WO2009018640A1 - Use of platelet aggregation and coagulation inhibitors isolated from bauhinia sp. - Google Patents

Abstract

The present invention relates to the use of peptidase inhibitors from Bauhinia sp. with anti-thrombotic activities, as well as to a pharmaceutical composition comprising them.

Description

USE OF PLATELET AGGREGATION AND COAGULATION INHIBITORS ISOLATED FROM BAUHINIA SP.

FIELD OF THE INVENTION

The present invention refers to the use of peptidase inhibitors isolated from Bauhinia sp. with antithrombotic properties, as well as a pharmaceutical composition comprising them for the treatment of diseases related to platelet aggregation as, for example, venous thrombosis, arterial thrombosis, deep venous thrombosis, atherosclerosis, systemic thromboembolism, pulmonary thromboembolism, ischemic, myocardial infarct, arrhythmias and unstable angina pectoris. BACKGROUND OF THE INVENTION

Nowadays, a series of anti-platelet agents is available for clinical use. Aspirin is the most extensive agent used in patients with ischemic cardiopathy. Its mechanism of action involves platelet aggregation inhibition induced by thromboxane A₂, through irreversible acetylation of cyclooxygenase. Moreover, it partially inhibits aggregation induced by ADP, collagen and thrombin. Nevertheless, in prolonged treatments, aspirin may cause gastrointestinal ulceration and salicilism characterized by encephalitis, vertigo and dizziness. Other collateral effects include nausea, vomit, dyspepsia, epigastric discomfort and pyrosis. Additionally, its concomitant ingestion with alcohol may cause gastrointestinal hemorrhage and bleeding time increase.

Others anti-platelet include ticlopidine and clopidogrel, which belongs to the same group of drugs, the thienopyridines. Its mechanism of action is through by the blockade of ADP linkage with its platelet receptor. Both efficacies are similar, but clopidogrel is associated with a minor occurrence of adverse effects. However, clopidogrel is also associated to allergic cutaneous reactions, particularly hives, "rash" and itch. Glycoprotein Ilb/IIIa antagonists inhibit the platelet aggregation final path through the blockage of the platelet surface Ilb/IIIa receptor linkage with fibrinogen, with Willebrand factor and with fibronectin. This class of agents is constituted by monoclonal antibodies, as abciximab, tiny peptides, as epitifibatide, and non- peptidic compounds, as tirofiban. Nevertheless, they present the inconvenience of only be administered by intravenous path.

Many factors, as nitrous oxide - NO, are straightly involved in the process of platelet aggregation in the wounded blood vessel. NO mediates many phenomena, as endothelial-dependent vessel loosening, cytotoxicity mediated by macrophages, platelet activation, adhesion and aggregation inhibition and regulation of basal blood pressure (Mollace et al. , 1991; Kiechele & Malinski, 1993; Shapira et al . , 1994).

Many inhibitors of platelet aggregation were characterized from snakes. These inhibitors show a RGD sequence in common and are potent inhibitors of platelet aggregation because they compete with fibrinogen due to their high affinity by GP Ilb/IIIa receptor (Huang et al. , 1987; Chao et al., 1989; Niewiarowski et al. , 1994; You et al. , 2006) .

The first factor Xa inhibitor found in leech was anistatin isolated from Haementeria officinalis salivary glandule (Tuszynski et al. , 1987), where other molecule named LAPP was found (Luch Antiplatelet Protein) ; and that inhibits platelet aggregation induced by collagen, since it blocks the platelet adhesion to collagen (Connolly et al., 1992) . Triadegin was isolated from the salivary glandule of Haementeria gilianni leech, which is a factor XIIIa inhibitor, and potent inhibitors were isolated from Theromyzon tessulatum species, the terostatin, which inhibits Xa factor, and teromina, which is a thrombin inhibitor (Salzet et al., 2000; Chopin et al., 2000) .

Nevertheless, the isolation of those substances is limited not only by difficulties in the obtainment of saliva from these animals, but also by the fact that they are present in very tiny quantities (Ciprandi, 2003) . Inhibitors of plant sources

Therefore, the use of inhibitors of plant sources can be an alternative to the use of inhibitors from animal sources, as long as peptidase inhibitors are abundant in storage organs, seeds and plant tissues (Ryan, 1989) . Many families of peptidase inhibitors were characterized; mainly serine peptidase inhibitors from Leguminosae, Solanaceae and Gramlneae (Olmeda, 1987, Pouvreau et al., 2003) .

In plants, inhibitors are generally storage proteins, nitrogen sources, and are one of the defense mechanisms of plants (Putzai et al., 1988) . These inhibitors, which are concentrated in plant's seeds, show the property to form stoichometric complexes with proteolytic enzymes in vivo and in vitro, this inhibition being strictly competitive (Ryan, 1978) . Plant inhibitors have natural function in blood coagulation and the variation of its specificity allows, usually, its application in laboratories (Thomaz et al., 1990) .

Inhibitors isolated from maize were used in the purification of XIIa coagulation factor through affinity chromatography (Rattnof et al . , 1985). In cabbage, inhibitors were identified for many peptidases with central role on homeostasis (Carter et al., 1990) . In the raw extract from bromeliad and pineapple, some components related as peptidases showed anti-inflammatory, anti- thrombotic and fibrinolytic activities (Maurer, 2001) .

Leguminous seeds are an excellent protein source, and the peptidase inhibitors found were the most studied in this class of compounds. The peptidase inhibitors isolated from plant seeds, like leguminous, are classified into families according to its primary structure, molecular mass and cysteine residues (Wenzel & Tscheche 1981; Richardson, 1991) .

The main families of plant inhibitors are: pumpkin, potatoes I and II, Bowman-Birk and Kunitz (Richardson, 1991) .

Kunitz type inhibitors are characteristics because they present their molecular weight around 20 KDa, containing four cysteine residues forming two disulfide bridges, and its reactive site shows an Arg residue in Pl that is located in a protein loop. They are encountered in seeds, storage organs and vegetative tissues of many plant species, having defense function against pathogens and insects (Richardson, 1991) .

The first inhibitor of this class was isolated from soybean seeds by Kunitz, 1945. This inhibitor, named soybean trypsin inhibitor, STI, is a typical representative of the class of Kunitz type inhibitors (Hoffman et al., 1984, Krauchenco et al. , 2004).

Kunitz type inhibitors were isolated by the present inventors from the seeds of Brazilian plants from Bauhinia genus (Oliva et al., 2000) . Bauhinias are trees or shrubs owing to Leguminosae family, Caesalpinoideae sub-family

(Hutchinson, 1972) .

Through comparative studies based on databank researches was possible to verify that the seed inhibitors from Bauhinia bauhinioides, as cruzipain inhibitor BbCl- from Bauhinia bauhinioides and human plasmatic kallikrein inhibitor BbKI- from Bauhinia bauhinioides , belongs to Kunitz family and show structural particularities (Oliva efc al., 1999; Oliveira, 2001). The Bauhinia sp. seeds have a high content of proteins related to other parts of the plant (Boulter et al., 1967) and a large quantity of these proteins correspond to peptidase inhibitors, generally trypsin inhibitors (Richardson, 1991) . In Bauhinia, Kunitz type serine peptidases inhibitors were isolated and sequenced from various species as Bauhinia variegata (Αndrade, 2003) , from B. rufa (Sumikawa et al., 2006,), from B. pentandra (Oliva et al., 1996), from B. forficata (Nakahata, 2001) and two inhibitors from B. bauhinioides (Oliva et al., 1999b; Oliveira et al., 2001) . The action was verified on enzymes involved on digestive process as trypsin and chymotrypsin and coagulation enzymes as human plasmatic kallikrein, thrombin, plasmin (Oliva et al., 2003) . "In vivo", the action of kallikrein inhibitor, BbKI, and of elastase and cruzipain inhibitor, BbCl, from B. bauhinioides, was verified in inflammation models induced by carragenin (Oliveira, 2004), in an isquemic and reperfusion model (Santomauro-Vaz, 2005) ; and for the study of effects of inhibitors on tumor development, were used BbCl, BbKi from B. bauhinioides (Nakahata, 2005), also embraced by the patent application PI 0601390-2, here incorporated in its entirely by reference.

These inhibitors, isolated by the present inventors, are used aiming to verify the specificity of blood coagulation enzymes, like plasmatic kallikrein, Xa and XIIa factors, enzymes involved on digestion processes like trypsin and chemotrypsin (Oliva et al. , 2000) . And with it, to provide a pharmaceutical composition comprising such inhibitors to treat diseases related with platelet aggregation. Therefore, as is described by the present invention, the use of peptidase inhibitors isolated from Bauhinla sp. for the treatment of diseases related to platelet aggregation constitutes a therapeutic alternative to the use of inhibitors from animal origin and others, besides presenting a better efficacy and specificity to diverse factors of platelet aggregation. BRIEF DESCRIPTION OF THE DRAWINGS

The following figures are part of the present specification and are herein included to illustrate specific aspects of the invention. The present invention can be better understood with reference to one or more of these figures, in combination with the detailed description of the preferred embodiment presented here.

Figure 1 shows the trypsin inhibition by the saline extract. 20 μL of trypsin (0,77 μM) and crescent doses of inhibitor were used and pre-incubated, during 10 minutes, at 37°C in a 50 mM Tris/HCl buffer containing 0,02% CaCl₂, in a final volume of 250 μL. The inhibitory activity was determined by the hydrolysis of 25 μL Bz-Arg-pNan (10 mM) as substrate. Figure 2 shows trypsin inhibition by ketonic extract. 20 μL of trypsin (0,77 μM) and crescent doses of the inhibitor were used and pre-incubated, during 10 minutes, at 37°C in 50 mM Tris/HCl buffer containing 0,02% CaCl₂, in a final volume of 250 μL. The inhibitory activity was determined by the hydrolysis of 25 μL Bz-Arg-pNan (10 mM) as substrate.

