Veterinary Parasitology 219 (2016) 44–52 Contents lists available at ScienceDirect Veterinary Parasitology journal h om epa ge: www.elsevier.com/ locate/vetpar Research paper BmTI-A, a Kunitz type inhibitor from Rhipicephalus microplus able to interfere in vessel formation Tatiane S. Soares a , Felipe Oliveira a , Ricardo J.S. Torquato a , Sergio D. Sasaki b , Mariana S. Araujo a , Thaysa Paschoalin c , Aparecida S. Tanaka a,∗ a Department of Biochemistry, Escola Paulista de Medicina, Universidade de Federal de São Paulo (UNIFESP), Rua 3 de Maio 100, 04044-020 São Paulo, SP, Brazil b Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, Rua Catequese, 242, 09090-400 Santo André, SP, Brazil c Department of Microbiology, Immunology and Parasitology, Escola Paulista de Medicina, Universidade de Federal de São Paulo (UNIFESP), 04023-062 São Paulo, SP, Brazil a r t i c l e i n f o Article history: Received 15 August 2015 Received in revised form 23 December 2015 Accepted 27 January 2016 Keywords: Tick Rhipicephalus microplus Kallikrein Plasmin Protease inhibitor Kunitz Angiogenesis a b s t r a c t Rhipicephalus microplus is an ectoparasite responsible for transmissions of babesiosis and anaplasmosis causing large losses to livestock production. To survive R. microplus tick produces several active molecules, such as protease inhibitors. This ectoparasite has been described as a rich source of serine protease inhibitors most of them are Kunitz-BPTI members named BmTIs which have no clear function yet. In the present work, we described the expression and functional characterization of rBmTI-A which showed to be similar to the native BmTI-A, a double-headed Kunitz-BPTI inhibitor, capable to inhibit trypsin, human neutrophil elastase (HNE), human plasma kalikrein (HuPK) and human plasmin. rBmTI-A was able to cause a decrease of HUVEC cell viability. Besides, the rBmTI-A showed to be a potent inhibitor of “in vitro” vessel formation. Our results suggested that BmTI-A may participate in the blood acquisition process interfering in the vessel formation during the tick parasite life stage, around 20 days. In conclusion, BmTI-A is a promising molecule to be used in the drug design and development of new method of R. microplus control. © 2016 Elsevier B.V. All rights reserved. 1. Introduction Rhipicephalus microplus is an ectoparasite, responsible for huge losses in cattle production, mainly by reducing the weight gain and milk production. R. microplus is the vector of infectious diseases such as babesiosis and anaplasmosis, which contribute for loss in livestock productions (Sauer et al., 1995). The control of R. microplus is performed mainly by acaricides, however with the emergence of resistant ticks (George et al., 2004) alternative control method need to be developed; vaccine can be an alternative which can combine efficacy and would avoid the excessive use of chemical products (Willadsen, 2004). Thereby, the identification of R. microplus impor- tant molecules in tick physiology can help in the development of alternative method to control tick infestations. Ticks are rich sources of serine proteinase inhibitors, mainly belonging to the BPTI-Kunitz type family. Several Kunitz inhibitors have been characterized in tick, among them: two inhibitors from ∗ Corresponding author. E-mail address: tanaka.bioq@epm.br (A.S. Tanaka). Ixodes scapularis, the factor Xa inhibitor, Ixolaris (Francischetti et al., 2002) and the FVIIa/tissue factor inhibitor, Penthalaris (Francischetti et al., 2004). In R. microplus, our group described several Kunitz inhibitors such as the boophilin, a trypsin, neu- trophil human neutrophil elastase (HNE) and thrombin inhibitor (Macedo-Ribeiro et al., 2008; Soares et al., 2012); BmCI, a chy- motrypsin inhibitor (Lima et al., 2010); several trypsin inhibitors, named BmTIs were also characterized (Sasaki et al., 2004; Sasaki and Tanaka, 2008). The first native BmTIs characterized was the inhibitor named BmTI-A which strongly inhibited trypsin, HNE, plasmin and human plasma kallikrein (HuPK) (Tanaka et al., 1999). BmTIs inhibitors pool contained BmTI-A used in a bovine immunization experiment followed by challenge with R. microplus showed high efficacy in tick control. But due to the complex com- position and low amount of purified BmTIs obtained in the native form it was not possible to produce a vaccine using this approach (Andreotti et al., 2002). Even though Kunitz proteins are known as potent inhibitors of trypsin, chymotrypsin, kallikrein, and plasmin and that they can control the host blood coagulation (Macedo-Ribeiro et al., 2008; Tanaka et al., 1999); little is known about their possible http://dx.doi.org/10.1016/j.vetpar.2016.01.021 0304-4017/© 2016 Elsevier B.V. All rights reserved.