Effects of Soybean Trypsin Inhibitor on Hemostasis

Soy bean trypsin inhibitor (SBTI) belongs to the family of serpins – serine protease inhibitors widely distributed in the nature [Silverman G.A. et al., 2004; 21]. Serpins participate in the regulation of proteopytic reactions underling very important physiological and pathological processes such as digestion [16], blood clotting [3, 14, 17], immunity [44] apoptosis [36, 42], inflammation [10], dystrophy [31], carcinogenesis [22, 11] and so on. Despite the apparent importance of the correction of imbalances of the proteolysis in pathology, in fact only the pancreatic trypsin inhibitor (aprotinin) is used more or less widely as a protease inhibitor drug (Trasisol, Contrical, Gordox etc) in the treatment of some diseases [24, 13, 40]. Being the animal protein aprotinin possesses substantial disadvantages [23, 12] and attempts to de‐ velop new drug on the base of plant or recombinant proteins or peptidomimetics are under‐ taken [35, 27]. SBTI is one of the candidates for such a role [34].


Introduction
Soy bean trypsin inhibitor (SBTI) belongs to the family of serpins -serine protease inhibitors widely distributed in the nature [Silverman G.A. et al., 2004;21].Serpins participate in the regulation of proteopytic reactions underling very important physiological and pathological processes such as digestion [16], blood clotting [3,14,17], immunity [44] apoptosis [36,42], inflammation [10], dystrophy [31], carcinogenesis [22,11] and so on.Despite the apparent importance of the correction of imbalances of the proteolysis in pathology, in fact only the pancreatic trypsin inhibitor (aprotinin) is used more or less widely as a protease inhibitor drug (Trasisol, Contrical, Gordox etc) in the treatment of some diseases [24,13,40].Being the animal protein aprotinin possesses substantial disadvantages [23,12] and attempts to develop new drug on the base of plant or recombinant proteins or peptidomimetics are undertaken [35,27].SBTI is one of the candidates for such a role [34].
Among proteolytic processes hemostasis is of special importance because of the high frequency of it's disturbances, accompanied many pathological processes and diseases [20,18].At first sight it looks strange to expect that plant protease inhibitor SBTI should influence hemostasis in humans because blood clotting represents the cascade of proteolytic reactions catalyzed by highly specific proteases [43].However, results of the very early studies support such possibility [26,41].The present study was aimed to investigate the influence of SBTI and aprotinin on hemostasis using modern bioinformatics approach and classical in vitro methods.The concrete purposes of the study included: 1. To establish the extent of structural homology, compare functional activities and evaluate potential targets of SBTI and aprotinin among the human proteases by bioinformatics (in silico) methods.

2.
To investigate the influence of trypsin, SBTI and aprotinin on some indexes of blood clotting, fibrilolysis and platelet aggregation as well as on the hemolytic activity of the complement in vitro.

3.
To elucidate the possibility of the regulation of the total proteolytic and tripsin-inhibiting activity of blood plasma in vivo by the consumption of soy foods enriched with soy protein isolate (SPI) possessing thermo-stable fraction of SBTI.

The study of aprotinin and SBTI in silico
In silico methods (bioinformatics) entails the creation and advancement of databases, algorithms, computational techniques to solve formal and practical problems arising from the management and analysis of biological data.One of the potential areas for exploiting these methods is computational drug design.We used in silico methods in our study for the comparison of structural homology, functional activities and evaluation of the potential targets of SBTI and aprotinin among the human proteases.
From fifty databases we had browsed in the INTRNET nine contained information on aprotinin and SBTI (Table 1).From these PDB (Protein Data Bank) and UniProt/Swiss-Prot were the most informative and we used these databases in our study.

The homology of the primary structures of SBTI and aprotinin
To investigate the homology of the primary structures of SBTI and aprotinin we used BLAST (Basic Local Alignment Search Tool) algorithm, sequence alignment editor Bio Edit 5.0.9 and got information on these proteins from Protein Data Bank and Uni Prot using FAS-TA (Table 2), MOL and PDB format-files.The extent of homology of the total sequences of SBTI and aprotinin, calculated by the method of the multiple paired alignment, makes up 10% (Figures 1 and 2).The low homology of the total sequences of should be attributed to the different lengths of polypeptide chains these proteins -58 amino acids in aprotinin [45] and 181 in SBTI [39].

