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Defibrinogenation by batroxobin and acylated batroxobin in rats.

The thrombin-like snake venom enzyme, batroxobin, was acylated by 4-amidinophenyl benzoate at the active site serine hydroxyl. From the enzymatically inactive benzoyl-batroxobin, batroxobin is generated with a half-life of deacylation of about 1 hour. The clotting activity of benzoyl-batroxobin in plasma is recovered with deacylation. The effect of batroxobin and benzoyl-batroxobin were studied following intravenous injection in rats. Compared to defibrinogenation with batroxobin, that obtained with benzoyl-batroxobin was much retarded. Batroxobin caused microthrombosis initially, which did not develop upon injection of benzoyl-batroxobin in equivalent doses.

Acylation↗

Defibrinogenating effect of batroxobin (Defibrase) in rats and inhibition of migration of human vascular smooth muscle cells by the plasma of batroxobin-treated rats in vitro.

The defibrinogenating effect of batroxobin (Defibrase) in male Wistar rats and the inhibitory effects of the plasma of batroxobin-treated rats on the migration of human vascular smooth muscle cells (SMCs) were investigated in vitro. At 1 h after a single intravenous injection of 3.0, 10.0 or 30.0 BU/kg batroxobin (ten rats in each group), the fibrinogen levels in the plasma of the rats decreased to 88.3, 66.2 and 16.5%, respectively, of that in the plasma of control saline-treated rats (261.0+/-26.7 mg/dl). When the plasma from the batroxobin-treated rats was added to Dulbecco's modified Eagle's medium at a concentration of 0.2% for a vascular SMC migration assay and incubated in a modified Boyden's chamber system at 37 degrees C for 24 h, significant inhibitory effects on vascular SMC migration were observed in the 10.0 (P<0.05) and 30.0 BU/kg (P<0.01) batroxobin-treated rats. The plasma of batroxobin-treated rats as well as standard rat fibrinogen induced vascular SMC migration in a fibrinogen content-dependent manner except the plasma of the 30.0 BU/kg batroxobin-treated rats. Moreover, the rat serum (0.1 approximately 5.0%) did not show any activity on vascular SMC migration in the present experimental system. These results indicate that the plasma fibrinogen significantly influences vascular SMC migration, and that the inhibitory effect of the plasma of batroxobin-treated rats on vascular SMC migration is related to the defibrinogenating action of batroxobin in vivo.

Animals↗

Modification of batroxobin with activated polyethylene glycol: reduction of binding ability towards anti-batroxobin antibody and retention of defibrinogenation activity in circulation of preimmunized dogs.

Amino groups of batroxobin (Bothrops atrox thrombic protease) were modified with 2,4-bis(O-methoxypolyethylene glycol)-6-chloro-s-triazine (activated PEG2). The modified batroxobin had the reduced binding ability towards anti-batroxobin antibody but retained its enzymic activity in vitro and in vivo. Administration of modified batroxobin in which 29% of the total amino groups in the molecule had been modified, to beagle dogs preimmunized with native batroxobin gave rise to a marked reduction of the fibrinogen level in plasma, accompanied with an increased level of fibrinogen (fibrin) degradation products, FDP. On the other hand, no reduction of fibrinogen level was observed when native batroxobin instead of modified batroxobin was injected to immunized dogs.

Animals↗

Defibrinogenation with benzoyl-batroxobin.

The B. moojeni thrombic protease, batroxobin, was acylated by 4-amidinophenyl benzoate at the active site serine hydroxyl. From the enzymatically inactive benzoyl-batroxobin, batroxobin is generated with a half-life of deacylation of about one hour. The clotting activity of benzoyl-batroxobin in plasma is recovered with deacylation. The effects of batroxobin and benzoyl-batroxobin were studied following intravenous injection in rats. Compared to defibrinogenation with batroxobin, that obtained with benzoyl-batroxobin was much retarded. Batroxobin caused microthrombosis initially, which did not develop upon injection of benzoyl-batroxobin in equivalent doses.

Acylation↗

[Effect of batroxobin on K+ channel activated by Ca2+ in primary culture rat cortex neurons of rat].

