title: In Vitro Mode of Action and Anti-thrombotic Activity of Boophilin, a Multifunctional Kunitz Protease Inhibitor from the Midgut of a Tick Vector of Babesiosis, Rhipicephalus microplus.
abstract: BACKGROUND
  Hematophagous mosquitos and ticks avoid host hemostatic system through expression of enzyme inhibitors targeting proteolytic reactions of the coagulation and complement cascades. While most inhibitors characterized to date were found in the salivary glands, relatively few others have been identified in the midgut. Among those, Boophilin is a 2-Kunitz multifunctional inhibitor targeting thrombin, elastase, and kallikrein. However, the kinetics of Boophilin interaction with these enzymes, how it modulates platelet function, and whether it inhibits thrombosis in vivo have not been determined.
  
  
  RESULTS
  Boophilin was expressed in HEK293 cells and purified to homogeneity. Using amidolytic assays and surface plasmon resonance experiments, we have demonstrated that Boophilin behaves as a classical, non-competitive inhibitor of thrombin with respect to small chromogenic substrates by a mechanism dependent on both exosite-1 and catalytic site. Inhibition is accompanied by blockade of platelet aggregation, fibrin formation, and clot-bound thrombin in vitro. Notably, we also identified Boophilin as a non-competitive inhibitor of FXIa, preventing FIX activation. In addition, Boophilin inhibits kallikrein activity and the reciprocal activation, indicating that it targets the contact pathway. Furthermore, Boophilin abrogates cathepsin G- and plasmin-induced platelet aggregation and partially affects elastase-mediated cleavage of Tissue Factor Pathway Inhibitor (TFPI). Finally, Boophilin inhibits carotid artery occlusion in vivo triggered by FeCl3, and promotes bleeding according to the mice tail transection method.
  
  
  CONCLUSIONS
  Through inhibition of several enzymes involved in proteolytic cascades and cell activation, Boophilin plays a major role in keeping the midgut microenvironment at low hemostatic and inflammatory tonus. This response allows ticks to successfully digest a blood meal which is critical for metabolism and egg development. Boophilin is the first tick midgut FXIa anticoagulant also found to inhibit thrombosis.
authors.affiliation: Vector Biology Section, Laboratory of Malaria and Vector Research (LMVR), National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, Maryland, United States of America.
authors.forename: Teresa C
authors.initials: TC
authors.lastname: Assumpção
authors.affiliation: Vector Biology Section, Laboratory of Malaria and Vector Research (LMVR), National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, Maryland, United States of America.
authors.forename: Dongying
authors.initials: D
authors.lastname: Ma
authors.affiliation: Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Daniella M
authors.initials: DM
authors.lastname: Mizurini
authors.affiliation: Protein Science Laboratory, Department of Biological Sciences, National University of Singapore, Singapore, Singapore.
authors.forename: R Manjunatha
authors.initials: RM
authors.lastname: Kini
authors.affiliation: Vector Biology Section, Laboratory of Malaria and Vector Research (LMVR), National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, Maryland, United States of America.
authors.forename: José M C
authors.initials: JM
authors.lastname: Ribeiro
authors.affiliation: Biology Centre of the Czech Academy of Sciences, Institute of Parasitology, České Budějovice, Czech Republic.
authors.forename: Michail
authors.initials: M
authors.lastname: Kotsyfakis
authors.affiliation: Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Robson Q
authors.initials: RQ
authors.lastname: Monteiro
authors.affiliation: Vector Biology Section, Laboratory of Malaria and Vector Research (LMVR), National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, Maryland, United States of America.
authors.forename: Ivo M B
authors.initials: IM
authors.lastname: Francischetti
mesh_terms: D000818:Animals; D002460:Cell Line; D015945:Factor XIa / Q000037:antagonists & inhibitors; D005343:Fibrinolytic Agents / Q000302:isolation & purification* / Q000378:metabolism*; D041981:Gastrointestinal Tract / Q000737:chemistry; D015870:Gene Expression; D006801:Humans; D007610:Kallikreins / Q000037:antagonists & inhibitors; D051379:Mice; D010974:Platelet Aggregation / Q000187:drug effects; D011480:Protease Inhibitors / Q000302:isolation & purification* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D048494:Rhipicephalus / Q000737:chemistry*; D013917:Thrombin / Q000037:antagonists & inhibitors; D013927:Thrombosis / Q000139:chemically induced / Q000517:prevention & control
chemical_list: D005343:Fibrinolytic Agents; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D007610:Kallikreins; D015945:Factor XIa; D013917:Thrombin