title: Simplagrin, a platelet aggregation inhibitor from Simulium nigrimanum salivary glands specifically binds to the Von Willebrand factor receptor in collagen and inhibits carotid thrombus formation in vivo.
abstract: BACKGROUND
  Among the several challenges faced by bloodsucking arthropods, the vertebrate hemostatic response against blood loss represents an important barrier to efficient blood feeding. Here we report the first inhibitor of collagen-induced platelet aggregation derived from the salivary glands of a black fly (Simulium nigrimanum), named Simplagrin.
  
  
  RESULTS
  Simplagrin was expressed in mammalian cells and purified by affinity-and size-exclusion chromatography. Light-scattering studies showed that Simplagrin has an elongated monomeric form with a hydrodynamic radius of 5.6 nm. Simplagrin binds to collagen (type I-VI) with high affinity (2-15 nM), and this interaction does not involve any significant conformational change as determined by circular dichroism spectroscopy. Simplagrin-collagen interaction is both entropically and enthalpically driven with a large negative ΔG, indicating that this interaction is favorable and occurs spontaneously. Simplagrin specifically inhibits von Willebrand factor interaction with collagen type III and completely blocks platelet adhesion to collagen under flow conditions at high shear rates; however, Simplagrin failed to block glycoprotein VI and Iα2β1 interaction to collagen. Simplagrin binds to RGQOGVMGF peptide with an affinity (K(D) 11 nM) similar to that of Simplagrin for collagen. Furthermore, Simplagrin prevents laser-induced carotid thrombus formation in vivo without significant bleeding in mice and could be useful as an antithrombotic agent in thrombosis related disease.
  
  
  CONCLUSIONS
  Our results support the orthology of the Aegyptin clade in bloodsucking Nematocera and the hypothesis of a faster evolutionary rate of salivary function of proteins from blood feeding arthropods.
authors.affiliation: Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Rockville, Maryland, United States of America.
authors.forename: Andrezza C
authors.initials: AC
authors.lastname: Chagas
authors.affiliation: Physical and Biochemistry Section, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, Maryland, United States of America.
authors.forename: Peter
authors.initials: P
authors.lastname: McPhie
authors.affiliation: Animal Surgery and Resources Core, National Heart Lung and Blood Institute, NIH, Bethesda, Maryland, United States of America.
authors.forename: Hong
authors.initials: H
authors.lastname: San
authors.affiliation: Laboratory of Malaria Immunology and Vaccinology, NIAID, NIH, Bethesda, Maryland, United States of America.
authors.forename: David
authors.initials: D
authors.lastname: Narum
authors.affiliation: Laboratory of Malaria Immunology and Vaccinology, NIAID, NIH, Bethesda, Maryland, United States of America.
authors.forename: Karine
authors.initials: K
authors.lastname: Reiter
authors.affiliation: Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Rockville, Maryland, United States of America.
authors.forename: Fuyuki
authors.initials: F
authors.lastname: Tokomasu
authors.affiliation: Centro de Pesquisas Aggeu Magalhães (CPqAM/FIOCRUZ) and Laboratório de Imunopatologia Keizo Asami. Universidade Federal de Pernambuco, Recife, Pernambuco, Brazil.
authors.forename: Fabio A
authors.initials: FA
authors.lastname: Brayner
authors.affiliation: Centro de Pesquisas Aggeu Magalhães (CPqAM/FIOCRUZ) and Laboratório de Imunopatologia Keizo Asami. Universidade Federal de Pernambuco, Recife, Pernambuco, Brazil.
authors.forename: Luiz C
authors.initials: LC
authors.lastname: Alves
authors.affiliation: Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Rockville, Maryland, United States of America.
authors.forename: José M C
authors.initials: JM
authors.lastname: Ribeiro
authors.affiliation: Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Rockville, Maryland, United States of America.
authors.forename: Eric
authors.initials: E
authors.lastname: Calvo
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002341:Carotid Artery Thrombosis / Q000517:prevention & control; D024061:Collagen Type III / Q000378:metabolism; D005260:Female; D057809:HEK293 Cells; D006801:Humans; D019476:Insect Proteins / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D010973:Platelet Adhesiveness; D010974:Platelet Aggregation; D010975:Platelet Aggregation Inhibitors / Q000378:metabolism*; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D012469:Salivary Glands / Q000737:chemistry*; D012843:Simuliidae / Q000378:metabolism*
chemical_list: D024061:Collagen Type III; D019476:Insect Proteins; D010975:Platelet Aggregation Inhibitors; D010980:Platelet Membrane Glycoproteins; D011994:Recombinant Proteins; C000604185:simplagrin; C028254:von Willebrand factor receptor