OBJECTIVE: To investigate the effect of batroxobin on K+ channel activated by Ca2+ in primary cultured cortex neurons of fetal SD rat. METHODS: The patch clamp technique of single channel recordings including cell-attach and inside-out mode was used. RESULTS: Extracellular batroxobin activated the Kca. In Ca2+ bath solution of cell-attach mode, Vp +30 mV, when the concentrations of batroxobin were 0.15, 0.25, 0.50, 0.75 and 1.0 mmol/L, the open probabilities of the channel were 0.013 +/- 0.002, 0.082 +/- 0.011, 0.131+/- 0.012, 0.211+/- 0.010 and 0.062 +/- 0.009 (P < 0.01), respectively. It appeared concentration-dependent within 0.75 mmol/ L. batroxobin. In Ca2+ free-bath solution of cell-attach mode, Vp+50 mV, when the concentrations of batroxobin were 0.15, 0.40, 0.60 and 1.0 mmol/L, the open probabilities of the channel were 0.013 +/- 0.001, 0.112 +/- 0.007, 0.193 +/- 0.010 and 0.301 +/- 0.009 (P < 0.05), respectively. In the 6 cases of inside-out mode patch clamp, Vp +40 mV, when the concentrations of batroxobin were 0, 0.25 and 0.50 mmol/L, the open probabilities of the channel were 0. 012 +/- 0.007, 0.011 +/- 0.009 and 0.013 +/- 0.008 (P > 0.05), respectively. There was no significant difference in open probabilities, average open/close times and amplitudes at different intracellular batroxobin concentrations. CONCLUSION: Batroxobin can affect the activation of the Kca channel through regulating the concentration of cytoplasmic free Ca2+. It may have a protective effect on neurons.

Animals↗

Organization of the gene for batroxobin, a thrombin-like snake venom enzyme. Homology with the trypsin/kallikrein gene family.

We have isolated and analyzed the gene for batroxobin, a thrombin-like snake venom enzyme. Three overlapping DNA segments containing the entire batroxobin gene were identified. Sequence analysis revealed that the batroxobin gene spans 8 kilobase pairs and contains five exons. Mature batroxobin is encoded by four separate exons, 2 to 5. The catalytic residues of batroxobin, His-41, Asp-86, and Ser-178, are encoded by separate exons, exons 2, 3, and 5, respectively. The exon/intron organization of the batroxobin gene is different from that of the prothrombin gene but very similar to those of the trypsin and kallikrein genes. These results indicate that batroxobin is not a member of the prothrombin family but one of the trypsin/kallikrein family. The snake venom gland is assumed to originate from the submaxillary gland. Therefore, batroxobin is expected to be a member of the glandular kallikrein family.

Amino Acid Sequence↗

Expression of cDNA for batroxobin, a thrombin-like snake venom enzyme.

The cloned cDNA for batroxobin has been expressed in E. coli. Batroxobin could only be obtained as intracellular aggregates of fusion proteins, fused with a small peptide. To obtain the mature batroxobin, the recognition sequence for thrombin was inserted between the peptide and the mature batroxobin. This fusion protein accumulated in an insoluble form and could easily be purified. After site-specific cleavage of the fusion protein with thrombin, recombinant batroxobin was isolated by preparative electrophoresis. Batroxobin with enzymatic activity was obtained by the refolding of recombinant batroxobin.

Amino Acid Sequence↗

[Role of natural killer cells in the antimetastatic effect of defibrinogenation with batroxobin].

The antimetastatic effects of defibrinogenation with batroxobin were investigated in normal mice and in mice with depressed or activated natural killer (NK) cell activity. Batroxobin inhibited the formation of lung metastases after intravenous inoculation of the F10 subline of B16 melanoma. Inhibition of NK activity by treatment of mice with anti-asialo GM1 antibody abrogated the antimetastatic effects of Batroxobin. Conversely, augmentation of NK cell activity by poly I:C plus treatment with batroxobin produced additive antimetastatic effects. Studies on the mechanism of interaction between Batroxobin and NK cells revealed that Batroxobin treatment did not affect splenic NK activity in vitro. From these data, it was found that the antimetastatic effects of batroxobin are dependent on the level of NK activity in the host.

Animals↗

Molecular cloning and sequence analysis of cDNA for batroxobin, a thrombin-like snake venom enzyme.

Determination of the nucleotide sequence of a cDNA for batroxobin, a thrombin-like enzyme from Bothrops atrox, moojeni venom, allowed elucidation of the complete amino acid sequence of batroxobin for the first time for a thrombin-like snake venom enzyme. The molecular weight of batroxobin is 25,503 (231 amino acids). The amino acid sequence of batroxobin exhibits significant homology with those of mammalian serine proteases (trypsin, pancreatic kallikrein, and thrombin), indicating that batroxobin is a member of the serine protease family. Based on this homology and enzymatic and chemical studies, the catalytic residues and disulfide bridges of batroxobin were deduced to be as follows: catalytic residues, His41, Asp86, and Ser178; and disulfide bridges, Cys7-Cys139, Cys26-Cys42, Cys74-Cys230, Cys118-Cys184, Cys150-Cys163, and Cys174-Cys199. The amino-terminal amino acid residue of batroxobin, valine, is preceded by 24 amino acids. This may indicate that the amino-terminal hydrophobic peptide (18 amino acids) is a prepeptide and that the hydrophilic peptide (6 amino acids), preceded by the putative prepeptide, is a propeptide.

Amino Acid Sequence↗

Antimetastatic effect of defibrinogenation with batroxobin depends on the natural killer activity of host in mice.

