Coagulation and fibrinolysis in capybara (Hydrochaeris hydrochaeris), a close relative of the guinea-pig (Cavia porcellus)

Leitão, D.P.S; Polizello, Ana Cristina Morseli; Rothschild, Z

doi:10.1016/s1095-6433(99)00166-x

Cited by 22 publications

(7 citation statements)

References 14 publications

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“…The fibrinolytic activity of MP was assessed on fibrin clots formed in Petri dishes, prepared with 0.95 % agarose solution containing 0.3 % fibrinogen and 1 mg/mL thrombin in 50 mM Barbital buffer, pH 7.8, according to Leitão et al [ 20 ]. Samples (25 μL) of phosphate-buffered saline (PBS, negative control), B. atrox crude venom (20 μg, positive control) or MP (4, 6, 8 and 10 μg) were added to cavities (5 mm diameter) made on the fibrin gel, and incubated at 37 °C for 24 hours.…”

Section: Methodsmentioning

confidence: 99%

Purification procedure for the isolation of a P-I metalloprotease and an acidic phospholipase A2 from Bothrops atrox snake venom

Menaldo¹,

Jacob-Ferreira²,

Bernardes³

et al. 2015

J Venom Anim Toxins Incl Trop Dis

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BackgroundSnake venoms are complex mixtures of inorganic and organic components, mainly proteins and peptides. Standardization of methods for isolating bioactive molecules from snake venoms is extremely difficult due to the complex and highly variable composition of venoms, which can be influenced by factors such as age and geographic location of the specimen. Therefore, this study aimed to standardize a simple purification methodology for obtaining a P-I class metalloprotease (MP) and an acidic phospholipase A2 (PLA2) from Bothrops atrox venom, and biochemically characterize these molecules to enable future functional studies.MethodsTo obtain the toxins of interest, a method has been standardized using consecutive isolation steps. The purity level of the molecules was confirmed by RP-HPLC and SDS-PAGE. The enzymes were characterized by determining their molecular masses, isoelectric points, specific functional activity and partial amino acid sequencing.ResultsThe metalloprotease presented molecular mass of 22.9 kDa and pI 7.4, with hemorrhagic and fibrin(ogen)olytic activities, and its partial amino acid sequence revealed high similarity with other P-I class metalloproteases. These results suggest that the isolated metalloprotease is Batroxase, a P-I metalloprotease previously described by our research group. The phospholipase A2 showed molecular mass of 13.7 kDa and pI 6.5, with high phospholipase activity and similarity to other acidic PLA2s from snake venoms. These data suggest that the acidic PLA2 is a novel enzyme from B. atrox venom, being denominated BatroxPLA2.ConclusionsThe present study successfully standardized a simple methodology to isolate the metalloprotease Batroxase and the acidic PLA2 BatroxPLA2 from the venom of B. atrox, consisting mainly of classical chromatographic processes. These two enzymes will be used in future studies to evaluate their effects on the complement system and the inflammatory process, in addition to the thrombolytic potential of the metalloprotease.

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Section: Methodsmentioning

confidence: 99%

Purification procedure for the isolation of a P-I metalloprotease and an acidic phospholipase A2 from Bothrops atrox snake venom

Menaldo¹,

Jacob-Ferreira²,

Bernardes³

et al. 2015

J Venom Anim Toxins Incl Trop Dis

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“…The fibrinolytic activity of BjSP was assessed on fibrin clots formed in Petri dishes, according to methodology of Leitão et al (2000). Different doses of BjSP (5, 10 and 20 μg in PBS to a final volume of 30 μL) were applied in cavities (5 mm diameter) made on the fibrin gel along with a negative control of phosphate-buffered saline (PBS, 30 μL) and positive controls of plasmin (5 μg) and B. jararaca venom (20 μg).…”

Section: Fibrinolytic Activitymentioning

confidence: 99%

BjSP, a novel serine protease from Bothrops jararaca snake venom that degrades fibrinogen without forming fibrin clots

