Biol. Chem. 2014; 395(9): 1027–1035
Bruno R. Salua, Rodrigo S. Ferreiraa, Marlon V. Brito, Tatiana F. Ottaiano, José Walber M.C. Cruz, Mariana Cristina C. Silva, Maria Tereza S. Correia, Patrícia M.G. Paiva, Francisco Humberto A. Maffei* and Maria Luiza Vilela Oliva*
CrataBL, a lectin and Factor Xa inhibitor, plays a role in blood coagulation and impairs thrombus formation
Abstract: Arterial thrombosis is an important complication of diabetes and cancer, being an important target for therapeutic intervention. Crataeva tapia bark lectin (CrataBL) has been previously shown to have hypoglycemiant effect and also to induce cancer cell apoptosis. It also showed inhibitory activity against Factor Xa (Kiapp = 8.6 μm). In the present study, we evaluated the anti-thrombotic properties of CrataBL in arterial thrombosis model. CrataBL prolongs the activated partial thromboplastin time on human and mouse plasma, and it impairs the heparin-induced potentiation of antithrombin III and heparin-induced platelet activation in the presence of low-dose ADP. It is likely that the dense track of positive charge on CrataBL surface competes with the heparin ability to bind to antithrombin III and to stimulate platelets. In the photochemically induced thrombosis model in mice, in the groups treated with 1.25, 5.0, or 10 mg/kg CrataBL, prior to the thrombus induction, the time of total artery occlusion was prolonged by 33.38%, 65%, and 66.11%, respectively, relative to the time of the control group. In contrast to heparin, the bleeding time in CrataBL-treated mice was no longer than in the control. In conclusion, CrataBL was effective in blocking a Bruno R. Salu and Rodrigo S. Ferreira contributed equally to this article. *Corresponding authors: Francisco Humberto A. Maffei, Departamento de Cirurgia e Ortopedia, Faculdade de Medicina de Botucatu, UNESP, 18618-970 Botucatu, SP, Brazil; and Centro de Estudos e Pesquisas, Hospital Santa Catarina, 01310-000 SP, Brazil, e-mail: [email protected]; and Maria Luiza Vilela Oliva, Departamento de Bioquímica, Universidade Federal de São Paulo, Rua três de Maio, 100, 04044-020 São Paulo, SP, Brazil, e-mail: [email protected] Bruno R. Salu, Rodrigo S. Ferreira, Marlon V. Brito, Tatiana F. Ottaiano, José Walber M.C. Cruz and Mariana Cristina C. Silva: Departamento de Bioquímica, Universidade Federal de São Paulo, Rua três de Maio, 100, 04044-020 São Paulo, SP, Brazil Maria Tereza S. Correia and Patrícia M.G. Paiva: Departamento de Bioquímica, Universidade Federal de Pernambuco, 50670-901 Recife, PE, Brazil
coagulation and arterial thrombus formation, without increasing bleeding time. Keywords: antithrombin III; blood coagulation; heparin; Kallikrein; Kunitz inhibitor; lectin; thrombosis. DOI 10.1515/hsz-2014-0127 Received February 13, 2014; accepted June 25, 2014
Introduction Vascular diseases lead to an increasing number of deaths as well as to the economic burden due to the high costs of health care and lost productivity; thus, it is important to search for new therapeutic drugs active against them (WHO, 2007). Thrombotic complications are common in diseases such as diabetes and cancer, making it desirable to develop drugs that could act simultaneously in these pathologies. One potential drug candidate is CrataBL, a bifunctional glycoprotein isolated from the bark of the tree Crataeva tapia. In addition to exhibit lectin activity, CrataBL also inhibits trypsin with a relatively weak affinity (Kiapp 43 μm) and more potently Factor Xa (Kiapp 8.6 μm), but it does not interfere directly with the activity of other blood coagulation enzymes such as plasma kallikrein, factor XIIa, factor Xia, or thrombin. CrataBL also does not inhibit plasmin or other serine proteinases, such as human tissue kallikrein, human neutrophil elastase, or chymotrypsin (Ferreira et al., 2013). The high-resolution crystal structures of two different crystal forms of glycosylated CrataBL were previously determined by us (Ferreira et al., 2013). We have also shown that, as a lectin, CrataBL binds only sulfated oligosaccharides, like heparin and its derivatives (Zhang et al., 2013). We also have observed that addition of CrataBL to the DU145 and PC3 cell lines leads to their apoptosis, with release of mitochondrial cytochrome c through activation
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1028 B.R. Salu et al.: A plant protein impairing thrombus formation of caspase-3 (Ferreira et al., 2013). More recently, hypoglycemic effect in a chronic rat diabetes model was described (da Rocha et al., 2013). These findings are points of departure for an investigation of the effects of CrataBL in an experimental animal model of arterial thrombosis, in order to evaluate its potential as an antithrombotic drug.