Using batroxobin, a thrombin-like enzyme found in snake venom, the effects of defibrinogenation on artificial lung metastasis in mice were studied. The role of natural killer (NK) cells in the inhibitory effects of defibrinogenation on metastasis was also investigated. Artificial lung metastasis experiments were performed by inoculating either B16-F10 cells or B16-BL/6 cells, highly metastatic strains of B16 melanoma cells, into C57BL/6 mice via the tail vein. The administration of batroxobin significantly inhibited lung metastasis, as did NK activity augmented by poly (I).poly (C) were administered, lung metastasis was more markedly inhibited. When NK activity was suppressed by administration of anti-(asialo GM1) antibody, lung metastasis was markedly increased. When batroxobin was administered with anti-(asialo GM1) antibody, no inhibitory effects on lung metastasis, such as those seen with batroxobin alone, were observed. The administration of batroxobin had no effect at all on spleen lymphocyte NK activity. These results indicated that defibrinogenation due to batroxobin inhibits lung metastasis, and these effects depend on NK activity of the host.

Animals↗

Inhibition of batroxobin, a serine proteinase from Bothrops snake venom, by derivatives of benzamidine.

Benzamidine derivatives which are competitive inhibitors of trypsin-like serine proteinases also inhibited the enzymatic activity of batroxobin, a thrombin-like snake venom proteinase. Structure-activity relationships showed that primary amides of 4-amidinophenyl-alpha-aminobutyric acid have pronounced, relatively selective antibatroxobin activity. Identical effects were found on batroxobin isolated from the venoms of Bothrops atrox or Bothrops moojeni. Esters containing a benzamidine moiety acylated the active centre serine hydroxyl of either batroxobin, however, the inhibition was temporary. Such compounds, especially 4-amidinophenyl esters of substituted benzoic acids, are a particularly useful tool for designing acyl-batroxobin intermediates with different deacylation rates. With 4-nitrophenyl 4'-guanidinobenzoate, the acyl enzyme was formed so rapidly that titration of the active site of batroxobin was possible. Irreversible inhibition of batroxobin was caused only by the selective thrombin inhibitor D-Phe-Pro-ArgCH2Cl.

Acylation↗

Haemostyptic effects of batroxobin with regard to hirudin treatment.

In contrast to thrombin the fibrinogen coagulant effect of the thrombin-like enzyme batroxobin in vitro and in vivo is not inhibited by the specific thrombin inhibitor hirudin. The haemostyptic effect of batroxobin has been studied in rats after bleeding had been induced by corresponding hirudin dosages. Dependent on batroxobin concentration bleeding time was shortened by local application of batroxobin containing solutions. Strong bleeding induced by i.v. injection of 5 mg r-hirudin/kg was stopped almost immediately when a batroxobin concentration of 40 BU/ml was used. Thrombin was less active to stop bleeding after r-hirudin administration than batroxobin.

Animals↗

Antithrombin and thrombolytic effects of a new antithrombin agent: angioscopic and angiographic comparison with heparin or batroxobin.

The antithrombotic effect of three different types of antithrombotic agents (antithrombin:argatroban, heparin, defibrinogenating agent:batroxobin) were evaluated in canine coronary and iliac arteries. An occlusive thrombus was produced by balloon injury. One of the three agents was infused intravenously at 1 hour after thrombus formation (heparin 250 U/kg, argatroban 0.5 mg/kg, batroxobin 0.5 U/kg) and the effect of thrombus size reduction was evaluated. On the contralateral side of the iliac artery, the preventive effect of these agents on thrombus formation was evaluated after balloon injury. In the iliac artery, angioscopic percent area obstruction by the thrombus before and 60 minutes after treatment reduced from 69% to 32% in the argatroban group, and from 64% to 51% in the batroxobin group (P < 0.0001 and P < 0.05, respectively). No significant change was observed in the heparin group. Angiography demonstrated the same trend. The percent area stenosis with thrombus at 60 minutes following balloon injury was 0.75% in the argatroban group, 18.9% in the heparin group (P < 0.05 vs argatroban), and 12.9% in the batroxobin group. Thrombus size at the treated site was smaller than that at the control site in all three groups (P < 0.05 vs control). In the coronary artery, angioscopic percent area obstruction by the thrombus before and 60 minutes after treatment reduced from 84% to 53% in the argatroban group, and from 86% to 68% in the batroxobin group (P < 0.0001 and P < 0.05, respectively). No significant change was observed in the heparin group. Angiography also demonstrated the same trend. The activated partial thromboplastin time (APTT) was prolonged to 189% of the control value with argatroban and to 1253% of the value with heparin (P < 0.0001). Fibrinogen was markedly reduced with batroxobin. These results showed that both the antithrombin agent and the defibrinogenating agent have a preventive effect on thrombus formation and the effect on thrombus size reduction, without marked prolongation of the APTT.

