International Journal of Biological Macromolecules 131 (2019) 127–133
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Secondary hemostasis studies of crude venom and isolated proteins from the snake Crotalus durissus terriﬁcus Ivancia D.L. Sousa a,1, Ayrton R. Barbosa a,1, Guilherme H.M. Salvador b, Breno E.F. Frihling c, Paula H. Santa-Rita c, Andreimar M. Soares d, Hilzeth L.F. Pessôa a, Daniela P. Marchi-Salvador a,⁎ a
Departamento de Biologia Molecular, CCEN, UFPB, João Pessoa, PB, Brazil Departamento de Física e Biofísica, Instituto de Biociências, UNESP, Botucatu, SP, Brazil Biotério da Universidade Católica Dom Bosco, PRPG, UCDB, Campo Grande, MS, Brazil d Fundação Oswaldo Cruz, Unidade de Rondônia, Fiocruz, Porto Velho, RO, Brazil b c
a r t i c l e
i n f o
Article history: Received 30 June 2018 Received in revised form 9 March 2019 Accepted 9 March 2019 Available online 10 March 2019 Keywords: Ophidism Brazilian rattlesnake Prothrombin Time Activated Partial Thromboplastin Time Anticoagulant proteins
a b s t r a c t Among the activities triggered by Crotalus durissus terriﬁcus snake venom, coagulation is intriguing and contradictory since the venom contains both coagulant and anticoagulant precursor proteins. This work describes the in vitro effects of crude venom and puriﬁed proteins from snake Crotalus durissus terriﬁcus as they affect coagulation factors of clotting pathways. Coagulant and/or anticoagulant activities of crude venom, and puriﬁed proteins were all analyzed directly in human plasma. Clots formed by crude venom and Gyroxin presented as ﬂexible hyaline masses in punctiform distribution. Clot formation time evaluation of isolated proteins with PT and APTT assays made it possible to infer that these proteins interfere in all coagulation pathways. However, regarding ophidism by C. d. terriﬁcus, Gyroxin acts directly, breaking down ﬁbrinogen to ﬁbrin and increasing the amount plasminogen activator, which results in the formation of thrombi. Crotoxin complex, Crotoxin A and Crotoxin B proteins can act in prothrombinase complex formation; Crotoxin B can inhibit prothrombinase complex formation by direct interaction with Factor Xa. Crotamine interacts with negatively charged regions of differing coagulation factors in all coagulation pathways, and possesses a whole set of activities causing dysfunction, activation and/or inhibition of natural anticoagulants and disturbing hemostasis. © 2019 Elsevier B.V. All rights reserved.
1. Introduction Snakebite envenoming is an important public health problem in rural areas of tropical and sub-tropical countries. The risk of snakebites is a daily concern, especially for rural and peri-urban communities where hundreds of millions of people depend on agriculture or subsistence hunting and gathering to survive . The World Health Organization (WHO) estimates that about 5.4 million snakebites occur each year, resulting in up to 2.7 million cases of envenoming. Each year, there are between 81,410 and 137,880 deaths and around three times as many amputations and other permanent disabilities. It is estimated that as many as 400,000 people a year are left permanently disabled after snakebites . Many snakebites go unreported, often because victims seek treatment from non-medical sources or do not have access to
⁎ Corresponding author at: Universidade Federal da Paraíba - UFPB, Centro de Ciências Exatas e da Natureza - CCEN, Departamento de Biologia Molecular - DBM, Laboratório de Cristalograﬁa de Proteínas - CPr-Lab, Campus I - Cidade Universitária, CEP 58051-900 João Pessoa, PB, Brazil. E-mail address: [email protected]
(D.P. Marchi-Salvador). 1 These authors contributed equally to this work.
https://doi.org/10.1016/j.ijbiomac.2019.03.059 0141-8130/© 2019 Elsevier B.V. All rights reserved.