Results CrataBL CrataBL was obtained following the methodology described by Araujo et al. (2012) and Ferreira et al. (2013). The maintenance of inhibitory properties was confirmed by the effect on Factor Xa (Figure 1) and its hemagglutinating activity.
Heparin-Sepharose affinity chromatography of CrataBL In a previous study, the interaction of CrataBL with carbohydrates was examined with surface plasmon resonance in order to provide better understanding of its binding interactions with various forms of glycosaminoglycans (GAGs). CrataBL binding to heparin is dependent of chain length and sulfation level (Zhang et al., 2013). The affinity chromatography was important to confirm the findings described by Zhang et al. (2013), as the binding to glycosaminoglycans is a novel property of plant proteinase inhibitors. This strong interaction was confirmed by liquid affinity chromatography as the
protein was eluted from heparin-Sepharose only with high concentration of 0.5 m NaCl (Figure 2), indicating that this interaction is strong and that the complex stability may occur also in vivo.
Influence of CrataBL and CrataBL plus heparin on human blood coagulation Figure 3A shows that the activated partial thromboplastin time (aPTT) was significantly prolonged after application of CrataBL at the concentrations of 0.025–0.20 mg and after CrataBL previously incubated with heparin (0.025 U). The concentration of heparin used in this experiment prolongs the aPTT approximately 7.5 times; however, despite the inhibitory effect of CrataBL on Factor Xa, no synergism with the glycosaminoglycan was observed. In contrast, CrataBL nullified the heparin effect on the intrinsic coagulation pathway, suggesting that the binding of CrataBL to heparin impairs the anticoagulation effects of heparin. Unlike heparin, CrataBL did not affect either the prothrombin time (PT) (data not shown) or thrombin time (TT), but it impaired the heparin-induced potentiation of antithrombin III (ATIII) (Figure 3B), at the used concentration.
Effect of CrataBL on the hydrolysis of the synthetic substrate by thrombin The effect of CrataBL on ATIII potentiation by heparin was also confirmed by the ATIII test. As shown in Figure 4,
0.6
0.20 0.15 0.10
PBS
0.4
0.2
1.0 M NaCl
0.25
0.5 M NaCl
0.8
0.3 M NaCl
0.30
A280
Residual activity (%)
1
0.05 0
0
5
10
15
20
25
30
CrataBL (µM)
0.00 0
Figure 1 Factor Xa inhibition. The inhibitory activity on Factor Xa (6.0 nm) was determined by measuring the hydrolysis of Boc-Ile-Glu-Gly-Arg-AMC (0.6 mm) in 0.02 mol/l Tris-HCl pH 7.4 buffer, 140 mm NaCl, 5.0 mm CaCl2, and 0.1% bovine serum albumin (w/v).
2
4
6
8
10
12
14
16
18
20
22
24
Fractions (1 mL)
Figure 2 Affinity chromatography of CrataBL on heparin-Sepharose. Pure CrataBL was applied to a heparin-Sepharose column (1 ml) equilibrated with 0.1 m PBS, pH 7.4. Bound protein was eluted with 0.1 m PBS, pH 7.4, 0.5 m NaCl.