Figure 3 shows the ionic exchange chromatography in a DEAE-Sephadex column A-50. 30 mL of sample from B. purpurea ketonic precipitate containing 5.13 mg (A₂₈o) were applied in a DEAE-Sephadex column (25 mL) equilibrated with 0,1 M Tris-HCl equilibration buffer, pH 8.0. The equilibration buffer solutions containing NaCl 0.15 M and 0.30 M were used to elute non-adsorbed substances. Chromatography was made under a 1,5 mL/min flux. 1 - non-adsorbed substance; 2 -material eluted with equilibration buffer containing 0.15 M NaCl; 3 - material eluted with equilibration buffer containing 0.30 M NaCl.

Figure 4 shows trypsin inhibition by non-adsorbed material in DEAE-Sephadex A-50 column. 20 μL trypsin (0,77 μM) and crescent doses of inhibitor were used and pre- incubated, during 10 minutes, at 37°C in 50 mM Tris-HCl buffer containing 0.02% CaCl₂, in a final volume of 250 μL. The inhibitory activity was determined by hydrolysis of 25 μL Bz-Arg-pNan (10 mM) as substrate. Figure 5 shows trypsin inhibition by the material eluted with equilibrium buffer containing 0.15 M NaCl from DEAE-Sephadex A-50 column. 20 μL trypsin (0.77 μM) and crescent doses of inhibitor were used and pre-incubated, during 10 minutes, at 37°C in 50 mM Tris-HCl buffer containing CaCl₂ 0.02%, in a final volume of 250 μL. The inhibitory activity was determined by hydrolysis of 25 μL Bz-Arg-pNan (10 mM) as substrate.

Figure 6 shows trypsin inhibition by the material eluted with equilibrium buffer containing 0.30 M NaCl from DEAE-Sephadex A-50 column. 20 μL trypsin (0.77 μM) and crescent doses of inhibitor were used and pre-incubated, during 10 minutes, at 37°C in 50 mM Tris-HCl buffer containing CaCl₂ 0.02%, in a final volume of 250 μL. The inhibitory activity was determined by hydrolysis of 25 μL Bz-Arg-pNan (10 mM) as substrate.

Figure 7 shows affinity chromatography in trypsin- Sepharose. 155 mL of non-adsorbed sample material in DEAE- Sephadex A-50 containing 0.358 mg (A₂₈₀) were applied in a trypsin-Sepharose column (09 mL) equilibrated with Tris/HCl equilibrium buffer 0.1 M, pH 8.0; The KCl/0.5 M HCl solution, pH 2.0, was used to elute proteins linked to trypsin. The chromatography was made under a flux of 1.5 mL/min.

Figure 8 shows trypsin inhibition by BpuTI inhibitor (eluted with a 0.5 M KC1/HC1 solution, pH 2.0) . 20 μL trypsin (0.77 μM) and crescent doses of inhibitor were used and pre-incubated, during 10 minutes, at 37⁰C in 50 mM Tris-HCl buffer containing CaCl₂ 0.02%, in a final volume of 250 μL. The inhibitory activity was determined by hydrolysis of 25 μL Bz-Arg-pNan (10 mM) as substrate. Figure 9 shows the reverse-phase chromatography of BpuTI inhibitor. Sample: 0.8 mg by A₂₈O of the active "pool" originated from trypsin-Sepharose affinity chromatography. Cis column from a HPLC system (4.6 mm x 15 cm) equilibrated with 0.1% TFA in water. Elution by linear gradient of acetonitrile in 0.1% TFA in water from 0 to 100% in 60 min.

Figure 10 .shows a gel electrophoresis in polyacrylamide gel (12%) of BpuTI. 1 - BpuTI, after chromatography on trypsine-Sepharose (20 μg) , non-reduced sample; 2 - BpuTI, after chromatography on trypsine- Sepharose (20 μg) , non-reduced sample; 3 - Standard of low molecular weight (15 μg) , reduced proteins; 4 - BpuTI, after chromatography on trypsine-Sepharose (20 μg) , reduced sample; 5 - BpuTI, after chromatography on trypsine- Sepharose (20 μg) , reduced sample.

Figure 11 shows the similarity of the BpuTI N-terminal region with trypsin inhibitors.

Figure 12 shows the CD spectrum of BpuTI inhibitor, 0,273 mg/mL, in aqueous solution. The spectrum was registered from 190 to 250 nm, with an average of thirty- two scanning, in a cylindrical cuvette of 1 mm of optical path. The content estimative of the secondary structure was calculated by CDPro program (Sreerama & Woody, 2000) .

Figure 13 shows the CD spectra of BpuTI inhibitor (0.273 mg/mL), as a pH function. PBA buffer was used, 10 mM, at pH 4.0; pH 6.0; pH 7.0; pH 9.0 and pH 10.0. The spectra were registered from 190 to 250 nm, as an average of eight scanning, in a cylindrical cuvette of 1 mm of optical path. The estimative of the secondary structure was calculated by CDPro program (Sreerama & Woody, 2000) .

Figure 14 shows the trypsin inhibition by BpuTI inhibitor (eluted with a 0.5 M KC1/HC1 solution pH 2.0) . 20 μL of trypsin (0.77 μM) and crescent doses of the inhibitor were used and pre-incubated, during 10 minutes, at 37 °C in 50 itiM Tris-HCl buffer containing CaCl₂ 0.02%, in a final volume of 250 μL. The inhibitory activity was determined by hydrolysis of 25 μL Bz-Arg-pNan (10 mM) as substrate.

Figure 15 shows the chymotrypsin inhibition by BpuTI inhibitor (eluted with a 0.5 M KC1/HC1 solution, pH 2.0) . 15 μL chymotrypsin (16 μM) and crescent doses of the inhibitor were used and pre-incubated, during 10 minutes, at 37°C in 50 mM Tris/HCl buffer containing 0.05 M NaCl, in a final volume of 250 μL. The inhibitory activity was determined by the hydrolysis of Suc-Phe-pNan 20 μL (50 mM) .

Figure 16 shows the HuPK inhibition by BpuTI inhibitor (eluted with a 0.5 M KC1/HC1 solution, pH 2.0) . 30 μL HuPK

(1.08 μM) and crescent doses of the inhibitor were used and pre-incubated, during 10 minutes, at 37°C in Tris/HCl buffer 50 mM containing 0.05 M NaCl, in a final volume of

250 μL. The inhibitory activity was determined by hydrolysis of 20 μL of HD-Pro-Phe-Arg-pNan (5 mM) .

Figure 17 shows the BpuTI influence (2.3 μM) on the time of partially activated thromboplastin (TTPA) . The time of partially activated thromboplastin was determined after a pre-incubation period of 2 minutes at 37 °C, from BpuTI (2.3 μM) , with 50 μL of human plasma, following the addition of 200 μL reagent. 1 - control; 2 - BpuTI (2.3 μM) .

Figure 18 shows the influence of BpuTI (2.3 μM) on the prothrombin time. The prothrombin time was determined after a BpuTI pre-incubation period of 2 minutes at 37°C (2.3 μM) , with 50 μL of human plasma, followed by the addition of 200 μL of reagent. 1 - control; 2 - BpuTI (2.3 μM) .

Figure 19 shows the platelet aggregation induced by ADP (20 μM) in platelet rich plasma suspension (PRP) . Trace 1 - control; Trace 2 - 2 μM BpuTI; Trace 3 - 4 μM BpuTI; Trace 4 - 6 μM BpuTI.

Figure 20 shows the platelet aggregation induced by collagen (2 μg/mL) in suspension of platelet rich plasma (PRP) . Trace 1 - control; Trace 2 - 2 μM BpuTI; Trace 3 - 4 μM BpuTI; Trace 4 - 6 μM BpuTI.

Figure 21 shows platelet aggregation induced by thrombin (1 uNHI/mL) in a washed platelet suspension (WP) . Trace 1 - control; Trace 2 - 2 μM BpuTI; Trace 3 - 4 μM BpuTI; Trace 4 - 6 μM BpuTI.

Figure 22 shows the platelet aggregation induced by ADP (20 μM) in suspension of platelet rich plasma (PRP) . Trace 1 - control; Trace 2 - 2 μM BbKI; Trace 3 - 4 μM BbKI; Trace 4 - 6 μM BbKI. Figure 23 shows platelet aggregation induced by collagen (2 μg/mL) in a suspension of platelet rich plasma (PRP) . Trace 1 - control; Trace 2 - 2 μM BbKI; Trace 3 - 4 μM BbKI; Trace 4 - 6 μM BbKI.

Figure 24 shows platelet aggregation induced by thrombin (1 uNHI/mL) in a washed platelet suspension (WP) . Trace 1 - control; Trace 2 - 2 μM BbKI; Trace 3 - 4 μM BbKI; Trace 4 - 6 μM BbKI.

Figure 25 shows platelet aggregation induced by ADP (20 μM) in a suspension of platelet rich plasma (PRP) . Trace 1 - control; Trace 2 - 2 μM BbCI; Trace 3 - 4 μM BbCI; Trace 4 - 6 μM BbCI.

Figure 26 shows platelet aggregation induced by- collagen (2 μg/mL) in suspension of platelet rich plasma (PRP) . Trace 1 - control; Trace 2 - 2 μM BbCI; Trace 3 - 4 μM BbCI; Trace 4 - 6 μM BbCI.

