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

Objective-Blood-sucking arthropods' salivary glands contain a remarkable diversity of antihemostatics. The aim of the present study was to identify the unique salivary anticoagulant of the sand fly Lutzomyia longipalpis, which remained elusive for decades. Methods and Results-Several L. longipalpis salivary proteins were expressed in human embryonic kidney 293 cells and screened for inhibition of blood coagulation. A novel 32.4-kDa molecule, named Lufaxin, was identified as a slow, tight, noncompetitive, and reversible inhibitor of factor Xa (FXa). Notably, Lufaxin's primary sequence does not share similarity to any physiological or salivary inhibitors of coagulation reported to date. Lufaxin is specific for FXa and does not interact with FX, Dansyl-Glu-Gly-Arg-FXa, or 15 other enzymes. In addition, Lufaxin blocks prothrombinase and increases both prothrombin time and activated partial thromboplastin time. Surface plasmon resonance experiments revealed that FXa binds Lufaxin with an equilibrium constant approximate to 3 nM, and isothermal titration calorimetry determined a stoichiometry of 1:1. Lufaxin also prevents protease-activated receptor 2 activation by FXa in the MDA-MB-231 cell line and abrogates edema formation triggered by injection of FXa in the paw of mice. Moreover, Lufaxin prevents FeCl3-induced carotid artery thrombus formation and prolongs activated partial thromboplastin time ex vivo, implying that it works as an anticoagulant in vivo. Finally, salivary gland of sand flies was found to inhibit FXa and to interact with the enzyme. Conclusion-Lufaxin belongs to a novel family of slow-tight FXa inhibitors, which display antithrombotic and anti-inflammatory activities. It is a useful tool to understand FXa structural features and its role in prohemostatic and proinflammatory events. (Arterioscler Thromb Vasc Biol. 2012;32:2185-2196.)

Defibrotide Interferes With Several Steps of the Coagulation-Inflammation Cycle and Exhibits Therapeutic Potential to Treat Severe Malaria

Objective-The coagulation-inflammation cycle has been implicated as a critical component in malaria pathogenesis. Defibrotide (DF), a mixture of DNA aptamers, displays anticoagulant, anti-inflammatory, and endothelial cell (EC)-protective activities and has been successfully used to treat comatose children with veno-occlusive disease. DF was investigated here as a drug to treat cerebral malaria. Methods and Results-DF blocks tissue factor expression by ECs incubated with parasitized red blood cells and attenuates prothrombinase activity, platelet aggregation, and complement activation. In contrast, it does not affect nitric oxide bioavailability. We also demonstrated that Plasmodium falciparum glycosylphosphatidylinositol (Pf-GPI) induces tissue factor expression in ECs and cytokine production by dendritic cells. Notably, dendritic cells, known to modulate coagulation and inflammation systemically, were identified as a novel target for DF. Accordingly, DF inhibits Toll-like receptor ligand-dependent dendritic cells activation by a mechanism that is blocked by adenosine receptor antagonist (8-p-sulfophenyltheophylline) but not reproduced by synthetic poly-A, -C, -T, and -G. These results imply that aptameric sequences and adenosine receptor mediate dendritic cells responses to the drug. DF also prevents rosetting formation, red blood cells invasion by P. falciparum and abolishes oocysts development in Anopheles gambiae. In a murine model of cerebral malaria, DF affected parasitemia, decreased IFN-gamma levels, and ameliorated clinical score (day 5) with a trend for increased survival. Conclusion-Therapeutic use of DF in malaria is proposed. (Arterioscler Thromb Vasc Biol. 2012; 32:786-798.)

The sialotranscriptome of Antricola delacruzi female ticks is compatible with non-hematophagous behavior and an alternative source of food

The hosts for Antricola delacruzi ticks are insectivorous, cave-dwelling bats on which only larvae are found. The mouthparts of nymphal and adult A. delacruzi are compatible with scavenging feeding because the hypostome is small and toothless. How a single blood meal of a larva provides energy for several molts as well as for oviposition by females is not known. Adults of A. delacruzi possibly feed upon an unknown food source in bat guano, a substrate on which nymphal and adult stages are always found. Guano produced by insectivorous bats contains twice the amount of protein and 60 times the amount of iron as beef. In addition, bacteria and chitin-rich fungi proliferate on guano. Comparative data on the transcriptome of the salivary glands of A. delacruzi is nonexistent and would help to understand the physiological adaptations of salivary glands that accompany different sources of food as well as the steps taken by the Acari toward haematophagy, believed to have evolved from scavenging dead animals. Annotation of the transcriptome of salivary glands from female instars of A. delacruzi collected on guano categorized 5.7% of the clusters of expressed genes as putative secreted proteins. They included abundantly expressed TIL-domain-containing proteins (possible anti-microbials), an abundantly expressed protein similar to a serum amyloid found in the sialotranscriptomes of Ornithodoros spp., a savignygrin, a family of mucin/peritrophin/cuticle-like proteins, anti-microbials and an HIV envelope-like glycoprotein also found in soft ticks. When comparing the transcriptome of A. delacruzi with those of blood-feeding female soft and hard ticks some notable differences were observed; they consisted of the following transcripts over- or under-represented or absent in the sialotranscriptome of A. delacruzi that may reflect its source of food: ferritin, mucins with chitin-binding domains and TIL-domain-containing proteins versus lipocalins, basic tail proteins, metalloproteases, glycine-rich proteins and Kunitz protease inhibitors, respectively. (C) 2012 Elsevier Ltd. All rights reserved.