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B.R. Salu et al.: A plant protein impairing thrombus formation 1029
Effects of CrataBL on the aPTT and PT of mice plasma
A heparin+CrataBL(0.2 mg) CrataBL(0.2 mg)
The anticoagulant effect of CrataBL was also assessed in mice. As observed for human plasma, CrataBL prolonged aPTT when compared to NaCl (Figure 5A). However, PT was not modified by CrataBL, in contrast to the reference drug heparin (Figure 5B).
heparin+CrataBL(0.1 mg) CrataBL(0.1 mg) heparin+CrataBL(0.05 mg) CrataBL(0.05 mg) heparin+CrataBL(0.025 mg) CrataBL(0.025 mg) heparin(0.025 U) NaCl(0.15 M)
Effect of CrataBL on platelet aggregation 0
3
6 R
9
12
B heparin+CrataBL(0.025 mg) CrataBL(0.025 mg) heparin(0.025 U) NaCl(0.15 M) 0
3
6 R
9
12
Figure 3 (A) Effect of CrataBL on aPTT, activated partial thromboplastin time. CrataBL (indicated by white boxes) was preincubated with heparin (indicated by black boxes) for 10 min, and subsequently, aPTT reagent was added. (B) Thrombin time (TT) in the presence of heparin, CrataBL alone, and CrataBL plus heparin. Samples were preincubated for 10 min, and human thrombin was added. The tests were performed in duplicate, and R is the ratio of the coagulation time in the presence of protein to a normal (control) sample.
CrataBL at the concentration of 8.0 μm did not exhibit any effects on platelet aggregation induced by ADP, epinephrine, arachidonic acid, collagen, fibrinogen, and thrombin (Figure 6A). Interestingly, CrataBL at 8.0 μm, neutralized the potentiating effect of heparin on platelet aggregation using ADP as agonist (Figure 6B, C). These results provide an evidence of the ability of CrataBL to impair the effects of heparin.
Thrombosis model The effect of CrataBL was examined in the alteration of thrombus formation time in mice. Figure 7 shows that the
A
CrataBL (mg) 20 000 heparin (U) NaCl (M)
15 000
0.05 0.11 0.14 0.22 0.26 0.36 0.55 1.1 12.5 0.15
RFU
0
10 000
4
8
12
8
12
R Serum Serum + CrataBL Serum + (heparin+CrataBL) Serum + heparin
5000 0 0
10
20
30
40
50
60
70
80
B
0.36
CrataBL (mg)
0.55
90
1.1
Time (min)
Figure 4 Effect of CrataBL on ATIII potentiation by heparin. Enzymatic activity of serum sample 70 μl was incubated with 18 μm substrate (Tosyl-Gly-Pro-Arg-AMC) in the presence or absence of CrataBL (1.25 μm), heparin 0.025 U, or CrataBL plus heparin preincubated for 10 min. RFU, Relative Fluorescence Unit (λexc = 380 nm and λem = 440 nm).
CrataBL did not interfere with thrombin hydrolysis of its specific synthetic substrate, but it was able to decrease the heparin anticoagulant activity.
heparin (U) NaCl (M)
12.5 0.15 0
4 R
Figure 5 Influence of CrataBL and heparin on the coagulation of mouse blood. Clotting tests (aPTT – A and PT – B) were performed as described under Materials and methods. The tests were done in duplicate, and R is the ratio of coagulation time in the presence of protein to a normal (control) sample. The data represent two independent experiments in triplicate.
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1030 B.R. Salu et al.: A plant protein impairing thrombus formation 0.15 M NaCl
A
8 µM CrataBL
150
Aggregation (%)
125 100 75 50 25 0 1
B
2
3
4
5
6
C ADP
Light transmission (%)
CrataBL + ADP
(Heparin + CrataBL) + ADP
100 Aggregation (%)
0
*** 80 60 40 20
P AD
P
+ rin pa he
(h
ep
ar
in
+
C
C
ra
ra
Time (min)
ta
ta
BL
BL
)+
+
1 min
AD
AD
P AD
100
P
0 Heparin + ADP
Figure 6 Evaluation of CrataBL, heparin, and heparin preincubated with CrataBL on platelet aggregation. (A) CrataBL (5 m) was incubated for 5 min at 37°C followed by the addition of platelet aggregation agonists (1) ADP, (2) epinephrine, (3) arachidonic acid, (4) collagen, (5) fibrinogen, or (6) thrombin. A 500-μl aliquot of the PRP suspension was placed in the aggregometer cuvettes, allowed to warm to 37°C for 5 min, followed by addition of (B) heparin (0.025 U), or heparin preincubated with 5 m CrataBL. Subsequently, platelets were stimulated with the agonist, 0.2 m ADP, for aggregation in PRP suspension under stirring. (C) Statistical data analysis of (B) experiment, p-value
Bruno R. Salua, Rodrigo S. Ferreiraa, Marlon V. Brito, Tatiana F. Ottaiano, José Walber M.C. Cruz, Mariana Cristina C. Silva, Maria Tereza S. Correia, Patrícia M.G. Paiva, Francisco Humberto A. Maffei* and Maria Luiza Vilela Oliva*
CrataBL, a lectin and Factor Xa inhibitor, plays a role in blood coagulation and impairs thrombus formation