Figure 27 shows platelet aggregation induced by thrombin (1 uNHI/mL) in a washed platelet suspension (WP) . Trace 1 - control; Trace 2 - 2 μM BbCI; Trace 3 - 4 μM BbCI; Trace 4 - 6 μM BbCI.

DETAILED DESCRIPTION OF THE INVENTION

The present invention refers to the use of peptidase inhibitors isolated from Bauhinia sp., preferably, from Baυhinia bauhinioid.es and Bauhinia purpurea var. corneri de Wit seeds for the treatment of diseases related to platelet aggregation.

In accordance to an aspect of the present invention, a pharmaceutical composition is provided comprising an effective amount of at least one serine peptidase inhibitor from Bauhinia sp. with inhibitory properties of platelet aggregation and coagulation. Such inhibitors are isolated from the entirety or part of plants from the genera Bauhinia sp. that comprises Bauhinia bauhinioides e a Bauhinia purpurea var. corneri de Wit, more preferably, the cruzipain inhibitor of Bauhinia bauhinioides (BbCl) , the human plasmatic kallikrein inhibitor of Bauhinia bauhinioides (BbKI) and the trypsin inhibitor of Bauhinia purpurea var. corneri de Wit (BpuTI) .

Typically, the pharmaceutical compositions of the present invention comprise at least one inhibitor in accordance to the present invention and at least one pharmaceutically acceptable carrier. Such pharmaceutically acceptable carriers include, but are not limited to aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and others of the art knowledge. The exact pH and concentration of various composition components can be adjusted in accordance with the current practice .

Preferably, pharmaceutical compositions of the present invention comprise at least one serine peptidase inhibitor from Bauhinia sp. , according to the present invention, in a concentration from 100 to 300 mg, more preferably from 150 to 200 mg, for the treatment of diseases related to platelet aggregation. Such diseases include but are not limited to venous thrombosis, arterial thrombosis, deep venous thrombosis, atherosclerosis, systemic thromboembolism, pulmonary thromboembolism, ischemic, myocardial infarct, arrhythmias and unstable angina pectoris. Nevertheless, it is to be emphasized that a specific dosage and treatment regimen to any particular patient will depend on many factors, including the activity of the specific compound used, age, body weight, global health, sex, food, administration time, excretion time, drug combination, the physician judgment and the gravity of the disease that is being particularly treated. The quantity of active ingredients will also depend on the particular compound and on the other therapeutic agent, if present, in the composition. Further, the present invention will be explained in a more detailed manner with the aid of the description of its preferred embodiment.

Isolation and purification of trypsin inhibitor from

Bauhinia purpurea var. corneri de Wit (BpuTI) seeds

The Bauhinia purpurea var. corneri de Wit. seeds (60 g) were triturated in a liquefier and the resulting flour was homogenized in 800 mL of saline solution (0.15 M NaCl) . The material was centrifuged at 4000 rpm for 20 minutes at 4⁰C and the supernatant named saline extract was used as the starting material for the achievement of the present invention.

During the purification steps, the proteic concentrations of the samples were estimated spectrophotometrically by its absorbance at 280 nm and assuming as an empiric value 1.0 mg/mL of protein for solutions that present absorbance equals to 1.0.

Quantitative determinations of proteins were also realized in accordance with Spector (1978) using Coomassie blue G-250 as colorant and bovine albumin as standard.

To the saline extract, cold acetone was slowly added, shaking continuously until it reached the final concentration of 80% of saturation. The mixture was kept in a cold chamber at 4⁰C for about 30 minutes. The acetone excess was removed with the aid of a syringe connected to a tube and then the material was centrifuged at 1000 rpm for 10 minutes at 4°C. The ketonic precipitate was kept at room temperature during 20 minutes for the complete elimination of acetone, resuspended in 500 mL of Tris/HCl 20 mM solution, pH 8.0, and centrifuged once again at 1500 rpm for 10 minutes. The supernatant was transferred to a clean flask and freezed for later use.

Dyspigmentation and purification by ionic exchange chromatography

DEAE-Sephadex A-50 column was prepared with 25 mL of resin and equilibrated with 100 mL of 0.1 M Tris/HCl equilibration buffer, pH 8.0, keeping the constant flux of 1.5 mL/min. 30 mL of ketonic extract that, after being resuspended in Tris/HCl (5.0 mg at A₂so) , showed conductivity of 1,6 mMHO and pH 7.0. The same was applied to the resin which was washed with equilibration buffer until the absorbance reading at 280 nm be lower than 0.030. Subsequently, the adsorbed proteins were eluted with 0.1 M Tris/HCl buffer, pH 8.0, containing 0.15 M NaCl and 2 mL fractions were collected until the absorbance reading at 280 nm lower than 0.030. Further, 0.1 M Tris/HCl buffer, pH 8.0, was used containing 0.30 M NaCl, with the same previous standards being adopted. The fractions with higher absorbances at 280 nm were pooled. The inhibitory activity over trypsin was accompanied by the hydrolysis of Bz-Arg- pNan and the fractions were accompanied by electrophoresis. Affinity chromatography purification in trypsin-Sepharose The non-adsorbed proteins fraction from ionic exchange chromatography in DEAE-Sephadex A-50 was chromatographed in an affinity column, following the method described by Oliva et al., 1999. The inhibitors were chromatographed in a column containing 9 mL of trypsin-Sepharose resin in 0.1 M Tris/HCl equilibration buffer, pH 8.0. The non-adsorbed material was pooled and the resin was washed with equilibration buffer until the absorbance reading at 280 nm be lower than 0.030. The resin was later washed with 0.1 M Tris/HCl, pH 8.0, containing 0.15 M NaCl. The retained inhibitor was eluted from the column by acidification with 0.5 M KC1/HC1, pH 2.0, and the collected fractions (1.0 mL/min) were immediately neutralized by the addition of 1.0 M Tris/HCl solution. The protein elution was followed by a photometric reading at 280 nm, and the inhibitory activity over trypsin from the non-adsorbed and the adsorbed material were monitored by hydrolysis of Bz-Arg-pNan and the fraction that showed the trypsin inhibitory activity was accompanied by electrophoresis. Reverse phase HPLC chromatography

The fraction from affinity chromatography Con A Sepharose was chromatographed in a C18 reverse-phase column in a Shimadzu HPLC system, model No. PPSQ-23 (Kyoto, Japan), equilibrated with 0.1% TFA solution in water (solvent A) and eluted with 90% acetonitrile gradient (0 to 100%) in 0.1% TFA (solvent B) as showed in the table below. Table I: Elution gradient for reverse phase chromatography

Solvent B: 90% acetonitrile, 0,1% trifluoroacetic acid in water.

Reduction and alkylation of cysteine residues About 300 nmol of inhibitor were dissolved in 0.3 mL of 0,25 M Tris/HCl buffer, pH 8.5, containing 6 M guanidine, 1 mM EDTA, 5 μL of β-mercaptoethanol and incubated for 2 h at 37 °C, under an hydrogen atmosphere.

The protein alkylation was made according to Friedman et al. method (1970), adding 5 μL of 4-vinylpyridine and incubating at 37⁰C for 90 min. Further, the sample was desalinized through reverse-phase chromatography in HPLC. Polyacrylamide gel electrophoresis (SD5-PA6E)

The purity and molecular weight analysis of the proteins was made by polyacrylamide gel electrophoresis in the presence of SDS, according to the method described by Laemmli (1970) . the gel was prepared using the following solutions: 30% acrylamide (30% acrylamide, 0.8% bisacrylamide) , 45% acrylamide (45% acrylamide, 1.2% bisacrylamide) , 10% SDS, TEMED, ammonium persulfate 200 mg/mL in water and 1 M Tris/HCl buffer, pH 8.8, pH 6.8. For the analysis, 10 μg of proteins from each sample were used, which were freeze-dried and resuspended in 25 μL of sample buffer (0.3 M Tris/HCl, 2% SDS, 20% glycerol, pH 6.68 containing bromophenol blue 0.012%) . The reduced samples were treated with the _. same amount of sample buffer containing DDT (dithiothreitol) 200 mg/mL. All the samples were centrifuged and heated in sand bath for 5 minutes at 100°C.

An electrophoretic standard marker of low molecular weight (LMW) at a concentration of 10 μg, was used as a reference for molecular weights from our purified proteins. The proteins used as molecular weight standard were phosphorylase b, bovine albumin, ovalbumin, carbonic anhydrase, cytochrome c, trypsin inhibitor and α- lactalbumin.

The electrophoresis was made on 0.025 M Tris buffer and 0.18 M glycine, containing 0.1% SDS. After the electrophoretic run, the gel was dyed with Blue Brilliant Comassie and the excess of dye was removed with ethanol solution, acetic acid and water (4.35:1:4.65 v/v/v) . The gel was conserved in 7% acetic acid. Protein N-terminal region se

The automatic sequen< deriving protein was made at UNIFESP Departamento de Bioquimica using a Shimadzu sequencer model PPSQ-23, using the Edman's degradation technique (1956) .

Circular dichroism - Determination of secondary structures

CD is observed when an optically active molecule, named chromophore, present absorption differences of circularly polarized light on the left and on the right. For that, this chromophore must be asymmetric. In the case of proteins, these chromophores are mainly the peptide links, the amino acid side chains and prosthetic groups, all with absorption in the ultraviolet region. Thus, the CD spectrum between 180 and 260 nm (far ultraviolet region) may identify different types of secondary structures as α- helix, β-sheets, β-loops and random structures (Sreerama & Woody, 1994) .

