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 ≈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 FeCl(3)-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. CONCLUSIONS: 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.

Repeated exposure to Lutzomyia intermedia sand fly saliva induces local expression of interferon-inducible genes both at the site of injection in mice and in human blood.

During a blood meal, Lutzomyia intermedia sand flies transmit Leishmania braziliensis, a parasite causing tegumentary leishmaniasis. In experimental leishmaniasis, pre-exposure to saliva of most blood-feeding sand flies results in parasite establishment in absence of any skin damages in mice challenged with dermotropic Leishmania species together with saliva. In contrast, pre-immunization with Lu. intermedia salivary gland sonicate (SGS) results in enhanced skin inflammatory exacerbation upon co-inoculation of Lu. intermedia SGS and L. braziliensis. These data highlight potential unique features of both L. braziliensis and Lu. intermedia. In this study, we investigated the genes modulated by Lu. intermedia SGS immunization to understand their potential impact on the subsequent cutaneous immune response following inoculation of both SGS and L. braziliensis. The cellular recruitment and global gene expression profile was analyzed in mice repeatedly inoculated or not with Lu. intermedia. Microarray gene analysis revealed the upregulation of a distinct set of IFN-inducible genes, an immune signature not seen to the same extent in control animals. Of note this INF-inducible gene set was not induced in SGS pre-immunized mice subsequently co-inoculated with SGS and L. braziliensis. These data suggest the parasite prevented the upregulation of this Lu. intermedia saliva-related immune signature. The presence of these IFN-inducible genes was further analyzed in peripheral blood mononuclear cells (PBMCs) sampled from uninfected human individuals living in a L. braziliensis-endemic region of Brazil thus regularly exposed to Lu. intermedia bites. PBMCs were cultured in presence or absence of Lu. intermedia SGS. Using qRT-PCR we established that the IFN-inducible genes induced in the skin of SGS pre-immunized mice, were also upregulated by SGS in PBMCs from human individuals regularly exposed to Lu. intermedia bites, but not in PBMCs of control subjects. These data demonstrate that repeated exposure to Lu. intermedia SGS induces the expression of potentially host-protective IFN-inducible genes.

Structure and function of a "yellow" protein from saliva of the sand fly Lutzomyia longipalpis that confers protective immunity against Leishmania major infection.

LJM11, an abundant salivary protein from the sand fly Lutzomyia longipalpis, belongs to the insect "yellow" family of proteins. In this study, we immunized mice with 17 plasmids encoding L. longiplapis salivary proteins and demonstrated that LJM11 confers protective immunity against Leishmania major infection. This protection correlates with a strong induction of a delayed type hypersensitivity (DTH) response following exposure to L. longipalpis saliva. Additionally, splenocytes of exposed mice produce IFN-γ upon stimulation with LJM11, demonstrating the systemic induction of Th1 immunity by this protein. In contrast to LJM11, LJM111, another yellow protein from L. longipalpis saliva, does not produce a DTH response in these mice, suggesting that structural or functional features specific to LJM11 are important for the induction of a robust DTH response. To examine these features, we used calorimetric analysis to probe a possible ligand binding function for the salivary yellow proteins. LJM11, LJM111, and LJM17 all acted as high affinity binders of prohemostatic and proinflammatory biogenic amines, particularly serotonin, catecholamines, and histamine. We also determined the crystal structure of LJM11, revealing a six-bladed β-propeller fold with a single ligand binding pocket located in the central part of the propeller structure on one face of the molecule. A hypothetical model of LJM11 suggests a positive electrostatic potential on the face containing entry to the ligand binding pocket, whereas LJM111 is negative to neutral over its entire surface. This may be the reason for differences in antigenicity between the two proteins.

SALO, a novel classical pathway complement inhibitor from saliva of the sand fly Lutzomyia longipalpis.

Blood-feeding insects inject potent salivary components including complement inhibitors into their host's skin to acquire a blood meal. Sand fly saliva was shown to inhibit the classical pathway of complement; however, the molecular identity of the inhibitor remains unknown. Here, we identified SALO as the classical pathway complement inhibitor. SALO, an 11 kDa protein, has no homology to proteins of any other organism apart from New World sand flies. rSALO anti-complement activity has the same chromatographic properties as the Lu. longipalpis salivary gland homogenate (SGH)counterparts and anti-rSALO antibodies blocked the classical pathway complement activity of rSALO and SGH. Both rSALO and SGH inhibited C4b deposition and cleavage of C4. rSALO, however, did not inhibit the protease activity of C1s nor the enzymatic activity of factor Xa, uPA, thrombin, kallikrein, trypsin and plasmin. Importantly, rSALO did not inhibit the alternative or the lectin pathway of complement. In conclusion our data shows that SALO is a specific classical pathway complement inhibitor present in the saliva of Lu. longipalpis. Importantly, due to its small size and specificity, SALO may offer a therapeutic alternative for complement classical pathway-mediated pathogenic effects in human diseases.