Angiography↗

Plasma levels and urinary excretion of batroxobin and its defibrinogenating effects in various animal species.

The plasma levels and urinary excretion of batroxobin administered to 6 species of animals were examined by an enzyme immunoassay method. Defibrinogenating effect of batroxobin was also studied in those species. The plasma levels of immunoreactive batroxobin disappeared exponentially in all the animals and differences in half-life were observed to occur according to species. The elimination half-life of immunoreactive batroxobin in the plasma was the largest in dogs, followed by rats, monkeys, guinea pigs, mice and rabbits. The extent of the defibrinogenating effect was also noted to vary according to the species, being greatest in dogs and then monkeys, mice, rats, guinea pigs and rabbits. Following the continuous infusion of batroxobin into dogs, its level in the plasma remained high over a considerable period of time and the defibrinogenating effect lasted in corresponding to its plasma level. The urinary excretion of immunoreactive batroxobin was quite small in these species, being 0.2-1.9% of the original dose.

Animals↗

Batroxobin against anoxic damage of rat hippocampal neurons in culture: morphological changes and Hsp70 expression.

Batroxobin, the thrombin-like enzyme, is used for therapeutic defibrination. We have found that batroxobin has good therapeutic effect in ischemic reperfusion rats and clinical practices in vivo. But we have not studied the neuroprotective effect of batroxobin on anoxic hippocampal neurons in vitro. The purpose of this study was to obtain further information on the mechanism of the batroxobin-induced neuroprotection and examine the neuroprotective effect on neurons exposed to anoxia. The effect of batroxobin on anoxic damages in cultured hippocampal neurons of neonatal rats was investigated by using morphological changes and heat shock protein 70 Kd (Hsp70) immunoreactive expression as indicators. The results indicate that batroxobin, besides its defibrination, may have a direct neuroprotective effect on anoxic damage of hippocampal neurons.

Animals↗

Enhancement of arterial thrombolysis with native tissue type plasminogen activator by pretreatment with heparin or batroxobin: an angioscopic study.

The enhancement of canine arterial thrombolysis with native tissue type plasminogen activator (nt-PA) obtained from human-derived normal cells by pretreatment with heparin or the defibrinogenating agent, batroxobin, was evaluated with angioscopy. The nt-PA, 0.25 mg/kg, was infused intravenously to lyse 1-hour-old thrombus (eight thrombosed arteries without medication, seven with nt-PA alone, seven with nt-PA and heparin, and seven with nt-PA plus batroxobin). Angioscopy provided a cross-sectional view of the vessel lumen with clear visualization of the thrombus. Thirty minutes after nt-PA infusion, the percent luminal obstruction decreased from 74 to 61 in nt-PA alone (p less than .025), from 77 to 37 in nt-PA plus heparin (p less than .005), and from 79 to 25 in nt-PA plus batroxobin (p less than .005). Fifteen minutes after drug infusion, plasma fibrinogen levels decreased to 89% of preinfusion value in nt-PA alone, to 84% in nt-PA plus heparin, and to less than 5% in nt-PA plus batroxobin. Thus rapid infusion of nt-PA alone provided slight thrombolytic effects. However, heparin and batroxobin showed marked enhancement of thrombolytic effects of nt-PA.

Angiography↗

Determination of batroxobin levels in the plasma of guinea pigs after epicutaneous and intravenous applications.

An enzyme immunoassay has been set up to measure batroxobin levels in plasma. This assay allowed the detection of amounts of batroxobin greater than 0.5 ng/ml plasma. After the epicutaneous application of 50 or 100 batroxobin units (BU)/kg body weight, no batroxobin could be detected in plasma samples taken 15, 30, 60, 120 or 240 min after the start of the experiment. In contrast, batroxobin could be detected in plasma samples after i.v. injection of 10 BU/kg body weight.

Animals↗

Functional characterization of recombinant batroxobin, a snake venom thrombin-like enzyme, expressed from Pichia pastoris.

A thrombin-like enzyme of Bothrops atrox moojeni venom, batroxobin, specifically cleaves fibrinogen alpha chain, resulting in the formation of non-crosslinked fibrin clots. The cDNA encoding batroxobin was cloned, expressed in Pichia pastoris and the molecular function of purified recombinant protein was also characterized. The recombinant batroxobin had an apparent molecular weight of 33 kDa by SDS-PAGE analysis and biochemical activities similar to those of native batroxobin. The purified recombinant protein strongly converted fibrinogen into fibrin clot in vitro, and shortened bleeding time and whole blood coagulation time in vivo. However, it did not make any considerable alterations on other blood coagulation factors. Several lines of experimental evidence in this study suggest that the recombinant batroxobin is a potent pro-coagulant agent.

Amino Acid Sequence↗