The CD measurements were made in a Jasco J-810 spectropolarimeter (Jasco Corporation, Japan) in a circular quartz cuvette with 1 mm of optical path. The spectrum were registered in the wavelength gap from 190 to 250 nm, at 25°C, with a measurement average of 8 scans using BpuTI (0.27 mg/mL) in sodium phosphate buffer, sodium borate and 10 mM sodium acetate (PBA), pH 4.0; pH 6.0; pH 7.0; pH 9.0 and pH 10.0. The estimative of the secondary structure was made using the CDPro software (Sreerama & Woody, 2000) and the CD data were expressed in molar ellipticity. INHIBITORY PROPERTIES Determination of inhibitory constants Inhibitory constants were determined by the value of dissociation constant of the enzyme-inhibitor complex (Kiapp) . Determinations were made following the Morrison's model, whose final equation was defined by Knight (1986), adapted to a program for computerized enzymatic kinetic graphics, the numeric value being calculated by Grafit software.

Enzymatic dosages and determinations of inhibitory constants

Chromogenic substrates, peptides derived from p- nitroanilide, were used mainly by the high sensibility of photometric detection (at A₄₀₅) of p-nitroaniline liberated, after enzymatic hydrolysis (Erlanger et al., 1961) in a Packard spectrophotometer (SpectraCount model) .

The substrates used in each experiment were the ones who presented the best specificity for tested enzymes. The substrates derived from p-nitroanilide were initially diluted following manufacturer instructions and posterior dilutions were made in an appropriate buffer for each assay.

Fluorogenic substrates, peptides derived from AMC were also used in some dosages due to the fact of being more sensitive than the chromogenic substrates derived from p- nitroanilide. Hydrolysis was monitored at 380 nm, excitation and at 460 nm, emission, wavelengths using a Packard spectrofluorometer (FluoroCount model) . These substrates were diluted in DMSO.

Determination of active trypsin concentration

Active trypsin concentration was determined by titration of its active site by p-nitrophenyl-p' -guanidine benzoate (NPGB) , in accordance with Chase & Shaw (1970) and Sampaio et al. (1984) . The assay was accomplished in a Spectronic Genesys 5 spectrophotometer using 0.06 M barbital buffer 900 μL, pH 8.3, to whom 100 μL of trypsin solution 1.0 mg/mL were added (in weight) followed by 5 μL of substrate (NPGB) 9.4 mM. The experiments were made in triplicate and the active trypsin concentration was calculated considering the obtained means. The titer of active trypsin was determined using substrate hydrolysis, estimating a photometric reading (at A₄₀₅) of p-nitroaniline liberated for 5 minutes.

The concentration of active trypsin was calculated multiplying the photometric reading value by 6.025 x 10^"5 factor, which is the result of the relation between p- nitrophenol molar extinction coefficient formed in the assay conditions, and the dilution of the reagents (Walsh, 1970) . At the assays of trypsin enzymatic activity, trypsin was used with a known concentration for the calculus of inhibitor's concentration.

Bz-Arg-pNan hydrolysis by bovine trypsin and determination of its inhibitory activity The trypsin enzymatic activity was determined on Bz- Arg-pNan substrate using 20 μL of trypsin (0.77 μM) that were pre-incubated at 37⁰C, with different inhibitor concentrations, in Tris/HCl buffer 0.1 M, pH 8.0, containing 0.02% CaCl₂ (v/v) and, at the end of 10 minutes, 25 μL of substrate were added (10.0 mM) in a final volume of 250 μL, following the incubation for 24 minutes at 37⁰C, interrupting the reaction with 50 μL of acetic acid 40%. Substrate hydrolysis by the enzyme was accompanied through photometric reading (A₄₀₅) of liberated p-nitroaniline. Inhibitory activity was calculated by the trypsin residual activity in the assay. This method was also used to locate the inhibitory activity during the purification processes of the inhibitors.

Suc-Ala-Ala-Pro-Val-pNan hydrolysis by swine pancreatic elastase (PPE) and determination of inhibitory activity PPE 20 μL (13 nM) were pre-incubated at 37°C, by 10 minutes, with crescent concentrations of the purified inhibition preparation in the assays made in 0.05 M Tris/HCl buffer, pH 8.0, containing 0.05 M NaCl. Following the addition of 20 μL of Suc-Ala-Ala-Pro-Val-pNan substrate (11 mM) , in a final volume of 250 μL, the reaction was interrupted, after 20 min of incubation, by the addition of 50 μL of acetic acid 40% (v/v) . The experiments were made in duplicate.

Substrate hydrolysis by the enzyme was monitored through photometric reading (at A₄₀₅) of the liberated p- nitroaniline. Inhibitory activity was calculated by the residual activity determination of PPE in the assay. H-D-Pro-Phe-Arg-pNan hydrolysis by human plasmatic kallikrein (HuPK) and determination of inhibitory activity HuPK was purified at the biochemistry laboratory from Universidade Federal de Sao Paulo from fresh blood, according to the procedures described by Oliva et al. (1982) .

HuPK used in the assay was previously titrated with EcTI, and at the moment of the assay HuPK 30 μL (1.08 μM) were pre-incubated, at 37°C, with crescent concentrations of the inhibitor in 0.05 M Tris/HCl buffer, pH 8.0, containing 0.05 M NaCl and, after 10 minutes, 20 μL of H-D- Pro-Phe-Arg-pNan substrate (5 mM) were added in a final volume of 250 μL, following the incubation by 20 minutes at 37°C. The reaction was interrupted by the addition of 50 μL of acetic acid 40% (v/v) .

Substrate hydrolysis by the enzyme was accompanied through photometric reading (A₄₀₅) of liberated p- nitroaniline . Inhibitory activity was calculated through the residual activity of HuPK in the assay.

Suc-Phe-pNan hydrolysis by chymotrypsin and determination of its inhibitory activity

The chymotrypsin used in the assay was previously titrated with EcTI and at the moment of the assay 35 μL of chymotrypsin (16 μM) were pre-incubated, at 37 °C, with crescent concentrations of the inhibitor in Tris/HCl buffer

0.1 M, pH 8.0, containing 0.02% CaCl₂ (v/v) and, after 10 minutes, 20 μL of Suc-Phe-pNan substrate (50 mM) were added to a final volume of 250 μL, following the incubation for

20 minutes at 37°C. The reaction was interrupted by the addition of 50 μL of acetic acid 40% (v/v) .

Substrate hydrolysis by the enzyme was monitored through photometric reading (A40₅) of the liberated p- nitroaniline. Inhibitory activity was calculated through the residual activity of chymotrypsin in the assay.

Determination of prothrombin time (PT)

PT was determined using the Simplastin Excel reagent, in accordance to the supplier instructions and through Coagulόmetro Organon Teknica. For control purposes was applied in a cuvette 50 μL of MiIIiQ water and 50 μL of plasma obtained at Departamento de Hematologia of

Universidade Federal de Sao Paulo, which was incubated at

37°C for 2 min without inhibitor. After incubation, 200 μL of reagent were added. The test in the presence of the inhibitor was made in a new cuvette with 50 μL of inhibitor and 50 μL of plasma, which was incubated at 37⁰C for 2 min and, after incubation, 200 μL of reagent were added.

The tests were made in duplicate and the results are expressed by the average of the determinations of each sample .

Time of partially activated thromboplastin (TPAT) determination

TPAT was determined using Simplastin Excel reagent, in accordance to the supplier instructions and through Coagulδmetro Organon Teknica.

The control was accomplished in a cuvette with 50 μL of MiIiQ water, 100 μL of reagent and 50 μL of plasma obtained in Departamento de Hematologia of Universidade Federal de Sao Paulo, which was incubated at 37°C for 3 min, without inhibitor. After incubation, CaCl2 100 μL

(0.025 M) were added.

The test in the presence of the inhibitor was made in a new cuvette with 50 μL of the inhibitor, 100 μL of reagent and 50 μL of plasma, which was incubated at 37 °C for 3 min, and, after the incubation, 100 μL of CaCl₂

(0.025 M) were added.

The tests were made in duplicate and the results are expressed by the average of determinations of each sample. Platelet aggregation

Aggregation curves were analyzed with an aggregometer that measures a combination of light absorption and dispersion. It constitutes a process that registers alterations in light transmission because, by the addition of agonist agents, there is a decrease due to conformational change of the platelet forms, which change from discoid to spherical. This is followed by a gradual increase in light transmission due to platelet aggregation that turns the medium clearer (Moreira & Bernard!, 2004) . Preparation of platelets rich plasma (PRP) and of platelets poor plasma (PPP)

40 mL of blood was slowly collected in 50 mL tubes containing trisodic citrate solution 3.8% (1:10) that were previously treated with silicon for 30 minutes. The supernatant (PRP) was obtained through the centrifugation of whole blood at 1000 rpm for 10 minutes at room temperature. The same was transferred with the aid of a pipette, for 25 mL tubes treated with silicon.

The whole blood was again submitted to a new centrifugation at 4000 rpm for 15 minutes at room temperature. After centrifugation the supernatant (PPP) was transferred for a 25 mL tube treated with silicon and the precipitate was discarded.

PRP (nearly 3 x 10⁵) and PPP (approximately 2 x 10³) platelets were quantified in a Coulter T8 90 device being used in a period of 3 hours, after blood collection. Determination of inhibitory activity on platelet aggregation (PRP)

Aggregation was monitored by a CHRONO-LOG Corporation, Whole-Blott aggregometer through the method described by Born and Cross (1963) .