health care. In June 2017, WHO added snakebite envenoming to its priority list of neglected tropical diseases (NTDs) [2,3]. In Brazil, whose population in 2016 was 207.7 million inhabitants , there were 173,630 cases of ophidism. For 150,468 cases, there was no genus identiﬁcation of the snake involved, and Crotalus accounted for 2188 of the 23,162 known cases of snake envenomation. Accidents with Crotalus and Lachesis genera presented the worst complications, with the highest lethality rates (0.73 and 0.77%, respectively) . The presenting symptoms and signs from Crotalus genus snakebites are both local and systemic. A lack of local symptoms (pain, edema) is a frequent feature, with only a tingling sensation. Systemic signs include difﬁculty in keeping eyes open, with a drowsy appearance (myasthenic face), blurred or double vision, malaise, nausea and headache are some of the manifestations, accompanied by generalized muscular pain and dark urine in the most severe cases . Snake venom consists of a complex mixture of pharmacologically and biochemically active substances (proteins, enzymes, peptides and inorganic compounds) which may have different physiological, hematological and neurological activities . Studying the activity triggered by envenoming contributes to discovering the many molecular mechanisms involved in physiological processes activated by the venom and
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enables development of new therapeutic agents for the treatment of various diseases [7,8]. The venom composition and the clinical and laboratory aspects caused by Crotalus durissus terriﬁcus and C. d. collilineatus ophidism are the most studied [9,10]. Melani et al.  characterized the molecular composition of C. d. terriﬁcus snake venom and described several toxic and pharmacological activities caused by its biomolecules. This snake venom is composed of procoagulant proteins (Convulxin and Gyroxin) and proteins that act systemically as anticoagulants such as Crotoxin complex, Crotoxin A, Crotoxin B and Crotamine . The systemic symptoms caused by Crotalus durissus terriﬁcus envenoming match symptoms observed in acquired coagulopathies [8,11,12]. There are several reports in the literature concerning disseminated intravascular coagulation (DIC) disease that can be compared with ophidism [13–16]. Coagulopathy or coagulation disorders may be inherited genetically or acquired; and must be diagnosed to be treated appropriately [17,18]. Coagulation tests such as Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT) can be applied to identify which clotting factors are compromised and to characterize the pathology [19–21]. The purpose of this study was to describe the in vitro effects of crude venom and puriﬁed proteins from Crotalus durissus terriﬁcus snake venom on coagulation factors of the extrinsic, intrinsic and common pathways in human plasma.
2. Experimental procedures 2.1. Citrated human plasma and coagulation kits Blood from healthy volunteers, those not using drugs that interfere with platelet aggregation or secondary hemostasis parameters, was collected in vacuum tubes containing sodium citrate (1:9). The blood was immediately centrifuged and the plasma was obtained. All experiments were performed with fresh pool of plasma. The Research Ethics Committee in the Health Science Center approved this study under #60251516.0.0000.5188 - Federal University of Paraíba, PB, Brazil. In order to evaluate the Prothrombin Time and Activated Partial Thromboplastin Time, we used the kits Coagulation-PT Ref. 730100-1C (batch #17A130) and Coagulation-APTT Ref. 731200-C (batch #17C171), made by Wama Diagnóstica, Brazil.
2.3. Coagulant activity Evaluation of the coagulation activity was performed according to a methodology previously described by Alvarado and Gutiérrez  (modiﬁed) using citrated human plasma. In order to conduct the coagulation experiments, a pool of fresh citrated human plasma was distributed to each vial and kept at 37 °C. Four different quantities of crude venom from the C. d. terriﬁcus snake (0.5, 1.0, 2.0 and 4.0 μg, solubilized in PBS) were added separately to each vial, and the clot formation time (in seconds) was registered. 