Batroxobin

History

Bothrops atrox was described by Carl Linnaeus as early as 1758, but batroxobin, the active compound in its venom, was first described only in 1954 by H. Bruck and G. Salem. In the years following, this first description of batroxobin was shown to have several uses in surgery. Because of the increasing interest in the properties of batroxobin, several studies on its hemostatic effect and coagulation have been published. More recently, in 1979, a German study showed the uses of batroxobin (reptilase clot retraction test) as a replacement test for the more commonly used thrombin time. Because the enzyme is unaffected by heparin, it is mostly used when heparin is present in blood. Recent studies emphasize more on improving its uses in surgery, mostly spinal surgery, and the uses as serine protease.

Available forms

Batroxobin is a protein of the serine protease family. Batroxobin is closely related in physiological function and molecular size to thrombin. Five subspecies for the Brazilian lancehead snake (Bothrops atrox) are found. Batroxobin obtained from certain subspecies exhibits the hemostatic efficacy, whereas the protein obtained from other subspecies exhibits the cleavage of fibrinogen. Some of the forms have hemostatic efficacy as main effect, where the other forms have degradation of fibrinogen as main effect. Batroxobin that is naturally extracted from the snake venom is mainly obtained from the snake Bothrops moojeni. But the concentration is low and it is difficult to purify the protein. Often the product remains polluted, this makes it harder to use for clinical purposes. Theoretically, the molecular weight of batroxobin should be around 25.5 kDa. Often, isolated batroxobin is heavier, around 33 kDa. The higher molecular weight is caused by a glycosylation modification during the secretion. The differences in weight result from different possible purification procedures, which can remove different sugar(chains) from the enzyme. Because the batroxobin isolated from venom is highly irregular in quality, it is now more often synthesized in organisms using Bothrops moojeni cDNA.

Structure

The structure and working mechanism of batroxobin extracted from the Bothrops moojeni have been thoroughly studied. It should be noted that various subspecies exist and the working mechanisms of each batroxobin differ. As such, the structure of Bothrops moojeni batroxobin is further elucidated. The structure of batroxobin has been studied by various research groups throughout the years. These studies have mostly been performed by biologically synthesizing batroxobin from Bothrops moojeni cDNA, and analyzing this product and using homology models based on other proteases, such as thrombin and trypsin, among others. One of the earlier studies from 1986 showed that the molecular weight is 25.503 kDa, 32.312 kDa with the carbohydrate, and it consists of 231 amino acids. The amino acid sequence exhibited significant homology with other known mammalian serine proteases, such as trypsin, thrombin, and most notably pancreatic kallikrein. It was therefore concluded that it is indeed a member of the serine protease family. Based on the homology, the disulfide bridges were identified and the structure was elucidated further. A later molecular modelling study from 1998 used the homology between glandular kallikrein from the mouse and batroxobin, which is about 40%, to propose a 3D structure for biologically active batroxobin. To date no definite 3D structure has been proposed.

Biological synthesis in micro-organisms

After the cDNA nucleotide sequence of batroxobin from Bothrops moojeni was determined back in 1986, a research group from the Kyoto Sangyo university successfully expressed the cDNA for batroxobin in E. Coli in 1990. The recognition sequence for thrombin was used to obtain mature batroxobin. The fusion protein which was obtained was insoluble and was easily purified. After cleaving the fusion protein, the recombinant batroxobin could be isolated by electrophoresis and it was then successfully refolded to produce biologically active batroxobin. This study showed that it was possible to produce batroxobin using micro-organisms, a method which was more promising than isolating the enzyme from extracted snake venom. In 2004, a research group from Korea produced batroxobin by expressing it in the yeast species Pichia pastoris. This recombinant enzyme had a molecular weight of 33 kDa and included the carbohydrate structure. This method of expressing it in Pichia pastoris turned out to be more effective, as the produced enzyme showed cleaving activity which was more specific than thrombin in some cases and was more specific than non-recombinant batroxobin. Therefore, synthesis using Pichia pastoris seems promising for producing high quality recombinant batroxobin.