Tertiary structures of SBTI and aprotinin
For the visualization of the 3D-structures of SBTI and aprotinin we used Chem Office 5.0 and Yasara 6.2.5 software.The files in MOL format, containing the information on the sequences of these protease inhibitors, were imputed into Chem Office 5.0 software and converted in PDB format.The obtained PDB files were imputed into Yasara 6.2.5 software.The results of the generation of the electronic 3D-structures of aprotinin and SBTI are presented in Figure 3. Apparently there there is similarity of 3D-structures of these proteins.
We failed to find molecular targets of SBTI and aprotinin because there was no necessary software in free excess.

Spectra of biological activity of SBTI and aprotinin
The exploiting the PASS software shows four potential activities of aprotinin and three for SBTI.Both SBTI and aprotinin may inhibit rennin as well as angiotensin-and endotheleinconverting enzymes according to the revealing of the possible biological activities of these protease inhibitors by ISIS/Draw 2.4 and PASS Professional chemical structure drawing programs (Figure 4).The possibility of exerting of such effects (drug-likeness) is nearly the same in both protease inhibitors.Aprotinin may inhibit neutral endopeptidase, also.For all the abovementioned activities probability of the activity (P a ) is higher than the probability of the lack of activity (P i ).SBTI and aprotinin should not reveal serious toxicity, mutagenic, carcinogenic and teratogenic effects according to in silico, in vitro and in vivo studies.

The study of aprotinin and SBTI in vitro
In silico methods testify the common features in the primary structure, 3D-structures, functional activities of t SBTI and aprotinin and allow to propose that SBTI should influence processes of hemostasis similar to aprotinin.To prove this assumption we studied the influence of trypsin, SBTI and aprotinin on blood clottting, fibrilolysis and platelets aggregation in vitro.

Trypsin-inhibitory activity of SBTI and aprotinin
We measured tyipsin-inhibitory activity of aprotinin, SBTI and low molecular weight protease inhibitors (ε-aminocapronic acid and D-lysine) by their ability to inhibit BAEE-esterase activity of the trypsin [33].The results are presented in the table 3.
Trypsin-inhibitory activity aprotinin, SBTI, ε-aminocapronic acid and D-lysine was measured as their ability to inhibit BAEE-esterase activity of trypsin.
Tyipsin-inhibitory activity, calculated per mg of inhibitor, decrease down in the order: aprotinin > SBTI > aminocapronic acid > D-lysine.Thus, tyipsin-inhibitory activity of aprotinin is 2-times higher than similar activity of SBTI.However, taking into account that molecular weight of SBTI (20100) is 3-times higher than molecular weight of aprotinin (6514), it should be concluded that the trypsine-inhibitory activity of SBTI, calculated per mole of the inhibitor, should be nearly 1,5-times higher than trypsine-inhibitory activity of aptotinin.Trypsine-inhibitory activity of ε-aminocapronic acid and D-lysine when calculated per mole of the inhibitor is negligibly low because of the low molecular weight.

The influence of SBTI and aprotinin on coagulation hemostasis
The following indexes, characterizing mainly the first and the second phases of blood clotting, were measured: prothrombin time (reflects activity of coagulation factors YII, V, X and II), activated partial thromboplastin time (reflects activity of coagulation factors YII and VIII), activated clotting time (a measure of the anticoagulation affects of heparin) and thrombin time (time of the formation of fibrin clot).Results are summarized in the table 4.  4).

The influence of SBTI and aprotinin on platelet aggregation
Results of the study of the influence of SBTI and aprotinin on platelets aggregation are presented in the table 5 and Figures 5-8.Table 5.The effects of aprotinin, SBTI and trypsin on the aggregation of platelets initiated by ADP (reversible, twophase and irreversible aggregation) and adrenalin (two-phase aggregation).MA -maximum aggregation (transmittance, %), Т МА -time of accomplishment of maximum aggregation (sec).
The effects of trypsin, SBTI and aprotinin on the aggregation of platelets initiated by ADP (reversible, two-phase and irreversible aggregation) and adrenalin (two-phase aggregation) are similar to their effects on blood coagulation and fibrinolysis.Trypsin increases platelet aggregation (the increase of the maximum of all types of aggregation and decrease of maxi-mum aggregation time of irreversible ADP-initiated aggregation).SBTI and aprotinin reveals anti-aggregation effect manifested by the decrease of maximum aggregation.So, the results of in vitro experiments proves the ability of SBTI to influence the hemostatsis predicted by the in silico studies.