2.4. Prothrombin Time (PT) Analysis of PT variations allowed detailing the action of proteins in the extrinsic and common pathways of the coagulation cascade, or on Factors I, II, V, VII and X [19–21]. The PT hemostasis clotting assays were used to determine clotting times in citrated human plasma upon addition of thromboplastin and calcium. The assays were performed at a constant temperature of 37 °C and used the methodology proposed by the manufacturer. To evaluate Prothrombin Time, vials containing citrated human plasma and PBS (reference plasma or test control) and the isolated proteins were incubated for 120 min at 37 °C. After incubation, Reagent 1 from Coagulation-PT clotting assay (thromboplastin extracted from rabbit brains in tricine buffer and calcium chloride) was added to each vial (individually) and the lab timer was triggered. The vial was homogenized and after 9 s observed at intervals of less than 1 s. The timer was immediately stopped and the clot formation time was recorded. The degree of anticoagulation was determined by the PT Ratio (R) according to Eq. (1). The INR value for each R-value, and prothrombin activity percentage (A%), were found in a conversion table that accompanied the clotting test (Coagulation-PT, batch #17A130, ISI = 1.16). The reduction evaluation for the prothrombin activity value (in percentage, rA%) for each sample analyzed was calculated according to Eq. (2). R¼
Coagulation time; in seconds; of the Sample Coagulation time; in seconds; of the Control
rA% ¼ 100%−A%
2.5. Activated Partial Thromboplastin Time (APTT) 2.2. Crude venom and isolated proteins Crude venom from the snake Crotalus durissus terriﬁcus was provided for these studies by the Dom Bosco Catholic University Ophidiarium - UCDB, Campo Grande, MS, Brazil. Puriﬁed proteins from C. d. terriﬁcus snake venom (Gyroxin, Convulxin, Crotamine, Crotoxin complex, Crotoxin A and Crotoxin B) were provided by the Oswaldo Cruz Foundation - Fiocruz, Rondônia, RO, Brazil. Gyroxin was puriﬁed in two chromatographic steps using gel ﬁltration on a Sephadex G-75 and Benzamidine-Sepharose 6B afﬁnity column according to Seki et al. . Convulxin, Crotamine and Crotoxin complex were isolated on a Sephadex G-75 gel ﬁltration column as described by Toyama et al. . Crotoxin A (or Crotapotin) was puriﬁed by liquid chromatography using a reverse phase C18 column, according to de Oliveira et al. . Crotoxin B (or basic Asp49-Phospholipase A2) was isolated by ion exchange chromatography on a CM-Sepharose column as described by Soares et al. . Lyophilized crude venom and isolated venom proteins from C. d. terriﬁcus were stored at −10 °C until coagulation experiments or until analyzing degrees of anticoagulation. The samples were solubilized in phosphate-buffered saline (PBS 10×, pH 7.4) and used immediately. All experiments for evaluating the clotting time or degree of anticoagulation were performed in triplicate.
APTT evaluation was used to detect coagulation factor deﬁciencies associated with the intrinsic (VIII, IX, XI and XII) and common (I, II, V and X) pathways of the coagulation cascade [19–21]. Determining the clotting time of the citrated human plasma (after calcium addition in the presence of ellagic acid) was performed at a constant temperature of 37 °C, using the Coagulation-APTT clotting assay and the methodology proposed by the manufacturer. To evaluate the APTT in both the plasma reference (control) and in the presence of the isolated proteins we used citrated human plasma with PBS, or isolated proteins that were incubated for 120 min. After incubation time, Reagent 1 from Coagulation-APTT clotting assay (ellagic acid and phospholipids from rabbit brains) was added, the mixture was then homogenized and incubated at 37 °C for 3 min. Calcium chloride was then added and the lab timer was triggered. The vials were homogenized and maintained at 37 °C and after 20 s, the vials were observed at intervals of less than 1 s. When the clot formation occurred, the chronometer was immediately stopped and the time was registered. 2.6. Statistical data analysis All results are expressed as mean ± standard error of the mean (SEM) and statistically analyzed employing One-way ANOVA test and
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3. Results 3.1. Crude venom from Crotalus durissus terriﬁcus snake In vitro, crude venom presented fast-acting coagulant activity. It was observed that the time for clot formation (in seconds) was inversely proportional to the quantity of crude venom (Fig. 1). The results for plasmatic clotting time in the presence of crude venom were compared to those obtained in the Coagulation-PT and Coagulation-APTT clotting assays. The plasmatic clotting time in presence of crude venom in the quantities 0.5 and 1.0 μg were similar to the control values for the Coagulation-APTT and Coagulation-PT clotting assays, not presenting signiﬁcant differences. However, in measures of crude venom evaluated at 2.0 and 4.0 μg, the clotting time was signiﬁcantly lower than the time using conventional clotting assays (Fig. 1). The physical appearance of the clots formed in the presence of the C. d. terriﬁcus crude venom (CV) was of a small ﬂexible, punctiform and adhesive mass on hyaline and more ﬂuid remaining plasma. On the other hand, the clots formed in the Coagulation-PT and Coagulation-APPT clotting assays presented as opaque, rigid and nonpunctiform shape. 3.2. Proteins isolated from Crotalus durissus terriﬁcus snake venom
