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. PRINCIPAL FINDINGS: 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.

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

Institute of Parasitology Academy of Sciences of the Czech Republic České Budjovice Czech Republic

Instituto de Bioquimica Médica Federal University of Rio de Janeiro Rio de Janeiro Brazil

Instituto de Ciências Biológicas e Naturais Universidade Federal do Triângulo Mineiro Uberaba Brazil

Laboratory of Immunogenetics National Institute of Allergy and Infectious Diseases National Institutes of Health Bethesda Maryland United States of America

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

Laboratory of Malaria and Vector Research National Institute of Allergy and Infectious Diseases National Institutes of Health Bethesda Maryland United States of America

Research Technology Branch National Institute of Allergy and Infectious Diseases National Institutes of Health Rockville Maryland United States of America

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