Carone

Menaldo

Sartim

et al. 2018

Toxicology and Applied Pharmacology

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Snake venom serine proteases (SVSPs) are commonly described as capable of affecting hemostasis by interacting with several coagulation system components. In this study, we describe the isolation and characterization of BjSP from Bothrops jararaca snake venom, a serine protease with distinctive properties. This enzyme was isolated by three consecutive chromatographic steps and showed acidic character (pI 4.4), molecular mass of 28 kDa and N-carbohydrate content around 10%. Its partial amino acid sequence presented 100% identity to a serine protease cDNA clone previously identified from B. jararaca venom gland, but not yet isolated or characterized. BjSP was significantly inhibited by specific serine protease inhibitors and showed high stability at different pH values and temperatures. The enzyme displayed no effects on washed platelets, but was able to degrade fibrin clots in vitro and also the Aα and Bβ chains of fibrinogen differently from thrombin, forming additional fibrinopeptides derived from the Bβ chain, which should be related to its inability to coagulate fibrinogen solutions or platelet-poor plasma. In the mapping of catalytic subsites, the protease showed high hydrolytic specificity for tyrosine, especially in subsite S1. Additionally, its amidolytic activity on different chromogenic substrates suggests possible effects on other factors of the coagulation cascade. In conclusion, BjSP is a serine protease that acts nonspecifically on fibrinogen, generating different Bβ fibrinopeptides and thus not forming fibrin clots. Its distinguished properties in comparison to most SVSPs stimulate further studies in an attempt to validate its potential as a defibrinogenating agent.

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“…The method reported by Leitão et al [28] was used with some modifications. A 1% (m/v) agarose solution in 0.01 mol/l Tris-HCl buffer (containing 0.15 mol/l NaCl, pH 7.8) was heated up to the formation of a transparent colloid.…”

Section: Fibrin Degradation Assaymentioning

confidence: 99%

TA-2, a thrombin-like enzyme from the Chinese white-lipped green pitviper (Trimeresurus albolabris)

Zhou

et al. 2012

Blood Coagulation &Amp; Fibrinolysis

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Through three chromatographic steps, a new thrombin-like enzyme (TLE), named TA-2, from the venom of the Chinese white-lipped green pitviper (Trimeresurus albolabris) has been isolated and purified to homogeneity. TA-2 was a single-chain glycoprotein with about 6% sugar, pI 3.9 and a molecular weight of 38.8 kD. Its N-terminal sequence (VVGGDECNIN) showed high sequence conformity with many other TLEs. In vitro, it coagulated bovine fibrinogen (108.6 NIH units/mg) and cleaved the Aα and Bβ chains of bovine fibrinogen-releasing fibrinopeptide A and B, but did not degrade bovine fibrin; displayed high stability at different temperature, pH, and presence of several divalent cations and inhibitors; also exhibited strong activity towards casein (192.3 units/mg) and high esterase activity upon Nα-p-tosyl-L-arginine methyl ester (11 units/mg); and behaved as a promoter to platelet aggregation induced by ADP or collagen. In vivo, TA-2 caused dose-dependent prolongation of bleeding time in mice, but had no hemorrhagic and edema-inducing activities even at high concentrations.

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Coagulation and fibrinolysis in capybara (Hydrochaeris hydrochaeris), a close relative of the guinea-pig (Cavia porcellus)

Cited by 22 publications

References 14 publications

Purification procedure for the isolation of a P-I metalloprotease and an acidic phospholipase A2 from Bothrops atrox snake venom

Purification procedure for the isolation of a P-I metalloprotease and an acidic phospholipase A2 from Bothrops atrox snake venom

BjSP, a novel serine protease from Bothrops jararaca snake venom that degrades fibrinogen without forming fibrin clots

TA-2, a thrombin-like enzyme from the Chinese white-lipped green pitviper (Trimeresurus albolabris)

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