The six proteins puriﬁed from the venom of C. d. terriﬁcus - Gyroxin, Convulxin, Crotamine, Crotoxin complex, Crotoxin A, and Crotoxin B were subjected to coagulant activity evaluations in citrated human plasma. According to Weitz et al. , in vitro, the blood of healthy individuals coagulates in 4–8 min. When citrate is added to prevent clotting, the re-calciﬁed plasma clots were formed in 2–4 min. Ten minutes, at the most, is the time necessary to describe a protein as capable of acting on the clotting factors and therefore cause clotting. Gyroxin caused plasma coagulation (Fig. 1), while Convulxin (in all evaluated quantities) induced clot formation in citrated human plasma after 10 min (approximate average of 650 s). After 2 h of incubation at a
constant temperature of 37 °C, the citrated human plasma showed no clot formation in the presence of the other proteins evaluated. A possible dysfunction in at least one of the coagulation factors involved in the extrinsic and common pathways (I, II, V, VII and X) was veriﬁed by Coagulation-PT clotting test through the measurement of Prothrombin Time (Fig. 2). While alterations in the activity of at least one of the coagulation factors involved in the intrinsic and common pathways of the secondary hemostasis (I, II, V, VIII, IX, X, XI and XII) were assessed by Coagulation-APTT clotting assay (Fig. 3). The PT and APTT averages for clot formation induced by the control (Coagulation-PT and Coagulation-APTT plus PBS) were respectively 15.7 and 37.3 s. The PT average for clot formation in the presence of Crotoxin A, Crotoxin B, Crotoxin complex and Crotamine were respectively 19.2, 23.6, 24.7 and 36.4 s. However, the APTT average for plasma coagulation in the presence of Crotoxin A, Crotoxin B, Crotoxin complex and Crotamine were respectively 74.8, 333.4, 243.0 and 380.0 s. In the Coagulation-APTT assay, there was clot formation caused by the presence of 4.0 μg of Crotoxin B only after 614 s of monitoring. The APTT clotting time was higher than the PT clotting time for all of the proteins evaluated. PT and APTT values, in the presence of Crotoxin A, were lower than values in the presence of other venom proteins. In the presence of all evaluated proteins, the APTT values showed more signiﬁcant changes than the PT values. At high quantities of Crotoxin B, Crotoxin complex and Crotamine, the APTT values increased N16, 10 and 14 times, respectively. The degree of anticoagulation in citrated human plasma in the presence of the Crotoxin complex, Crotoxin A, Crotoxin B and Crotamine after 2 h of incubation and at a constant 37 °C was calculated using Eq. (1), and the R-value (R), International Normalized Ratio (INR) and percentage of prothrombin activity (A%) was described in Table 1. The INR value for healthy people ﬂuctuates between 1.0 and 1.08, and corresponds to 100% prothrombin activity . Prothrombin activity reduction (rA%) was calculated according Eq. (2). All amounts of the evaluated proteins reduced prothrombin activity. The citrated human plasma in the presence of Crotoxin B showed greater viscosity; however, no clot formation was observed. In all of the tested proteins, the rA% values were directly proportional to the quantity evaluated. The A% of citrated human plasma in the presence of Crotoxin A showed less reduction (considering the average of the values obtained for all measured quantities) in relation to the other proteins evaluated and less discrepancy between the measured quantities. When citrated human plasma was incubated with Crotoxin complex in quantities of 2.0 and 4.0 μg, it became more ﬂuid in relation to the
Dunnett's multiple comparisons test. The results were considered nonsigniﬁcant (ns) at 5% or greater probability of error. p values b0.05 were considered signiﬁcant (★: p b 0.01; ★★: p b 0.001; ★★★: p b 0.0001 and ★★★★: p ≪ 0.0001). All results were evaluated using GraphPad Prism software, version 6.0 (GraphPad Software Inc., San Diego, CA, USA).
Fig. 1. Coagulant activity evaluation of citrated human plasma in the presence of crude venom and Gyroxin from Crotalus durissus terriﬁcus snake venom. Coagulation-PT and Coagulation-APTT coagulation assays were used as control. Control assays are shown on the right. Results are expressed as mean ± standard error of the mean (SEM) (n = 3). and were considered non-signiﬁcant (ns) at p N 0.05; p values b0.05 were considered signiﬁcant (★ or ☆: p b 0.01; ★★ or ☆☆: p b 0.001; ★★★ or ☆☆☆: p b 0.0001 and ★★★★ or ☆☆☆☆: p ≪ 0.0001) (★PT and ☆APTT).