PRP aggregation was induced by ADP (10 μM) and collagen (2 μg/mL) . In a typical experiment, to 450 μL of PRP was added 5 μL of ADP (I mM) or 1 μL of collagen (1 mg/mL) and the final volume was adjusted with saline solution to 500 μL. For the analysis of BpuTI, BbKI, BbCI and EcTI effects, 10 μL of crescent concentrations of inhibitors were incubated during 5 minutes at 37°C with 450 μL of PRP, the volume being adjusted with saline solution. After this period the agonists were added at concentrations described above. The platelet aggregation in the absence of inhibitors was made at the beginning and at the end of each experiment. Preparation of washed platelets (WP)

The PRP obtained by the previous procedure was transferred with the aid of a pipette whose tips were treated with silicon, to 25 mL tubes previously treated with silicon. EDTA 2% is added (EDTA/PRP 1:20) and the suspension is centrifuged at 2500 rpm for 15 min.

The supernatant was discarded and the obtained precipitate was resuspended in 10 mL of buffer described at Table II, again centrifuged at 2500 rpm for 15 min. This procedure was repeated once more.

The obtained precipitate was resuspended in 2 mL of the buffer "Tyrode Buffer" (Table III) and the platelet number was quantified with a Coulter T8 90 device. In all experiments about 3 x 10⁵ - 3.4 x 10⁵ platelets were used. Table II - Tyrode Solution 1, pH 6.5.

Table III - Tyrode solution 2, pH 7.4

Inhibitor activity determination on platelet aggregation in washed platelets (WP)

The WP aggregation was induced by thrombin (1 uNHI/mL) . In a typical experiment, to 450 μL of WP was added 25 μL of thrombin and the final volume was adjusted with saline solution to 500 μL. For the analysis of BpuTI, BbKI and BbCI effects, 10 μL of crescent concentrations of the inhibitor were incubated during 5 minutes at 37⁰C with 450 μL of WP, the volume being adjusted with saline solution. After this period, thrombin was added with the concentration described above. The platelet aggregation in the absence of inhibitors was made at the beginning and at the end of each experiment. RESULTS

Partial purification and quantification of trypsin inhibitor from Bauhinia purpurea var. corneri de Wit (BpuTI) seeds

The methodology used for inhibitor purification followed the procedures described above.

The first step of purification made was the protein extraction from Bauhinia purpurea var. corneri de Wit (BpuTI) seeds with 0.15 M NaCl solution followed by precipitation with acetone at 80% (v/v) , which eliminated part of the pigment and concentrated proteins. This ketonic precipitate was used as the starting material for the proposed study.

Figure 1 shows the bovine trypsin inhibition by the saline extract and Figure 2 the inhibition after precipitation with 80% acetone (v/v) .

The quantification of trypsin inhibition in the saline extract and in the material after precipitation by acetone is presented at Table IV. Assuming as inhibition unit (IU) the quantity of the inhibitor that inhibits 1 μg of trypsin was possible to determine the yield and the purification of the inhibitor at this step.

Table IV - Partial purification and extraction of trypsin inhibitor

IU/mL - Inhibition unit per mL, S. A. - Specific activity, Purif. - Purification, IU - Inhibitor quantity able to inhibit 1 μg of trypsin, Proteins - Determined by Loewry et al. , 1961 Method. Ionic-exchange chromatography in DEAE-Sephadex A-50

The ketonic precipitate was applied in a DEAE-Sephadex column A-50. The chromatography was developed as described in the methods above. This chromatography was accomplished in unfavorable conditions for the inhibitor linkage to the resin, enabling the elimination of the pigment still present. The chromatographic profile of the three obtained fractions at this step is showed at Figure 3.

Inhibitory activity of bovine trypsin was detected in all fractions as Figures 4, 5 and 6 shows respectively. Assuming the stoichometry of the reaction we can verify that the non-adsorbed fraction showed a higher inhibitor concentration (nearly 95 μg/mL) while the eluted fractions with 0.1 M Tris/HCl pH 8.0 containing 0.15 M NaCl and 0.1 M Tris/HCl pH 8.0 containing 0.30 M NaCl showed 22.0 μg and 3.0 μg of inhibitor, respectively. Table V shows the purification characteristics of 30 mL of the ketonic precipitate at this step.

Table V - Trypsin inhibitor purification by ionic exchange chromatography in DEAE-Sephadex A-50.

IU/mL - Inhibition unit per mL, S. A. - specific activity, Purif. - Purification, IU - Inhibitor amount able to inhibit 1 μg of trypsin, Proteins - Determined by the Loewry et al . , 1961 method. * - Eluted material with equilibrium buffer containing 0.15 M NaCl. ** - Eluted material with equilibrium buffer containing 0.30 M NaCl. Affinity chromatography on trypsin-Sepharose

The non-adsorbed material in DEAE-Sephadex A-50 was submitted to affinity chromatography in trypsin-Sepharose equilibrated with equilibrium buffer 0.1 M Tris/HCl, pH 8.0. The column was washed with equilibrium buffer to eliminate unspecific proteins. Afterwards, the protein fractions linked to trypsin were eluted with a 0.5 M KC1/HC1 solution, pH 2.0, with immediate neutralization, as the chromatographic profile shows (Figure 7) .

In this step was possible to eliminate proteins that were still contaminating the preparation. Figure 8 shows the trypsin inhibitory curve and Table VI the purification results of 48 mL of saline extract from seeds. Table VI - Trypsin inhibitor purification from Bauhinia purpurea seeds

IU/mL - Inhibition unit per mL, S. A. - specific activity, Purif. - Purification, IU - Inhibitor amount able to inhibit 1 μg of trypsin, Proteins - Determined by Loewry et al., 1961 method. * - Eluted material with equilibrium buffer containing 0.15 M NaCl. ** - Eluted material with equilibrium buffer containing 0.30 M NaCl. *** - Eluted proteins from affinity column with a 0.5 M KC1/HC1 solution, pH 2.0. Reverse-phase chromatography in HPLC system The eluted fraction with a 0.5 M KC1/HC1 solution, pH 2.0, from affinity chromatography on Trypsin-Sepharose was submitted to reverse-phase chromatography in u-Bondapack C18 column and the N-terminal region of the eluted protein at 33,18 minutes (Figure 9) was determined. The reverse-phase chromatography, in Cia column with an acetonitrile gradient revealed only one homogeneous peak. Structural characteristics of BpuTI inhibitor

The sample electrophoretic profile of affinity chromatography in trypsin-Sepharose was analyzed in native conditions and, after the treatment with 2-mercaptoethanol

(Figure 10) . The procedure shows the inhibitor homogeneity.

From the calibration curve with proteins with known molecular weights we can estimate the BpuTI mass as around 20 KDa. The inhibitor mobility did not modify with DDT treatment .

The gel-polyacrylamide electrophoresis (SDS-PAGE 12%, Figure 10) made with native inhibitor and after treatment with 2-mercaptoethanol showed single bands and with the same mobility, indicating that the inhibitor is composed by only one polypeptydic chain, similar to other isolated inhibitors from the Bauhinia family (Oliva et al., 2000; Batista et al._r 2001) . Characteristics from BpuTI primary structure The sequence from the N-terminal region (10 residues) was obtained through the A protein automatic sequencing. Figure 11 shows the BpuTI inhibitor similarity with members of the family of inhibitors from Kunitz type plants. Particularly with the inhibitor isolated from B. ungulata (BuXZI) this inhibitor shows 90% of identity. BpuTI - DIVLDTDGEP

The N-terminal region shows similarity with inhibitors from Kunitz family, mainly with the inhibitors isolated from Bauhinia and in this group inhibitors were identified with two disulfide bridges (BuXI and BVTI from B. ungulata and B. variegata, respectively, (Oliva et al., 1999a), inhibitor with just one disulfide bridge (gBrTI from B. rufa, Sumikawa et al., 2006), inhibitor with just one cysteine residue (BbKI from B. bauhinioid.es, Oliva et al., 1999b) and Araύjo et al., 2005) and inhibitors whose structure does not show the cysteine residue (BbCI from B. bauhinioides, Oliveira et al., 2001 and Araύjo et al., 2005; BrTI from B. rufa, Nakahata, 2005) . BpuTI has the major structural identity with Factor Xa inhibitor from Bauhinia ungulata (BuXI) [Bauhinia ungulata Factor Xa inhibitor] which shows the 4 cysteine residues (Oliva et al. , 2003) .

DETERMINATION OF SECONDARY STRUCTURES OF BpuTI PROTEIN Circular dichroism (CD - Circular Dichroism)

Studies of BpuTI secondary structure were made through circular dichroism measurements (CD) . The BpuTI CD spectrum is characterized by an accentuated negative band with a minimum around 200 nm, and by a low intensity positive band with a maximum at 230 nm. This positive band is the interference of the aromatic amino acid side chains on CD from peptide links that probably are due to the aromatic amount in this inhibitor. Otherwise, positive and negative bands at the 200 nm region are representative of β-sheet and disordered structures, respectively. Otherwise, the whole characteristic bands suggest that these inhibitors belong to a subgroup of the rich-β protein class (high content of β-conformation) , named β-II (Manavalan & Johnson, 1983, Wu et al. , 1992) . The CD spectrum of this subclass shows an intense negative band around 200 nm, similar to the protein spectrum from disordered structures. CDPro program was used to estimate the content of the inhibitor secondary structure that showed α-helix 6%, β- sheet 40%, β-loop 22% and disordered 30% (Figure 12) .