Abstract: Arterial thrombosis is an important complication of diabetes and cancer, being an important target for therapeutic intervention. Crataeva tapia bark lectin (CrataBL) has been previously shown to have hypoglycemiant effect and also to induce cancer cell apoptosis. It also showed inhibitory activity against Factor Xa (Kiapp = 8.6 μm). In the present study, we evaluated the anti-thrombotic properties of CrataBL in arterial thrombosis model. CrataBL prolongs the activated partial thromboplastin time on human and mouse plasma, and it impairs the heparin-induced potentiation of antithrombin III and heparin-induced platelet activation in the presence of low-dose ADP. It is likely that the dense track of positive charge on CrataBL surface competes with the heparin ability to bind to antithrombin III and to stimulate platelets. In the photochemically induced thrombosis model in mice, in the groups treated with 1.25, 5.0, or 10 mg/kg CrataBL, prior to the thrombus induction, the time of total artery occlusion was prolonged by 33.38%, 65%, and 66.11%, respectively, relative to the time of the control group. In contrast to heparin, the bleeding time in CrataBL-treated mice was no longer than in the control. In conclusion, CrataBL was effective in blocking a Bruno R. Salu and Rodrigo S. Ferreira contributed equally to this article. *Corresponding authors: Francisco Humberto A. Maffei, Departamento de Cirurgia e Ortopedia, Faculdade de Medicina de Botucatu, UNESP, 18618-970 Botucatu, SP, Brazil; and Centro de Estudos e Pesquisas, Hospital Santa Catarina, 01310-000 SP, Brazil, e-mail: [email protected]; and Maria Luiza Vilela Oliva, Departamento de Bioquímica, Universidade Federal de São Paulo, Rua três de Maio, 100, 04044-020 São Paulo, SP, Brazil, e-mail: [email protected] Bruno R. Salu, Rodrigo S. Ferreira, Marlon V. Brito, Tatiana F. Ottaiano, José Walber M.C. Cruz and Mariana Cristina C. Silva: Departamento de Bioquímica, Universidade Federal de São Paulo, Rua três de Maio, 100, 04044-020 São Paulo, SP, Brazil Maria Tereza S. Correia and Patrícia M.G. Paiva: Departamento de Bioquímica, Universidade Federal de Pernambuco, 50670-901 Recife, PE, Brazil
coagulation and arterial thrombus formation, without increasing bleeding time. Keywords: antithrombin III; blood coagulation; heparin; Kallikrein; Kunitz inhibitor; lectin; thrombosis. DOI 10.1515/hsz-2014-0127 Received February 13, 2014; accepted June 25, 2014
Introduction Vascular diseases lead to an increasing number of deaths as well as to the economic burden due to the high costs of health care and lost productivity; thus, it is important to search for new therapeutic drugs active against them (WHO, 2007). Thrombotic complications are common in diseases such as diabetes and cancer, making it desirable to develop drugs that could act simultaneously in these pathologies. One potential drug candidate is CrataBL, a bifunctional glycoprotein isolated from the bark of the tree Crataeva tapia. In addition to exhibit lectin activity, CrataBL also inhibits trypsin with a relatively weak affinity (Kiapp 43 μm) and more potently Factor Xa (Kiapp 8.6 μm), but it does not interfere directly with the activity of other blood coagulation enzymes such as plasma kallikrein, factor XIIa, factor Xia, or thrombin. CrataBL also does not inhibit plasmin or other serine proteinases, such as human tissue kallikrein, human neutrophil elastase, or chymotrypsin (Ferreira et al., 2013). The high-resolution crystal structures of two different crystal forms of glycosylated CrataBL were previously determined by us (Ferreira et al., 2013). We have also shown that, as a lectin, CrataBL binds only sulfated oligosaccharides, like heparin and its derivatives (Zhang et al., 2013). We also have observed that addition of CrataBL to the DU145 and PC3 cell lines leads to their apoptosis, with release of mitochondrial cytochrome c through activation
Brought to you by | Georgetown University Authenticated Download Date | 5/24/15 1:41 AM
1028 B.R. Salu et al.: A plant protein impairing thrombus formation of caspase-3 (Ferreira et al., 2013). More recently, hypoglycemic effect in a chronic rat diabetes model was described (da Rocha et al., 2013). These findings are points of departure for an investigation of the effects of CrataBL in an experimental animal model of arterial thrombosis, in order to evaluate its potential as an antithrombotic drug.