The influence of SBTI and aprotinin on the hemolytic activity of the complement
Limited cascade proteolysis underlies the activation of the complement system, which includes nearly 20 individual proteins [30,37].Majority of these proteins belongs to the serine protease family (subunits C1r and C1s, С3/С5-cinvertase of the classical way of activation, factors I and D) [15].While complement system does not have direct relation to hemostasis and targeted on alien cells and microorganisms, in some conditions complement system may play a role in the disruption of intrinsic cells of the organism and among them blood cells [15,37].Because of this we investigated the influence of trypsin, SBTI and aprotinin on the hemolytic activity of the complement assuming that SBTI and aprotinin may retard hemolysis of red blood cells by inhibiting activation of some components of the complement system.Results are presented in the table 6 and Figures 9-10.
The inhibiting effect of trypsin should be attributed to the disruption of С3 component of the complement system.
Thus, the results obtained show that aprotinin and SBTI in the abovementiioned concentrations do not influence the hemolytic activity of the complement.

The influence of the consumption of soy priotein isolate by healthy persons on the indexes of proteolysis in the blood serum
Soya is cultivated as a foodstuff, having favorable effect on human health, more than 3000 years [28].Within the last decades soy foods have started to be used in Europe, USA and in Russia as dietotherapy means at a number of diseases [6,29].In particular, we have shown the cholesterol-lowering effect of soy protein, resulting from the consumption of SPI enriched cookies by persons with modern hypelipidemia [7,9].The consumption of soy foods is followed by the antioxidant effect [Santana et al., 2008, 8, 9] due to the high antioxidant content in these foods [2,9].Antioxidant effect is important for the treatment of diseases followed by the development of the oxidative stress [1,38].
The other possible use of soy foods in dietotherapy may be the correction of disturbances of proteolytic processes.Activation of the tissue proteolysis accompanies different diseases and pathological processes [34].Soy protein foods contain Bouman-Birk type trypsin inhibitor -SBTI [4].This protease inhibitor has been officially recognized as a component of foodstuff [25].There are data showing the possibility of absorbtion of SBTI in the intestin after the consumption of soy protein foods [22].So, it seams reasonable to assume that the con-sumption of soy foods will follow anti-proteolytic effect.Because of this it was interesting to elucidate whether prolonged consumption of the cookies enriched with soy protein isolate (SPI) will influence total proteolytic and trypsin-inhibiting activity of the blood plasma in humans or not?

Tripsin-inhibiting activity of SPI
According to the characteristics, specified by manufacturer, protein content of SPI consists of 90%.From this 10% belongs to protease inhibitors [32].Within the processing of soy beans into SPI the former are expoused to high temperatures, which inactivates the major part of soy proteins.However, about 5-20 % of SBTI are represented by the thermostable fraction [19,22].The measurement of the trypsin-inhibiting activity of SPI shows that trypsin-inhibiting activity of SPI consists of 1,4±0,1 IU/mg of SPI.For example 1 mg of pure SBTI possesses activity equal nearby 60 IU.Thus, the active SBTI makes up 2,7% from SPI mass.

The influence of the consumption of SPI by healthy persons on the total proteolytic and trypsin inhibiting activity of blood serum
30 adult people aged 35-67 years without the expressed signs of chronic diseases consumed cookies, enriched with SPI (30% protein content), for two months [7].Fasting blood samples were drawn before and after the dietary treatment.Serum samples were frozen and analyzed for total proteolytic (BAEE-esterase activity) and tripsin-inhibiting activity [33].Daily intake of 100g of cookies corresponds to consumption of 30g of SPI and about 0,85g of the active SBTI (nearly 52 00 IU).The total consumption of SPI for two months consists of 1,8 kg or 50 g of an active SBTI.Twenty-eight participants (19 females and 9 males) could complete the trial.Results are presented in the table 7.  Total proteolytic activity of blood serum was measures as serum BAEE-esterase activity and trypsin-inhibitory activity by inhibiting by serum of BAEE-esterase activity of trypsin.