BpuTI, BuXI, BbKI and BbCI CD spectra are very similar. Additionally, the estimative for secondary structures of these inhibitors are similar to the determined fractions for SBTI, that shows α-helix 2%, β- sheet 38%, β-loop 23% and disordered structure 37% (Tetenbaum & Miller, 2001) . Conformational stability as a function of pH

Figure 13 shows CDs spectrum of BpuTI inhibitor as a function of pH. The samples were incubated on the following pHs: pH 4.0; pH 6.0; pH 7.0; 9.0; pH 10.0 at 25°C. We can observe that significant changes on CD spectrum of the inhibitors did not happen.

The absence of significant differences on CD spectrum at different pHs (Figure 14) shows the conformational stability of this inhibitor, and this stability is characterized by Kunitz type inhibitors (De Carolli, 2003, Araύjo et al., 2005), probably due to the β-trefoil conformation showed by this protein group (Mukhopadhyay, 2000) .

Functional characterization of BpuTI inhibitor Trypsin inhibition

BpuTI showed high affinity for trypsin. The inhibition constant was determined as 9.8 x 10^"9 M. Through the inhibition curves, where growing concentrations of inhibitors were used, was possible to define the stoichometry of the reaction as 1:1, by the linear extrapolation to 100% of enzymatic inhibition (Figure 14) . Inhibition of other serine peptidases The dissociation constant of the inhibitor-enzyme complex was determined by the measurement of the residual enzymatic activity, after the inhibitor pre-incubation with bovine trypsin and other peptidases, the K_iapp values being identified using the equation described by Morrison (Knight, 1986) for the "slow tight binding" model.

BpuTI inhibited chymotrypsin activity (Ki 5.7 x 10 M, Figure 15) and HuPK (Ki 2.7 x 10^"8 M, Figure 16) and did not show and action over elastase activity (Table VII) .

Table VII - BpuTI action on serine peptidase activity

Ki (inhibition constant) , HuPK (human plasmatic kallikrein) , PPE (swine pancreatic elastase) , WJ (no inhibition) .

The Ki value (M) was determined using specific substrates for each enzyme. Bz-Arg-pNan (trypsin) , HD-Pro- Phe-Arg-pNan (HuPK) , Suc-Phe-pNan (chymotrypsin) . Like trypsin and chymotrypsin, the blood coagulation enzymes are serine peptidases but, as opposed to trypsin, peptidases from coagulation cascade, during the evolution course, acquired a high degree of specificity and cleave only a limited quantity of peptide linkages involving basic amino acid residues. Based on this fact, we can explain why few serine peptidase inhibitors isolated from plants block the action of enzymes involved on blood coagulation (Andrade, 2003) . The results show that inhibitors isolated from Bauhinia can act in different biologic models, in which proteolytic enzymes perform any action.

Action of inhibitors isolated from plants in physiological models

BpuTI action was analyzed in vitro in models similar to physiological conditions as blood coagulation and platelet aggregation and its effect was compared to BbCI and BbKI isolated in our laboratory.

Coagulation times

Prothrombin (TP) and partially activated thromboplastin (TTPA) times were analyzed. The partially activated thromboplastin time, which measures the factors involved in the intrinsic path or in the common path was affected by BpuTI inhibitor (Figure 17) and the prothrombin time (TP) , which is used as an instrument and as a quantitative test for coagulation factors on extrinsic and common coagulation paths, was not altered by BpuTI (Figure 18) .

BpuTI influence on platelet aggregation the BpuTI influence at different concentrations (2, 4 and 6 μM) was analyzed on platelet aggregation of platelets rich plasma (PRP) induced by ADP (20 μM) , collagen (2 μg/mL) and on aggregation of washed platelets induced by thrombin (1 uNHI/mL) .

In the aggregation induced by ADP (Figure 19) and by collagen (Figure 20), in relation with control, we observed that BpuTI (2 and 4 μM) does not interfere in this processes. On the other hand, at 6 μM concentration an inhibition of 22% and 12% on platelet aggregation induced by ATP and collagen, respectively, was observed.

During the aggregation induced by thrombin in washed platelets we observed that BpuTI (2 and 4 μM) promoted platelet aggregation (Figure 21) . Influence of BbKI on platelet aggregation

The influence of BbKI at different concentrations (2,

4 and 6 μM) was analyzed on platelet aggregation of platelets rich plasma (PRP) induced by ADP (20 μM) and collagen (2 μg/mL) and the aggregation of washed platelets induced by thrombin (1 uNHI/mL) .

On Figure 22, we can observe that BbKI inhibited aggregation induced by ADP when concentrations of 4 and 6 μM were used (46% and 85%, respectively) . BbKI induced 32% (4 μM) and 100% (6 μM) the aggregation induced by collagen (Figure 23) .

On aggregation induced by thrombin, in washed platelets, was observed that BbKI (2 μM) did not cause interference on the process. However, with the increase on concentration (4 and 6 μM) , the platelet aggregation was completely inhibited (Figure 24) . Influence of BbCI on platelet aggregation

The influence of BbCI at different concentrations (2, 4 and 6 μM) was studied on platelet aggregation of platelets rich plasma (PRP) induced by ADP (20 μM) , collagen (2 μg/mL) and on the aggregation of washed platelets induced by thrombin (1 uNHI/mL) .

On Figure 25, we can observe that BbCI inhibited the aggregation induced by ADP when used at concentrations of 4 and 6 μM (25% and 43%, respectively) . BbCI inhibited 42% (4 μM) and 100% (6 μM) aggregation induced by collagen (Figure

26) .

On aggregation induced by thrombin, in washed platelets, we can observe that BbCI on the three concentrations used (2, 4 and 6 μM) inhibited significantly platelet aggregation (Figure 27).

Blood coagulation is affected when the time of thromboplastin partially activated is measured, which indicates that is the contact stage that is inhibited in the presence of BpuTI (Figure 17) . Similar results were observed by Andrade, 2003, that studied BuXI inhibitor (factor X inhibitor of blood coagulation isolated from Bauhinia ungulata) . BpuTI has homologous sequences to BuXI and, in this case, both plasmatic kallikrein and factor Xa can be inhibited once that BuXI is also a potent inhibitor of Factor Xa. Nevertheless, BpuTI action on the activity of this enzyme over synthetic substrates was not yet evaluated. The degree of interference of inhibitors over plasmatic kallikrein and on Factor Xa seems to be directly related with coagulation prolongation, since BpuTI does not interfere on TTPA, reflecting a weaker interaction with HuPK (Ki_app, 8.0 x 10^~8 M, Oliva et al. , 2003).

Many platelet aggregation inhibitors were studied, demonstrating the importance and the action of different compounds in promoting or inhibiting this aggregation, "in vitro" or "in vivo" (Moreira & Bernardi, 2004), as the actual anti-platelet agents as, for example, aspirin and ticlopidin, and many others endovenous such as the oral ones, show some limitations (Hynes, 1987; Hirsh & Weitz 1999) .

The platelet aggregation is induced by different known agonists, and the mechanism responsible for the effect of each one has been discussed in many works (Lundblad & White, 2005; Murugappa & Kunapuli 2006) . In the present invention, we seek to use various agonists to induce aggregation, enabling to verify the inhibitory effect over aggregation mediated by different pathways.

BpuTI at the concentrations of 2 and 4 μM did not affect platelet aggregation induced by ADP (Figure 19) . On the other hand, an inhibition of aggregation when this inhibitor is used at a concentration of 6 μM is observed. The effect is not as significant as the ones demonstrated by BbKI and BbCI, whose inhibition was evidenced in a well defined fashion in accordance to the curves (Figures 22 and 25) . The maximum inhibitory effect on aggregation speed by these inhibitors was observes at the concentration of 6 μM, although the inhibition is significant at the concentration of 4 μM. The profile presented at Figure 19 shows that the BpuTI inhibitory effect is slow, and this type of response may indicate that the inhibitor is interacting with the receptor, while in the platelet aggregation response induced by ADP, the BbKI and BbCI effects is fast.

Beyond the inhibition of aggregation induced by collagen BbKI and BbCI (Figures 23 and 26) provoke a prolongation on the aggregation latency time, since this effect is more evident with BbKI.

On the other hand, BpuTI, BbKI and BbCI, in high concentrations (Figures 19; 20; 22; 23; 25; 26), show a potent inhibitory effect. Moreover, the BbKI effect is highlighted, whose action altered both aggregation speed and latency time to begin aggregation induced by collagen. Hence, the anti-aggregator effect of these proteins might not be only a consequence of the specific characteristics of its actions over peptidases, although many groups have been demonstrating the enhancement of the effect of peptidases on platelet aggregation (Ishii-Watabe et al., 2000; Ervin & Peerschke, 2001) .

The results showed the effects of BbKI and BbCI on the blockage of platelet activation pathways (Figures 22 to 27) . This property turn these inhibitors extremely interesting for the production of a drug for the treatment of diseases that involve platelet disfunction because, to block all activation pathways, the association of various medicines is necessary, and it can be dangerous and, probably, not much efficient. The way to block all these pathways would be the blockage of the last activation step, common to all, that is the receptor exposition, Glycoprotein Ilb/IIIa molecule

(GPIIb/IIIa) that suffers a modification in its spatial configuration on the platelet surface, where it will bind to a fibrinogen molecule forming a bridge between two platelets and so on, creating a mesh of links, resulting on platelet aggregation (Chung et al., 2004) . BbCI and BbKI interfere on aggregation by different pathways and also interact with the platelet receptor. This interaction was demonstrated by Nakahata (2005) in its inhibition studies of cell adhesion mediated by fibronectin and vitronectin. In this study, Nakahata demonstrated the best interaction of these inhibitors with vitronectin. Moreover the interference of BbCI on cell adhesion mediated by vitronectin is more effective than that demonstrated by BbKI. This effectiveness is also observed on the washed platelet aggregation inhibition induced by thrombin. The prolongation of the beginning of aggregation, the latency time observed with BbCI permits the supposition that inhibitors can modify the thrombin linkage to the receptor, turning the signal generation in response to thrombin a bit slower. We cannot also exclude the possibility of these inhibitors to form with thrombin a complex that would not affect the enzyme action over synthetic substrates and fibrinogen, but that would hinder its linkage to platelets. BpuTI does not inhibit the aggregation induced by thrombin. The activity enhancement effect is more clearly observed (Figure 21) in this model, being possible to observe the dose-effect relation, indicating that, in this case, this inhibitor does not interfere on thrombin linkage to the receptors.