Ticks (Acari: Ixodidae) associated with small terrestrial mammals in the state of Minas Gerais, southeastern Brazil

From June 2005 to November 2010, 43 small mammals encompassing 6 species of Didelphimorphia, 8 species of Rodentia, and 1 species of Lagomorpha were found parasitized by ticks in the state of Minas Gerais, southeastern Brazil. Nine tick species, in total 186 specimens, were identified as follows: Amblyomma cajennense (larvae and nymphs) on opossums and rodents; Amblyomma ovale (nymphs) on rodents; Amblyomma parvum (nymphs) on rodents; Amblyomma coelebs (nymphs) on opossums; Amblyomma dubitatum (nymph) on opossums; Ixodes amarali (females, nymphs, and larvae) on opossums and rodents; Ixodes loricatus (male, females, nymph) on opossums; Ixodes schulzei (female) on rodents; and Haemaphysalis leporispalustris (female) on rabbits. Most of the tick-host associations found in the present study have never been recorded in the literature; those include three new host records for I. amarali, four for A. cajennense, one for A. dubitatum, two for A. ovale, and one for A. coelebs. In addition, we provide the first record of A. coelebs in the state of Minas Gerais.

Epidemiology of Brazilian spotted fever in the Atlantic Forest, state of Sao Paulo, Brazil

The tick-borne bacterium Rickettsia rickettsii is the aetiological agent of Brazilian spotted fever (BSF). The present study evaluated tick infestations on wild and domestic animals, and the rickettsial infection in these animals and their ticks in 7 forest areas adjacent to human communities in the Sao Paulo Metropolitan Area (SPMA). The results were compared to ecological traits of each sampled area. Two main tick species, Amblyomma aureolatum and Rhipicephalus sanguineus, were collected from dogs. The major ticks found on small mammals and birds were Ixodes loricatus and Amblyomma longirostre, respectively. Both anti-R. rickettsii antibodies and R. rickettsii-infected ticks were detected on dogs from only 2 areas in the southern part of the SPMA, which were considered to be endemic for BSF; the remaining 5 areas were considered to be non-endemic. Ecologically, the BSF-endemic areas clearly differed from the non-endemic areas by the presence of significantly more degraded forest patches in the former. The present results corroborate historical observations that have indicated that all human cases of BSF in the SPMA were contracted in the southern part of this metropolitan area. However, not all forest patches in the southern part of the SPMA were shown to be associated with BSF endemism.

Ecological implications on the aggregation of Amblyomma fuscum (Acari: Ixodidae) on Thrichomys laurentius (Rodentia: Echimyidae), in northeastern Brazil

We investigated the Amblyomma fuscum load on a pullulating wild rodent population and the environmental and biological factors influencing the tick load on the hosts. One hundred and three individuals of Thrichomys laurentius were caught in an Atlantic forest fragment in northeastern Brazil, as part of a longitudinal survey on ticks infesting non-volant small mammals. Ticks (n = 342) were found on 45 individuals and the overall mean intensity of infestation was 7.6 ticks per infested rodent. Ticks were highly aggregated in the host population and the negative binomial distribution model provides a statistically satisfactory fit. The aggregated distribution was influenced by sex and age of the host. The microhabitat preference by T. laurentius probably increases contact opportunities between hosts and aggregated infesting stages of the ticks and represents important clues about the habitat suitability for A. fuscum.

Exposure of small mammals to ticks and rickettsiae in Atlantic Forest patches in the metropolitan area of Recife, North-eastern Brazil

Between December 2007 and March 2009, small mammals were captured in 6 Atlantic Forest patches in Brazil. We assessed tick-host associations and whether they differ among forest strata, sites, seasons, and host age classes or between sexes. Moreover, we assessed the exposure of animals to Rickettsia spp. In total, 432 animals were captured and 808 ticks were found on 32-9% of them. Significant differences were found among host species, collection sites, and forest strata; microhabitat preference was a strong risk factor for tick infestation. The highest tick density rates were recorded in forest fragments settled in rural areas; 91.3% of the ticks were collected from animals trapped in these forest fragments. A high prevalence (68.8%) of antibodies to Rickettsia spp. was detected among animals. This study suggests that disturbed Atlantic Forest fragments provide an environment for ticks and small mammals, which are highly exposed to rickettsiae. It also indicates that forest patches settled in rural areas are usually associated with higher small mammal diversity as well as with higher tick density rates.