title: Plasmodium falciparum infection induces expression of a mosquito salivary protein (Agaphelin) that targets neutrophil function and inhibits thrombosis without impairing hemostasis.
abstract: BACKGROUND
  Invasion of mosquito salivary glands (SGs) by Plasmodium falciparum sporozoites is an essential step in the malaria life cycle. How infection modulates gene expression, and affects hematophagy remains unclear.
  
  
  RESULTS
  Using Affimetrix chip microarray, we found that at least 43 genes are differentially expressed in the glands of Plasmodium falciparum-infected Anopheles gambiae mosquitoes. Among the upregulated genes, one codes for Agaphelin, a 58-amino acid protein containing a single Kazal domain with a Leu in the P1 position. Agaphelin displays high homology to orthologs present in Aedes sp and Culex sp salivary glands, indicating an evolutionarily expanded family. Kinetics and surface plasmon resonance experiments determined that chemically synthesized Agaphelin behaves as a slow and tight inhibitor of neutrophil elastase (K(D) ∼ 10 nM), but does not affect other enzymes, nor promotes vasodilation, or exhibit antimicrobial activity. TAXIscan chamber assay revealed that Agaphelin inhibits neutrophil chemotaxis toward fMLP, affecting several parameter associated with cell migration. In addition, Agaphelin reduces paw edema formation and accumulation of tissue myeloperoxidase triggered by injection of carrageenan in mice. Agaphelin also blocks elastase/cathepsin-mediated platelet aggregation, abrogates elastase-mediated cleavage of tissue factor pathway inhibitor, and attenuates neutrophil-induced coagulation. Notably, Agaphelin inhibits neutrophil extracellular traps (NETs) formation and prevents FeCl3-induced arterial thrombosis, without impairing hemostasis.
  
  
  CONCLUSIONS
  Blockade of neutrophil elastase emerges as a novel antihemostatic mechanism in hematophagy; it also supports the notion that neutrophils and the innate immune response are targets for antithrombotic therapy. In addition, Agaphelin is the first antihemostatic whose expression is induced by Plasmodium sp infection. These results suggest that an important interplay takes place in parasite-vector-host interactions.
authors.affiliation: Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America; Department of Pathology, University of Virginia, Charlottesville, Virginia, United States of America.
authors.forename: Michael
authors.initials: M
authors.lastname: Waisberg
authors.affiliation: Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Alvaro
authors.initials: A
authors.lastname: Molina-Cruz
authors.affiliation: Instituto de Bioquimica Médica, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Daniella M
authors.initials: DM
authors.lastname: Mizurini
authors.affiliation: Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Nidhi
authors.initials: N
authors.lastname: Gera
authors.affiliation: Instituto de Ciências Biológicas e Naturais, Universidade Federal do Triângulo Mineiro, Uberaba, Brazil.
authors.forename: Beatriz C
authors.initials: BC
authors.lastname: Sousa
authors.affiliation: Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Dongying
authors.initials: D
authors.lastname: Ma
authors.affiliation: Instituto de Bioquimica Médica, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Ana C
authors.initials: AC
authors.lastname: Leal
authors.affiliation: Instituto de Bioquimica Médica, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Tainá
authors.initials: T
authors.lastname: Gomes
authors.affiliation: Institute of Parasitology, Academy of Sciences of the Czech Republic, České Budjovice, Czech Republic.
authors.forename: Michalis
authors.initials: M
authors.lastname: Kotsyfakis
authors.affiliation: Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: José M C
authors.initials: JM
authors.lastname: Ribeiro
authors.affiliation: Research Technology Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, United States of America.
authors.forename: Jan
authors.initials: J
authors.lastname: Lukszo
authors.affiliation: Research Technology Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, United States of America.
authors.forename: Karine
authors.initials: K
authors.lastname: Reiter
authors.affiliation: Genomics Unit, Research Technology Section, Rocky Mountain Labs, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, United States of America.
authors.forename: Stephen F
authors.initials: SF
authors.lastname: Porcella
authors.affiliation: Instituto de Ciências Biológicas e Naturais, Universidade Federal do Triângulo Mineiro, Uberaba, Brazil.
authors.forename: Carlo J
authors.initials: CJ
authors.lastname: Oliveira
authors.affiliation: Instituto de Bioquimica Médica, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Robson Q
authors.initials: RQ
authors.lastname: Monteiro
authors.affiliation: Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Carolina
authors.initials: C
authors.lastname: Barillas-Mury
authors.affiliation: Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Susan K
authors.initials: SK
authors.lastname: Pierce
authors.affiliation: Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Ivo M B
authors.initials: IM
authors.lastname: Francischetti
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000852:Anopheles / Q000378:metabolism / Q000469:parasitology*; D002942:Circular Dichroism; D004487:Edema / Q000209:etiology / Q000378:metabolism / Q000517:prevention & control; D005260:Female; D006487:Hemostasis / Q000502:physiology*; D006790:Host-Parasite Interactions; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007303:Insect Vectors; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000276:immunology*; D010963:Plasmodium falciparum / Q000472:pathogenicity*; D012469:Salivary Glands / Q000378:metabolism / Q000469:parasitology; D012471:Salivary Proteins and Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance; D013927:Thrombosis / Q000517:prevention & control*
chemical_list: D019476:Insect Proteins; D012471:Salivary Proteins and Peptides