In conjunction, the data here presented demonstrate that the effects of inhibitory action of the activity of proteolytic enzymes reflected on mechanisms of chemical mediation on cellular events of platelet aggregation. Each one of these agents shows properties with specific profiles, which grant them different clinic- pharmacologically characteristics .

This way, the use of such inhibitors appears to be interesting, according to the present invention, for the elaboration of a pharmaceutical composition targeted to the treatment of diseases related to platelet aggregation, according to the results presented above.

Moreover, it should be evidenced that, according to a preferred aspect of the present invention, the effective inhibitor concentration occurs in levels above 6 μM. Thus, the proportionally equivalent dosage in a drug preparation would be from 100 to 300 mg, preferably from 150 to 200 mg. Many modifications and variations of the description of the invention here presented are evident for those skilled in the art, without deviate from the ambit and spirit of the invention. BIBLIOGRAPHIC REFERENCES

Andrade SA. Coagulacao sanguinea: Acao de substratos e de inibidores peptidicos. Sao Paulo, 2003 (Doctorate Thesis - Escola Paulista de Medicina) .

Andrade SA, Santomauro-Vaz EM, Lopes AR, Chudzinski- Tavassi AM, Juliano MA, Terra WR, Sampaio UM, Sampaio CA, Oliva ML. Bauhinia proteinase inhibitor-based synthetic fluorogenic substrates for enzymes isolated from insect midgut and caterpillar bristles. Biol Chem. 2003 Mar; 384 (3) :489-92

Araύjo APU, Hansen D, Vieira DF, Oliveira C, Santana LA, Beltramini LM, Sampaio CA, Sampaio MU, Oliva ML. Kunitz-type Bauhinia bauhinioides inhibitors devoid of disulfide bridges: isolation of the cDNAs, heterologous expression and structural studies. Biol Chem. 2005; 386(6), 561-8.

Batista IF, Nonato MC, Bonfadini MR, Beltramini LM,

Oliva ML, Sampaio UM, Sampaio CA, Garratt RC. Preliminary crystallographic studies of EcTI, a serine proteinase inhibitor from Enterolobium contortisiliquum seeds. Acta

Crystallogr D Biol Crystallogr. 2001; 57 (Pt 4), 602-4.

Born GV and Cross MJ. The aggregation of blood platelets. J Physiol. 1963 Aug; 168:178-95.

Boulter D, Thurman DA, Derbyshire E. A disc eletrophoretic study of globulin proteins of legume seeds with reference to their systematics. New Phytol 1967; 66: 27-36.

Carter TH, Everson BA, Ratnoff OD. Cabbage seed protease inhibitor: a slow, tight-binding inhibitor of trypsin with activity toward thrombin, activated Stuart factor (factor Xa) , activated Hageman factor (factor XIIa) , and plasmin. Blood. 1990 Jan 1; 75(1), 108-15.

Chao BH, Jakubowskitt JA, Savage BE, Chow P, Marzec

UM, Harker LA, and Maraganore JM. Agkistrodon piscivorus piscivorus platelet aggregation inhibitor: a potent inhibitor of platelet activation. Medical Sciences. 1989; 86, 8050-8054.

Chase T, Shaw E. Titration of trypsin, plasmin and thrombin with p-nitrophenyl-p-guanidinobenzoate HCl. Meth Enzymol. 1970; 19, 20-7. Chopin V, Salzet M, Baert J, Vandenbulcke F, Sautiere PE, Kerckaert JP, Malecha J. Therostatin a novel clotting factor Xa inhibitor from the rhynchobdellid leech Theromyzon tessulatum. J. Biol Chem 2000; 275(42), 32. 701- 7. Chung AW, Radomski A, Alonso-Escolano D, Jurasz P, Stewart MW, Malinski T, Radomski MW. Platelet-leukocyte aggregation induced by PAR agonists: regulation by nitric oxide and matrix metalloproteinases. Br J Pharmacol. 2004; 143(7), 845-55. Ciprandi A, Horn F, Termignoni C. Saliva de animais hematόfagos: fontes de novos anticoagulantes . Rev Bras hematol e hemoter. 2003; 25(4), 250-262.

Connolly TM, Jacobs JW, Condra C. An inhibitor of collagen- stimulated platelet activation from the salivary glands of the Haementeria officinalis leech. I. Identification isolation and characterization. J. Biol Chem. 1992; 267(10), 6.893-8.

De Carolli FP. Aςao de BbKI e peptideos relacionados a sua estrutura em fungos e bacterias patogenicas . Sao Paulo, 2003 (Master Thesis - Escola Paulista de Medicina) .

Edman P. Preparation of phenyl thiohydaltoins from some amino acids. Acta Chem Scand. 1956; 10: 761-5.

Erlanger BF, Kokowsky N, Cohen N. Preparation and properties of two new chromogenic substrates of trypsin. Arch. Biochem. Biophys. 1961; 95, 271-8.

Ervin AL, Peerschke EI. Platelet activation by sustained exposure to low-dose plasmin. Blood Coagul. Fibrinolysis. 2001; 12(6), 415-25.

Friedman M, Krull LH, Cavinss J. The chromatographic determination of cystine and cysteine residues in proteins as s-β- (4-pyridythylcysteine) . J Biol Chem. 1970, 10; 245(15)3868-71.

Hirsh J, Weitz J. New antithrombotic agents. The Lancet. 1999; 353, 1431-1436.

Hoffman LM, Sengupta-Gopalanm C, Paaren HE. Structure of soyben Kunitz trypsin inhibitor mRNA determined from cDNA by using oligodeoxynucleotide primers. Plant MoI Biol 1984; 3, 111-117.

Huang TF, Wu YJ, Ouyang C. Characterization of a potent platelet aggregation inhibitor from Agkistrodon rhodostoma snake venom. Biochim Biophys Acta. 1987; 11; 925(3) : 248-57.

Hutchinson J L. The genera flowering plants (angiospermae) . Oxford University Press, 1972, vol. 1. p. 516. Hynes R. Integrins: a family of cell surface receptors. Cell 1987; 48, 549-54.

Ishii-Watabe A, Uchida E, Mizuguchi H, Hayakawa T. On the mechanism of plasmin-induced platelet aggregation. Implications of the dual role of granule ADP. Biochem Pharmacol. 2000; 59(11), 1345-5.

Kiechele FL, Malinski T. Nitric oxide: biochemistry, pathophysiology, and detection. Am J Clin Pathol, 1993; 100, 567-75.

Knight CG. The characterization of enzymes inhibiton. In: BARRETT, A J. & SALVESEN, G., eds . Proteinase inhibitors. 1986; Elsevier, p. 23-51. Kraucherico S, Nagem RAP, Silva JA, Marangoni S, Polikarpov I. Three-dimensional structure of an unusual Kunitz (STI) type trypsin inhibitor from Copaifera langsdorffi. Biochimie 2004; 167-172. Kunitz M. Crystallization of a trypsin inhibitor from soybean. 1945, Science 101, 668-669.

Laemmli UK. Cleavage of structural proteins during the assembly of head of bacteriophage T4. 1970; Nature, 227, 680-685. Loewry AG, Gallant JA, Dunathan K. Fibrinase. IV. Effect on fibrin solubility. J Biol Chem. 1961; 236, 2648- 55.

Lundblad RL, White GC 2nd. The interaction of thrombin with blood platelets. Platelets. 2005; 16(7), 373-85. Manavalan P, Johnson WC Jr. Sensitivity of circular- dichroism to protein tertiary structure class. Nature, 1983; 305, 5937, 831-832.

Maurer HR. Bromelain: biochemistry, pharmacology and medical use. Cell MoI Life Sci. 2001; 58(9), 1234-45. Mollace V, Salvemini D, Anggard E, Vane J. Nitric oxide from vascular smooth muscle cells: regulation of platelet reactivity and smooth muscle cell guanylate cyclase. Br J Pharmacol, 1991; 104, 633-8.

Moreira HW, Bernard! PSM. Analise dos tragados de ondas da agregacao palquetaria em pacientes com doencas cardiovasculares em uso do acido acetil salicilico comparados a doadores de sangue. Rev. Bras. Hematol.

Hemoter. 2004; 26(4), 239-244.

Mukhopadhyay D. The molecular evolutionary history of a winged bean alpha-chymotrypsin inhibitor and modeling of its mutations through structural analyses. J MoI Evol. 2000; 50(3), 214-23.

Murugappa S, Kunapuli SP. The role of ADP receptors in platelet function. Front Biosci. 2006; 11, 1977-86.

Nakahata AM. Acao dos inibidores de peptidases isolados das sementes de Bauhinia bauhinioides e enterolobium contortisiliquum sobre tumores. Sao Paulo, 2005 (Doctorate Thesis - Escola Paulista de Medicina) .

Nakahata AM. Estudos das propriedades de peptideos sinteticos contendo seqϋencia RGD baseado no inibidor isolado das sementes de B. rufa. Sao Paulo, 2001. (Master Thesis - Escola Paulista de Medicina) .