Fig. 2. Prothrombin Time evaluation of citrated human plasma in the presence of Crotoxin A, Crotoxin B, Crotoxin complex and Crotamine proteins puriﬁed from Crotalus durissus terriﬁcus snake venom, which presented no clot formation after 2 h of incubation under constant temperature 37 °C. Control assay is shown on the right. Results are expressed as mean ± standard error of the mean (SEM) (n = 3) and were considered nonsigniﬁcant (ns) at p N 0.05; p values b0.05 were considered signiﬁcant (★: p b 0.01; ★★: p b 0.001; ★★★: p b 0.0001 and ★★★★: p ≪ 0.0001).
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Fig. 3. Activated Partial Thromboplastin Time analyzed in citrated human plasma in the presence of Crotoxin A, Crotoxin B, and Crotamine proteins puriﬁed from Crotalus durissus terriﬁcus snake venom, which presented no clot formations after 2 h of incubation under constant temperature 37 °C. Control assay is shown on the right. Results are expressed as mean ± standard error of the mean (SEM) (n = 3) and were considered non-signiﬁcant (ns) at p N 0.05; p values b0.05 were considered signiﬁcant (☆: p b 0.01; ☆☆: p b 0.001; ☆☆☆: p b 0.0001 and ☆☆☆☆: p ≪ 0.0001).
control (reference) plasma. This corroborated TP results indicating N40% reduction in prothrombin activity for all quantities evaluated.
4. Discussion The coagulant activity of crude Crotalus durissus terriﬁcus snake venom and Gyroxin was analyzed in citrated human plasma and the results showed that the time of clot formation decreased in the presence of more crude venom or Gyroxin used. This corroborates with Barrio , who reported that this protein is the toxin responsible for the coagulant activity of Crotalus durissus terriﬁcus snake venom. Clots formed by the presence of C. d. terriﬁcus crude venom exhibited as punctual (small thrombi) and sticky, that easily adhered to the inner walls of the vials (in vitro). In blood vessels (in vivo) these sticky clots may cause blockage or limitation of blood ﬂow leading to ischemia and, in more severe cases, necrosis, which may corroborate with the systemic symptoms observed in patients affected by crotaline envenomation. The fact that clots formed by crude venom are mostly hyaline can be explained by the presence of Gyroxin [7,22,28,29]. Gyroxin promotes unusual breakage of ﬁbrinogen to ﬁbrinopeptide A, yielding a soluble form of ﬁbrin that is more susceptible to the action of ﬁbrinolytic agents [22,28–30]. Bucaretchi et al.  reported that the complete lack of blood clotting in severe cases of rattlesnake ophidism could be due to ﬁbrinogen consumption by Gyroxin. However, the incomplete clotting observed in citrated human plasma may be related to the venom's overall composition, due to the presence of anticoagulant proteins such as Crotoxin A, Crotoxin B, Crotoxin complex and Crotamine [7,8,23,24,31–33].
Vargaftig  stated that Convulxin is an aggregating protein similar to thrombin, von Willebrand factor, collagen and ﬁbrinogen. Polgar et al.  described Convulxin as a potent activator of platelet thrombotic action, but which does not interfere with coagulation cascade factors. Convulxin cannot be classiﬁed as a coagulant protein; however, in our studies it induced clot formation. The clot formation by Convulxin was only observed after 10 min. This effect was only noticed because we used citrated plasma, since sodium citrate exerts its anticoagulant effect through reversible chelation of circulating divalent cations, including Ca2+ and Mg2+, and sequestration of these ions from their normal physiological function. In contrast, if we had used EDTA as an anticoagulant, an irreversible inhibitor of clotting factor activity, clot formation cannot be observed, since the activity of Convulxin would be reversed in the presence of EDTA . In our analyses, Crotoxin A showed signiﬁcant anticoagulant activity. Crotoxin complex showed strong anticoagulant