Regular Article The Kallikrein Inhibitor from Bauhinia bauhinioides (BbKI) shows antithrombotic properties in venous and arterial thrombosis models Marlon V. Brito a , Cleide de Oliveira a , Bruno R. Salu a , Sonia A. Andrade b , Paula M.D. Malloy a , Ana C. Sato a , Cristina P. Vicente c , Misako U. Sampaio a , Francisco H.A. Maffei d, ⁎, Maria Luiza V. Oliva a, ⁎⁎ a Departamento de Bioquímica, Universidade Federal de São Paulo, Rua três de Maio, 100, 04044-020 São Paulo, Brazil b Laboratório de Bioquímica e Biofísica, Instituto Butantan, Av Vital Brazil, 1500, 05503-900 São Paulo, Brazil c Universidade Estadual de Campinas, Instituto de Biologia, Rua Charles Darwin, s/n, 13083-863 Campinas, Brazil d Departamento de Cirurgia e Ortopedia, Faculdade de Medicina de Botucatu, UNESP, 18618-970, Botucatu, Brazil abstract article info Article history: Received 5 December 2013 Received in revised form 31 January 2014 Accepted 25 February 2014 Available online 1 March 2014 Keywords: Blood coagulation Kallikrein Kunitz inhibitor Thrombosis Trypsin inhibitor Tumor cells The Bauhinia bauhinioides Kallikrein Inhibitor (BbKI) is a Kunitz-type serine peptidase inhibitor of plant origin that has been shown to impair the viability of some tumor cells and to feature a potent inhibitory activity against human and rat plasma kallikrein (K iapp 2.4 nmol/L and 5.2 nmol/L, respectively). This inhibitory activity is possi- bly responsible for an effect on hemostasis by prolonging activated partial thromboplastin time (aPTT). Because the association between cancer and thrombosis is well established, we evaluated the possible antithrombotic activity of this protein in venous and arterial thrombosis models. Vein thrombosis was studied in the vena cava ligature model in Wistar rats, and arterial thrombosis in the photochemical induced endothelium lesion model in the carotid artery of C57 black 6 mice. BbKI at a concentration of 2.0 mg/kg reduced the venous thrombus weight by 65% in treated rats in comparison to rats in the control group. The inhibitor prolonged the time for total artery occlusion in the carotid artery model mice indicating that this potent plasma kallikrein inhibitor prevented thrombosis. © 2014 Elsevier Ltd. All rights reserved. Introduction Legume seeds are sources of proteinase inhibitors that interact with varying degrees of specificity with different proteinase classes ac- cording to standard enzyme-substrate reactions. They may contribute to elucidating biochemical processes involved in coagulation, inflamma- tion, formation/repression of tumors, and/or as potential therapeutic agents in specific situations [1]. Legumes in the genus Bauhinia (Caesalpinoideae), colloquially known as cow paw because of the shape of its leaves, are widely distrib- uted in most of the tropical regions worldwide including Africa, Asia, and South America [2]. Several compounds have been isolated from this genus and used in different disease models [3–7]. A 18 kDa protein isolat- ed from Bauhinia bauhinioides seeds [8,9], named Bauhinia bauhinioides Kallikrein Inhibitor (BbKI) contains only one cysteine residue localized at C-terminal and shows similarity to Kunitz-plant inhibitors, classified in the group of Bauhinia-type I inhibitors [1]. BbKI blocks the activity of human (K iapp 2.4 nmol/L) and rat (K iapp 5.2 nmol/L) plasma kallikreins, trypsin (K iapp 2.0 nmol/L), chymotrypsin (K iapp 2.6 nmol/L), and plasmin (K iapp 33.1 nmol/L). Additionally, it is the unique inhibitor, so far, isolated from plants that inhibits the tissue kallikrein activity [1,6,10,11]. Plasma and/or tissue kallikreins are directly involved in tumor progression through increased expression and unregulated proteolysis [12–16], or indirectly involved through the generation of kinins and/or activation of other peptidases [17–20]. Recombinant BbKI showed specificity in the inhibition of tumor cell viability [21]. In addition, kallikrein inhibition is possibly responsible for an effect on hemostasis by prolonging activated partial thromboplastin time (aPTT) in rat and human plasma [6]. Because cancer and thrombosis are associated, we studied BbKI in venous and arterial thrombosis models under the hypothesis that the same substance could display both antitumor and anti-thrombotic activities. Materials and Methods Agents Unfractionated heparin (UFH) (liquemine® - Roche), human plasma kallikrein (HuPK) (EC 3.4.21.34) (0.042 μmol/L) purified according to the procedure previously described by Oliva [22]. Trypsin (EC 3.4.21.4) Thrombosis Research 133 (2014) 945–951 Abbreviations: aPTT, activated partial thromboplastin time; BAPA, Nα-benzoyl-D- L-arginine-p-nitroanilide; BbKI, Bauhinia bauhinioides Kallikrein Inhibitor; rBbKI, Bauhinia bauhinioides Kallikrein Inhibitor recombinant form; HuPK, human plasma kallikrein; PT, prothrombin time; UFH, Unfractionated heparin. ⁎ Correspondence to F.H.A. Maffei, Departamento de Bioquímica, Universidade Federal de São Paulo, Rua três de Maio, 100, 04044-020 São Paulo, Brazil. Tel.: +55 11 5576 4445. ⁎⁎ Corresponding author. Tel.: +55 11 5576 4445. E-mail addresses: fhmaffei@uol.com.br (F.H.A. Maffei), olivaml.bioq@epm.br (M.L.V. Oliva). http://dx.doi.org/10.1016/j.thromres.2014.02.027 0049-3848/© 2014 Elsevier Ltd. All rights reserved. Contents lists available at ScienceDirect Thrombosis Research journal homepage: www.elsevier.com/locate/thromres