Both factor Xa inhibitors delayed the formation of microparticles by delaying thrombin generation. The thrombogenic effect of the microparticles were studied in vivo in a modified arterio-venous shunt model in the rat. In this model, the increase in the thrombus weigh due to microparticles or phospholipids did not differ significantly (33% and 23% respectively). In these conditions, prothrombinase activity seemed to play a lesser role in the thrombogenic effect than phospholipids. Nevertheless, factor Xa inhibitors were efficient and inhibited thrombus formation in a dose-dependent manner.These results demonstrate that platelet-derived microparticles display a potent prothrombotic effect in vivo and show that factor Xa inhibitors are potent antithrombotic compounds when thrombosis was induced by microparticles.

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