activity, chieﬂy in the intrinsic pathway of the coagulation cascade. Crotoxin B presented very strong anticoagulant activity in both coagulation pathways. Crotoxin is a heterodimeric complex formed by two subunits, an acidic component called Crotoxin A and another represented by a basic phospholipase A2 or Crotoxin B [31,32]. The acidic part of the complex has no neurotoxic action, acting as a nontoxic and non-enzymatic natural inhibitor of Crotoxin B, increasing toxin lethality and decreasing the catalytic activity of Crotoxin B [37,38]. The basic component presents considerable neurotoxic and anticoagulant activities . The Crotoxin complex crystal structure revealed the complete interaction site between Crotoxin A and B. The α and β chains of Crotoxin A participate in almost all contacts with Crotoxin B. The α-helix 1, the short helix, preceding and including the Ca2+ binding loop, the loop preceding the β-wing, and the C-terminal loop of Crotoxin B are regions involved in Crotoxin complex stabilization, and are situated in its canonical front-face  (Fig. 4a). The speciﬁc anticoagulant sites proposed by Kini  of the two subunits of the Crotoxin complex are arranged side by side, assisting in a cluster formation with a greater interaction surface with Factor Xa inhibiting the formation of the prothrombinase complex and, as a consequence preventing coagulation. Crotoxin A derives from a PLA2-like precursor and, in solution, is a monomer made up of three disulﬁde-linked polypeptide chains (α, β, and γ) [39,40] (Fig. 4b). Faure et al.  reported that Crotoxin A interacts in a synergistic manner with Crotoxin B to form the Crotoxin complex. In addition, Faure and Saul  emphasized that random association of CA and CB isoforms  generate Crotoxin complex variants with different enzymatic, toxic and pharmacological activities. Almost all PLA2 isolated from snake venoms that exhibit anticoagulant activity are basic proteins that inhibit coagulation through various mechanisms: i. hydrolysis and destruction of pro-coagulant phospholipids; ii. competition (antagonist effect) with clotting proteins for lipid surface binding; and iii. interaction with FXa, preventing formation of the FXa/FVa complex, and delaying thrombin formation [39,43]. Additionally, Kini  classiﬁed the anticoagulant potency exerted by the PLA2s as strong, weak or absent and proposed a molecular mechanism (speciﬁc anticoagulant site) of interaction between snake venom PLA2s and plasma components.
Table 1 Ratio value (R) calculated for the Crotoxin A, Crotoxin B, Crotoxin complex, and Crotamine proteins puriﬁed from Crotalus durissus terriﬁcus snake venom; R corresponds to the International Normalized Ratio (INR), Prothrombin activity, in percentage (A%). Isolated proteins
Quantity 0.5 μg
Crotoxin A Crotoxin B Crotoxin complex Crotamine
1.08 1.15 1.36 2.30
1.09 1.17 1.41 2.56
81.7 76.8 58.0 27.6
1.21 1.34 1.40 2.30
1.24 1.39 1.46 2.56
71.9 58.2 55.4 27.6
1.21 1.38 1.62 2.34
1.24 1.44 1.72 2.62
71.9 56.0 45.9 27.0
1.38 2.15 1.91 2.36
1.44 2.37 2.08 2.64
56.0 30.1 35.7 26.8
The calculated R-value was converted to the International Normalized Ratio (INR) and the relative percentage of prothrombin activity (A%) was obtained.
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Fig. 4. Crystal structures of Crotoxin A (blue), Crotoxin complex and Crotoxin B (green) and its sequence alignment. The proposed anticoagulant sites, which are responsible to interact to the Factor Xa are highlighted in red and depicted as sticks. Monomeric crystal structure of Crotoxin A, PDBID: 3R0L (a); dimeric crystal structure of Crotoxin complex, PDBID: 3R0L (b); tetrameric crystal structure of Crotoxin B, PDBID: 2QOG (c). Alignment of primary sequences from Crotoxin A, Crotoxin B (from Crotoxin complex), Crotoxin B1 and B2 isoforms (from Crotoxin B) (d). The structural ﬁgures were generated using the Pymol v.1.3 program .