Niewiarowski S, McLane MA, Kloczewiak M, Stewart GJ. Disintegrins and other naturally occurring antagonists of platelet fibrinogen receptors. Semin Hematol. 1994; 31(4), 289-300.

Oliva MLV, Andrade AS, Batista IFC, Sampaio MU, Juliano M, Fritz H, Auerswald EA, Sampaio CAM. Human plasma kallikrein and tissue kallikrein binding to a substrate based on the reactive site of a FXa inhibitor isolated from Bauhinia ungulata seeds. Immunopharmacol . 1999; 45, 141- ^'l49.

Oliva, MLV, Andrade, SA., Juliano, MA, Juliano, L, Sampaio, MU and, Sampaio, CAM. Kinetic Characterization of Factor Xa Binding Using a Quenched Fluorescent Substrate Based on the Reactive Site of Factor Xa Inhibitor from Bauhinia ungulata Seeds. Curr. Med. Chem. 2003; 10 (13) :1085-93.

Oliva MLV, Grisolia D, Sampaio MU, Sampaio CAM. Properties of highly purified human plasma kallikrein. Agents and Actions 1982; 9, 52-7.

Oliva MLV, Mendes CR, Juliano MA, Chagas JR, Rosa JC, Greene LJ, Sampaio MU and Sampaio CAM. Characterization of a tissue kallikrein iinhibitor isolated from Bauhinia bauhinioides seeds: inhibition of the hydrolysis of kininogen related substrates. Immunopharmacology 1999; 45, 163-169.

Oliva MLV, Sallai RC, Sampaio CAM, Fritz H, Auerswald EA, Tanaka AS, Torquato RJS, Sampaio MU. Bauhinia serine proteinase inhibitors: effect on factor X, factor XII and plasma kallikrein. Immunopharmacology 1996; 32, 85-7. Oliva MLV, Souza-Pinto JC, Batista IFC, Araύjo MS, Silveira VF, Auerswald EA, Mentele R, Exkerskorn C, Sampaio MU, Sampaio CAM. Leucaena leucocephala serine proteinase inhibitor: primary structure and action on bllod coagulation, kinin release and rat paw edema. Biochimica et Biophysica Acta 2000; 1477, 64-74.

Oliveira C. Investigagao da acao anti-inflamatόria dos inibidores isolados das sementes de B. bauhinioides. Sao Paulo, Doctorate Thesis - Universidade Federal de Sao Paulo. Sao Paulo, 2004. (Doctorate Thesis - Escola Paulista de Medicina) .

Oliveira, C, Santana L. A., Carmona A. K., Cezari M. H., Sampaio M. U., Sampaio CA. , Oliva M. L. V., Structure of Cruzipain and Cruzain Inhibitors Isolated from Bauhinia bauhinioides Seeds. Biological Chemistry 2001, 382(5) 847- 852, 2001.

Olmeda A, Trivellin AM. The treatment of Dupuytren's contracture by radical aponeurectomy. Ital J Orthop Traumatol. 1986, 12 (3) : 305-14.

Pouvreau L, Gruppen H, van Koningsveld GA, van den Broek LAM, Voragem AGJ. The most abundant protease inhibitor in potato tube (cv. Elkana) is a protease inhibitor from the Kunitz family. J. Agric. Food Chem. 2003, 51, 501-505.

Putzai A, Grant G, Stewart JC, Watt WB. Isolation of soybean trypsin inhibitors by affinity chomatography on anhydrotrypsin-Sepharose 4B. Anal. Biochem 1988; 172, 108- 112.

Rattnoff OD. The biology and pathology of the initial coagulation reactions. Prog. Hematol 1966; 5, 204-245.

Ratnoff OD, Lederman MM, Krill C, Pass LM, Ross CE. Hemophilia and the acquired immunodeficiency syndrome. Ann Intern Med. 1985; 102 (3) : 412

Richardson M., Seed storage proteins: the enzymes inhibitors. In methods in plants biochemistry, Academic Press, New York, 1991; 5, 259-305. Ryan, CA. Proteinase inhibitor in plant leaves: A biochemical model for pest-induced natural plant protection. TIBS. 1978; 3, 48-150.

Ryan, C. A. Proteinase inhibitor gene families: strategies for transformation to improve plant defenses against herbivores .Bioessays . 1989; 10(1), 20-4.

Salzet, M; Chopin, V; Baert, J; Matias, I; Malecha, J. Theromin a novel leech thrombin inhibitor. J. Biol Chem. 2000; 275(40), 30, 774-80.

Sampaio CAM, Sampaio UM, Prado S. Active titration of horseb urinary kallikrein. Hoppe-Seyler ' s Z Physiol Chem. 1984; 365, 297-302.

Santomauro-Vaz EM. Agao de inibidores isolados de plantas brasileiras na sindrome da isquemia e reperfusao. Sao Paulo, 2005 (Doctorate Thesis - Escola Paulista de Medicina) .

Shapira S, Kadar T, Weissman BA. Dose-dependent effect of nitric oxide synthase inhibition following transient forebrain ischemia in gerbils. Brain Res. 1994; 668:80-4.

Spector T. Refinement on the coomassie blue method of protein quantitation. A single and linear spectrophotometry assay for 0.5 to 50 μg of protein. Analytical Biochemistry. 1978; 86: 144-146.

Sreerama N, Woody RW. Protein secondary structure from circular dichroism spectroscopy. Combining variable selection principle and cluster analysis with neural network, ridge regression and self-consistent methods. J

MoI Biol. 1994;30;242 (4) :497-507.

Sreerama N., Woody R. W. Estimation of protein secondary structure from circular dichoism spectra: comparasion of CONTIN, SELCON and CDSSTR. Methods with an expanded reference set. Analytical Biochemistry, 2000; 287:

252-260.

Sumikawa JT, Nakahata AM, Fritz H, Mentele R, Sampaio MU, Oliva ML. A Kunitz-type glycosylated elastase inhibitor with one disulfide bridge. Planta Med. 2006; 72(5), 393-7. Tao J, Johansson JS, Hayenes DH. Stimulation of dense tubular Ca2+ uptake in human platelets by cAMP. Biochem Biophys Acta 1992; 1105 (1), 29-39.

Tetenbaum J, Miller LM. A new spectroscopic approach to examining the role of disulfide bonds in the structure and unfolding of soybean trypsin inhibitor. Biochemistry. 2001; 9, 40(40), 12215-9.

Tchuszynski GP, Rothman VL, Murphy A, Knudsen KA. Role of thrombospondin in hemostasis and cell adhesion. Semin Thromb Hemost. 1987; 13(3), 361-8. Walsh CT, Hildebrand JG, Spector LB. Succinyl phosphate. Its nonenzymatic hydrolysis and reaction with coenzyme A. J Biol Chem. 1970 Nov 10; 245 (21) : 5699-708.

Wenzel HR, Tchesche H. Cleavage of peptide 4- nitroaniline substrates with varying chain lenght by human leukocyte elastase. Hoppe Seylers Z Physiol Chem, 1981; 362 (6), 829-31.

Wu J, Yang, JT, Wu CSC. Beta-II conformation of all- beta proteins can be distingued from unordered form by circular-dichroism. Analytical Biochemistry, 1992; v. 200, n. 2, p. 359-364.

Claims

1. Use of Bauhinia sp. peptidase inhibitors, wherein it is used for the preparation of a pharmaceutical composition comprising at least one of the aforesaid inhibitors which have inhibitory activity on platelet aggregation and on coagulation for the treatment of diseases related to platelet aggregation.

2. Use according to claim 1, wherein the aforesaid inhibitors are isolated from Bauhinia bauhinioides or Bauhinia purpurea var. corneri de Wit.

3. Use according to claim 2, wherein the aforesaid inhibitors are Bauhinia bauhinioides cruzipain inhibitor (BbCl) , Bauhinia bauhinioides human plasmatic kallikrein inhibitor (BbKI) and Bauhinia purpurea var. corneri de Wit trypsin inhibitor (BpuTI) .

4. Use according to any one of the preceding claims, wherein the aforesaid diseases related to platelet aggregation include, but are not limited, to venous thrombosis, arterial thrombosis, deep venous thrombosis, atherosclerosis, systemic thromboembolism, pulmonary thromboembolism, ischemic, myocardial infarct, arrhythmias and unstable angina pectoris.

5. Pharmaceutical composition for the treatment of diseases related to platelet aggregation comprising peptidase inhibitors isolated from Bauhinia sp. , wherein it comprises an effective amount of at least one of the aforesaid inhibitors and at least one pharmaceutically acceptable carrier.

6. Pharmaceutical composition according to claim 5, wherein the aforesaid inhibitors are Bauhinia bauhinioides cruzipain inhibitor (BbCl) , Bauhinia bauhinioides human plasmatic kallikrein inhibitor (BbKI) and Bauhinia purpurea var. corneri de Wit trypsin inhibitor (BpuTI) .

7. Pharmaceutical composition according to claim 5 or 6, wherein the aforesaid diseases related to platelet aggregation include but are not limited to venous thrombosis, arterial thrombosis, deep venous thrombosis, atherosclerosis, systemic thromboembolism, pulmonary thromboembolism, ischemic, myocardial infarct, arrhythmias and unstable angina pectoris.

8. Pharmaceutical composition according to any one of the claims 5 to 7, wherein the aforesaid inhibitors are at a concentration of 100 to 300 mg.

9. Pharmaceutical composition according to any one of the claims 5 to 8, wherein the aforesaid inhibitors are at a concentration from 150 to 200 mg.