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.

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...

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Main Authors: Teresa C Assumpção (Author), Dongying Ma (Author), Daniella M Mizurini (Author), R Manjunatha Kini (Author), José M C Ribeiro (Author), Michail Kotsyfakis (Author), Robson Q Monteiro (Author), Ivo M B Francischetti (Author)
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Published: Public Library of Science (PLoS), 2016-01-01T00:00:00Z.
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100 1 0 |a Teresa C Assumpção  |e author 
700 1 0 |a Dongying Ma  |e author 
700 1 0 |a Daniella M Mizurini  |e author 
700 1 0 |a R Manjunatha Kini  |e author 
700 1 0 |a José M C Ribeiro  |e author 
700 1 0 |a Michail Kotsyfakis  |e author 
700 1 0 |a Robson Q Monteiro  |e author 
700 1 0 |a Ivo M B Francischetti  |e author 
245 0 0 |a 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. 
260 |b Public Library of Science (PLoS),   |c 2016-01-01T00:00:00Z. 
500 |a 1935-2727 
500 |a 1935-2735 
500 |a 10.1371/journal.pntd.0004298 
520 |a 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. METHODOLOGY/PRINCIPAL FINDINGS: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. CONCLUSION/SIGNIFICANCE: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. 
546 |a EN 
690 |a Arctic medicine. Tropical medicine 
690 |a RC955-962 
690 |a Public aspects of medicine 
690 |a RA1-1270 
655 7 |a article  |2 local 
786 0 |n PLoS Neglected Tropical Diseases, Vol 10, Iss 1, p e0004298 (2016) 
787 0 |n http://europepmc.org/articles/PMC4706430?pdf=render 
787 0 |n https://doaj.org/toc/1935-2727 
787 0 |n https://doaj.org/toc/1935-2735 
856 4 1 |u https://doaj.org/article/ba3e1106f28645379f8830423cbc6335  |z Connect to this object online.