Several studies, such as those performed by Diaz et al.  with a basic PLA2 (myotoxin IV) isolated from Bothrops asper snake venom, Kerns et al.  with a basic PLA2 (CM-IV) isolated from Naja nigricollis snake venom, and Mounier et al.  with human group IIA basic PLA2 (hGIIA) suggest that phospholipases A2 inhibit prothrombinase complex formation (composed by Va and Xa Factors in the presence of Ca2+). Such studies reported that basic PLA2s inhibit prothrombinase activity in the absence of phospholipid pro-coagulant through direct interaction with Factor Xa [44–46]. In addition, Mounier et al.  and Kini  described that Crotoxin B inhibit the prothrombinase complex and they speciﬁcally bind to Factor Xa in a similar way to that observed for myotoxin IV, CM-IV, and the hGIIA proteins. Tryptophan residues at positions 31 and 70 (numbered according to Renetseder et al. ) are structurally conserved in all Crotoxin B isoforms. These residues play an important role in stabilizing both the Crotoxin complex , and the quaternary structure of Crotoxin B [49,50]. In contrast, α-helices 2 and 3, the β-wing, and the end of the C-terminal loop regions are all exposed on the surface of the oligomers (Fig. 4c). The four or ﬁve positively charged residues present in region between 53 and 78 residues of Crotoxin B isoforms (Fig. 4d) compose the speciﬁc anticoagulant site proposed by Kini . Indeed, all these sites are structurally arranged in a favorable manner to the Crotoxin B/Xa factor interaction. Analysis of surface-exposed residues by online PISA software (http://www.ebi.ac.uk/msd-srv/prot_int/cgi-bin/ piserver)  has indicated that 16 out of 18 positive charged residues
from monomers A, B, C and D responsible for the electrostatic interactions are exposed to the solvent and can allow electrostatic interactions with the Factor Xa. On the other hand, the structure of Crotoxin A  showed an incomplete speciﬁc anticoagulant site , composed of 12 residues and, of these, only one is positively charged (Fig. 4d). Because it has only one basic residue available for interaction with Factor Xa, it is possible that this protein has less interaction with this factor, which gives it a weaker anticoagulant action or act through another anticoagulant mechanism. Crotamine is a basic polypeptide composed of 42 amino acid residues [23,33]. The crystallographic structure of Crotamine revealed that all charged as well as hydrophobic residues are exposed to the solvent; this asymmetric surface-charge distribution can enable this polypeptide to complex with various target proteins . In extrinsic pathway evaluations, Crotamine was the protein that most delayed the clotting time after the addition of the coagulant Reagent 1 from Coagulation-PT clotting assay. Unlike the other evaluated proteins, the high degree of anticoagulation observed for PT was dose independent. This marked anticoagulant action of Crotamine may be related to its ability to interact in an electrostatic and non-speciﬁc manner with negatively charged regions of the different coagulation factors in all coagulation pathways. The partial or total blood incoagulability observed in severe cases of Crotalus durissus terriﬁcus envenomation is triggered by hemostasis deregulation. This is due to the action of proteins that inhibit thrombin or activate anti-thrombin III, and inactivate coagulation Factors IXa, Xa, XIa
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and XIIa. However, analysis of our results allows us to infer that crotalic envenomation triggers a secondary coagulopathy called disseminated intravascular coagulation (DIC). One of the ways of triggering DIC is when Factors III and VIIa are complexed. This complex activates Factor X, triggering the common coagulation pathway. In the common pathway, prothrombin is converted into thrombin [21,53,54]. The presence of thrombin hydrolyzes ﬁbrinogen into ﬁbrin, increasing levels of plasminogen activator inhibitor (PAI), and it directly activates Factors V and VIII [17,21,54]. Crotoxin B can replace Factor Va in a normal prothrombinase complex, which results in an inactive Crotoxin B/FXa complex, preventing the formation of thrombin. Non-activation of plasminogen generates ﬁbrin accumulation [21,53,54]. Crotoxin A, Crotoxin complex, and Crotamine proteins can act towards dysfunction and/or inhibition of natural anticoagulants, destabilizing hemostasis and resulting in hemorrhagic manifestations. On the other hand, Gyroxin, a thrombin-like protein, acts directly in an unusual breakdown of ﬁbrinogen to ﬁbrin [22,28–30] increasing PAI levels, which consequently form microthrombi in vivo. When thrombin formation or its neutralization by the anti-thrombin system exceeds hemostasis control mechanisms, a paradox is created in which hemorrhage and thrombosis occur at the same time. 5. Conclusion The in vitro coagulant activity of the crude Crotalus durissus terriﬁcus snake venom is caused by the presence of Gyroxin, which promotes the formation of hyaline clots having a punctiform distribution, dot shape and sticky texture. As measured using PT and APTT hemostasis coagulation assays, the clot formation time for citrated human plasma were altered in the presence of Crotoxin A, Crotoxin B, the Crotoxin complex, and the Crotamine proteins. This allows us to suggest that these proteins interfere with secondary hemostasis. Crotoxin B and Crotoxin complex can inhibit prothrombinase complex formation through direct interaction with Factor Xa and Crotamine can likely interacts with the negatively charged regions of the different coagulation factors, consequently acting in all coagulation pathways.
  
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