Results CrataBL CrataBL was obtained following the methodology described by Araujo et al. (2012) and Ferreira et al. (2013). The maintenance of inhibitory properties was confirmed by the effect on Factor Xa (Figure 1) and its hemagglutinating activity.
Heparin-Sepharose affinity chromatography of CrataBL In a previous study, the interaction of CrataBL with carbohydrates was examined with surface plasmon resonance in order to provide better understanding of its binding interactions with various forms of glycosaminoglycans (GAGs). CrataBL binding to heparin is dependent of chain length and sulfation level (Zhang et al., 2013). The affinity chromatography was important to confirm the findings described by Zhang et al. (2013), as the binding to glycosaminoglycans is a novel property of plant proteinase inhibitors. This strong interaction was confirmed by liquid affinity chromatography as the
protein was eluted from heparin-Sepharose only with high concentration of 0.5 m NaCl (Figure 2), indicating that this interaction is strong and that the complex stability may occur also in vivo.
Influence of CrataBL and CrataBL plus heparin on human blood coagulation Figure 3A shows that the activated partial thromboplastin time (aPTT) was significantly prolonged after application of CrataBL at the concentrations of 0.025–0.20 mg and after CrataBL previously incubated with heparin (0.025 U). The concentration of heparin used in this experiment prolongs the aPTT approximately 7.5 times; however, despite the inhibitory effect of CrataBL on Factor Xa, no synergism with the glycosaminoglycan was observed. In contrast, CrataBL nullified the heparin effect on the intrinsic coagulation pathway, suggesting that the binding of CrataBL to heparin impairs the anticoagulation effects of heparin. Unlike heparin, CrataBL did not affect either the prothrombin time (PT) (data not shown) or thrombin time (TT), but it impaired the heparin-induced potentiation of antithrombin III (ATIII) (Figure 3B), at the used concentration.
Effect of CrataBL on the hydrolysis of the synthetic substrate by thrombin The effect of CrataBL on ATIII potentiation by heparin was also confirmed by the ATIII test. As shown in Figure 4,
0.6
0.20 0.15 0.10
PBS
0.4
0.2
1.0 M NaCl
0.25
0.5 M NaCl
0.8
0.3 M NaCl
0.30
A280
Residual activity (%)
1
0.05 0
0
5
10
15
20
25
30
CrataBL (µM)
0.00 0
Figure 1 Factor Xa inhibition. The inhibitory activity on Factor Xa (6.0 nm) was determined by measuring the hydrolysis of Boc-Ile-Glu-Gly-Arg-AMC (0.6 mm) in 0.02 mol/l Tris-HCl pH 7.4 buffer, 140 mm NaCl, 5.0 mm CaCl2, and 0.1% bovine serum albumin (w/v).
2
4
6
8
10
12
14
16
18
20
22
24
Fractions (1 mL)
Figure 2 Affinity chromatography of CrataBL on heparin-Sepharose. Pure CrataBL was applied to a heparin-Sepharose column (1 ml) equilibrated with 0.1 m PBS, pH 7.4. Bound protein was eluted with 0.1 m PBS, pH 7.4, 0.5 m NaCl.