Discussion
The disturbances of the hemostasis accompany many pathological processes and diseases [20,18].Both blood clotting and fibrinolysis represents the cascade of proteolytic reactions catalyzed by highly specific proteases [43].To correct disturbances of the fibrinolysis as well as imbalances of the other proteolytic processes the animal trypsin inhibitor aprotinin is used as a drug (Trasisol, Contrical, Gordox etc) [24,13,40].The effectiveness of the aprotinin in the treatment of some diseases has been questioned recently because of it's substantial disadvantages [23,12].Following consultation with the German Federal Institute for Drugs and Medical Devices, the U.S. Food and Drug Administration, Health Canada, and other health authorities, the producer of Trasilol -Bayer announced in 2007 that it has elected to temporarily suspend worldwide marketing of Trasylol® (aprotinin injection) until final results from the Canadian BARTtrial can be compiled, received and evaluated.Information regarding the decision has been posted to Bayer's websites [5].Because of this attempts to develop new protease inhibitor drug are undertaken [35,27].One of the candidates for such a role is plant protease inhibitor -SBTI.
In the present study we exploited the advantages of the modern bioinformatics for establishing the extent of structural homology, comparing functional activities and evaluating potential targets of SBTI and aprotinin among the human proteases.The results of in silico study testifies apparent homology of SBTI and aprotinin manifested by the common features in the primary structure, 3D-structures, functional activities of these proteins and allow us to propose that SBTI should influence processes of hemostasis similar to aprotinin.The investigation of the influence of SBTI and aprotinin on coagulation and thrombocyte hemostasis by in vitro methods prove this assumption and show that both proteins inhibit blood clotting, fibrinolusis and platelet aggregation which is evident from the increase of prothrombin time, activated partial thromboplastin time, activated clotting time, thrombin time and inhibition of fibrinolysis.We investigated the influence of SBTI and aprotinin on the hemolytic activity of the complement assuming that SBTI and aprotinin may retard hemolysis of red blood cells by inhibiting activation of some components of the complement system.However, both inhibitors do not influence hemolysis time.
While the major part of SBTI is disrupted by heating, nearly 20% of inhibitor are thermostable and remains active in soy foods [19].Part of the consumed SBTI are absorbed in the intestine after the consumption of soy foods [22].Because of this we elucidated whether prolonged consumption of the cookies, enriched with SPI, will influence total proteolytic and trypsininhibiting activity of the blood plasma in humans or not?Daily intake of 100g of cookies corresponds to consumption of about 0,85g of the active SBTI.The total consumption of SBTI for the study period consists of 50 g of an active inhibitor.The consumption of 100 g of cookies daily for two months was followed by 18% decrease of total proteolytic activity of blood serum and by 21% increase of trypsin-inhibiting activity.The results obtained testify possibility of the soft regulation of proteolysis in vivo by the consumption of soy foods.
At first, the ability of SBTI to inhibit blood clotting was shown more than half a centaury ago [26,41].However, these results had no any practical consequences and in fact were forgot-ten for a long time.Our study represent new attempt to revive interest to SBTI as possible protease inhibiting drug.

Figure 1 .
Figure 1.The screen of the Bio Edit 5.0.9 with the results of calculation of homology of Aprotinin and SBTI.The identical amino acids are marked with black and similar with gray.Amino acid sequence are represented in FASTA format.

Figure 2 .
Figure 2. Homological regions of Aprotinin and SBTI (according to Bio Edit 5.0.9).The identical amino acids are marked with black and similar with gray.

Figure 4 .
Figure 4. Spectra of the biological activities of aprotinin and SBTI according to PASS software.Drug-Likeness -probability of exerting the effect.Probability of the activity (P a ) and the lack of activity (P i ).The average accuracy of the prognosis makes up more than 85%.

Table 4 .
The influence of trypsin, SBTI and aprotinin on the indexes of hemostasis in vitro.The final concentration of trypsin in the incubation medium consisted of 0,01%, SBTI and aprotinin -0,1%.. Prothrombin time decreases 33-35% in the presence of trypsin from 20±0,9 to 13±0,9 sec, increases insignificantly in the presence of aprotinin and increases 1,5-times up to 31±0,9 sec in the presence of SBTI.Activated partial thromboplastin time sharply decreases by trypsin from 36±1,7 sec in the control samples to 3,0+0,9 sec and increases 3-times by aprotinin (113±1,8 sec) and SBTI (105±2,7 sec).The changes of the activated clotting time under the influence of trypsin, aprotinin and SBTI are similar.Thrombin time decreases nearly 7-fold in the presence of trypsin from 16±0,9 to 2,5±0.5 sec and increases on 50% by aprotinin to 24±0,9 sec.SBTI completely blocked the formation of fibrin clot at least within 10 min.Similar effects trypsin and it's inhibitors exerts on fibrinolysis.Euglobulin lysis time (factor XII-callicrein-dependent fibrinolysis) decreases 60% by trypsin from 400±35 to 182±7 sec.Aptotinin and SBTI in the abovementioned concentrations entirely inhibit the lysis of fibrin clot (Table

Figure 10 .
Figure 10.The hemolytic activity of the complement in the presence of different concentrations of trypsin in vitro.The lag-phase is not shown.

Table 1 .
Databases containing the information on aprotinin and SBTI.

Table 2 .
Designation of aminoacids in FASTA format.*-symbol in FASTA format corresponds to the gap of unlimited length

Table 7 .
Total proteolytic and trypsin-inhibitory activity of blood serum of healthy persons before and after two months consumption of soy protein isolate.