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B.R. Salu et al.: A plant protein impairing thrombus formation 1029
Effects of CrataBL on the aPTT and PT of mice plasma
A heparin+CrataBL(0.2 mg) CrataBL(0.2 mg)
The anticoagulant effect of CrataBL was also assessed in mice. As observed for human plasma, CrataBL prolonged aPTT when compared to NaCl (Figure 5A). However, PT was not modified by CrataBL, in contrast to the reference drug heparin (Figure 5B).
heparin+CrataBL(0.1 mg) CrataBL(0.1 mg) heparin+CrataBL(0.05 mg) CrataBL(0.05 mg) heparin+CrataBL(0.025 mg) CrataBL(0.025 mg) heparin(0.025 U) NaCl(0.15 M)
Effect of CrataBL on platelet aggregation 0
3
6 R
9
12
B heparin+CrataBL(0.025 mg) CrataBL(0.025 mg) heparin(0.025 U) NaCl(0.15 M) 0
3
6 R
9
12
Figure 3 (A) Effect of CrataBL on aPTT, activated partial thromboplastin time. CrataBL (indicated by white boxes) was preincubated with heparin (indicated by black boxes) for 10 min, and subsequently, aPTT reagent was added. (B) Thrombin time (TT) in the presence of heparin, CrataBL alone, and CrataBL plus heparin. Samples were preincubated for 10 min, and human thrombin was added. The tests were performed in duplicate, and R is the ratio of the coagulation time in the presence of protein to a normal (control) sample.
CrataBL at the concentration of 8.0 μm did not exhibit any effects on platelet aggregation induced by ADP, epinephrine, arachidonic acid, collagen, fibrinogen, and thrombin (Figure 6A). Interestingly, CrataBL at 8.0 μm, neutralized the potentiating effect of heparin on platelet aggregation using ADP as agonist (Figure 6B, C). These results provide an evidence of the ability of CrataBL to impair the effects of heparin.
Thrombosis model The effect of CrataBL was examined in the alteration of thrombus formation time in mice. Figure 7 shows that the
A
CrataBL (mg) 20 000 heparin (U) NaCl (M)
15 000
0.05 0.11 0.14 0.22 0.26 0.36 0.55 1.1 12.5 0.15
RFU
0
10 000
4
8
12
8
12
R Serum Serum + CrataBL Serum + (heparin+CrataBL) Serum + heparin
5000 0 0
10
20
30
40
50
60
70
80
B
0.36
CrataBL (mg)
0.55
90
1.1
Time (min)
Figure 4 Effect of CrataBL on ATIII potentiation by heparin. Enzymatic activity of serum sample 70 μl was incubated with 18 μm substrate (Tosyl-Gly-Pro-Arg-AMC) in the presence or absence of CrataBL (1.25 μm), heparin 0.025 U, or CrataBL plus heparin preincubated for 10 min. RFU, Relative Fluorescence Unit (λexc = 380 nm and λem = 440 nm).
CrataBL did not interfere with thrombin hydrolysis of its specific synthetic substrate, but it was able to decrease the heparin anticoagulant activity.
heparin (U) NaCl (M)
12.5 0.15 0
4 R
Figure 5 Influence of CrataBL and heparin on the coagulation of mouse blood. Clotting tests (aPTT – A and PT – B) were performed as described under Materials and methods. The tests were done in duplicate, and R is the ratio of coagulation time in the presence of protein to a normal (control) sample. The data represent two independent experiments in triplicate.
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1030 B.R. Salu et al.: A plant protein impairing thrombus formation 0.15 M NaCl
A
8 µM CrataBL
150
Aggregation (%)
125 100 75 50 25 0 1
B
2
3
4
5
6
C ADP
Light transmission (%)
CrataBL + ADP
(Heparin + CrataBL) + ADP
100 Aggregation (%)
0
*** 80 60 40 20
P AD
P
+ rin pa he
(h
ep
ar
in
+
C
C
ra
ra
Time (min)
ta
ta
BL
BL
)+
+
1 min
AD
AD
P AD
100
P
0 Heparin + ADP
Figure 6 Evaluation of CrataBL, heparin, and heparin preincubated with CrataBL on platelet aggregation. (A) CrataBL (5 m) was incubated for 5 min at 37°C followed by the addition of platelet aggregation agonists (1) ADP, (2) epinephrine, (3) arachidonic acid, (4) collagen, (5) fibrinogen, or (6) thrombin. A 500-μl aliquot of the PRP suspension was placed in the aggregometer cuvettes, allowed to warm to 37°C for 5 min, followed by addition of (B) heparin (0.025 U), or heparin preincubated with 5 m CrataBL. Subsequently, platelets were stimulated with the agonist, 0.2 m ADP, for aggregation in PRP suspension under stirring. (C) Statistical data analysis of (B) experiment, p-value