Reactions and mechanism of action

As described earlier, batroxobin is an enzyme which has a serine protease activity on its substrate, fibrinogen. A serine protease cleaves a protein at the position of a serine, to degenerate a protein. Batroxobin is comparable to the enzyme thrombin, which is also a serine protease for fibrinogen. Fibrinogen is an important protein for hemostasis, because it plays a critical role in platelet aggregation and fibrin clot formation. Normally when one is wounded, thrombin cleaves the fibrinogen, which forms clots. As a result, the wound is ‘closed’ by these clots and recovery of the epithelial cells of the skin can take place. This is the natural process necessary for tissue repair. The venom batroxobin also induces clots, but does this with or without tissue damage. This is because batroxobin isn’t inhibited by specific cofactors like thrombin is. These clots can block a vein and hinder blood flow.

The differences between thrombin and batroxobin in binding fibrinogen

Fibrinogen is a dimeric glycoprotein, which contains two pairs of Aα-, Bβ- peptide chains and y- chains. There are two isoforms of this fibrinogen, one with two yA-chains (yA/yA) and one with a yA-chain and a y’-chain (yA,y’) When fibrinogen is cleaved by thrombin, it releases fibrinopeptide A or B. Thrombin acts on two exosites to fibrinogen. Exosite 1 mediates the binding of thrombin to the Aα- and Bβ-chains, and exosite 2 causes an interaction with a second fibrinogen molecule at the C-terminus of the y’-chain. Consequently, when thrombin binds a yA/yA fibinogen only exosite 1 is occupied, and when it binds yA/y’ both exosites are bound tightly. So fibrinogen yA/y’ is a competitor to yA/yA, which decrease the amount of clotting. yA/y’ binds with a factor 20-fold greater than yA/yA. There are also clotting inhibitors like antithrombin and heparin cofactor II, which prevent clotting when it isn’t necessary. In contrast, batroxobin isn’t inhibited by antithrombin and heparin cofactor II. Batroxobin also has a high Kd value for binding both forms yA/yA and yA/y’. The bindings sites of batroxobin and thrombin partially overlap, but there are some differences. The fibrin-bound batroxobin retains catalytic activity and is a more potent stimulus for fibrin aggregation than fibrin-bound thrombin. This is probably due to the more lipophilic character of exosite 1, that binds fibrinogen more tightly. Fibrinogen is the sole substrate for batroxobin, whereas thrombin has multiple substrates. This is probably due to the Natrium-binding pocket that thrombin contains.

Toxicokinetics

Toxicokinetic studies have been performed on various animal species, namely dogs, mice, guinea pigs, rabbits, rats and monkeys. The research was performed by using immunoassays to obtain the plasma and urinary levels of batroxobin. They also measured the levels of fibrinogen.

Exposure

Normally the venom is directly injected into the bloodstream by the snake. In the experiments performed they also used intravenous injection of batroxobin. They used a total dose of 2 BU/kg (in dogs also 0.2 BU/kg) given during a time of 30 minutes, three times a day. In the graph below you can see the plasma concentrations of batroxobin after administration.

Distribution

All the species showed a large Vd (Volume of Distribution). The value of the plasma in animals was around 50ml/kg on average. In dogs and monkeys the value of the Vd was very low compared to other species, namely 1.5 times the value of plasma in other animals. So the batroxobin is distributed mainly through veins and little is taken up by tissues. In the other species this value was around four times higher. This might be because batroxobin is more easily taken up by the reticuloendotherial system in those species.

Excretion

Batroxobin is excreted by the liver, kidney and spleen. The excretion of batroxobin can be detected by small metabolite molecules in the urine. With use of the immunoassay only 0.2 - 1.9% of the dose was detected in the urine. The amount of radioactivity of 125I-batroxobin was 69% in rats and 73% in dogs within 48 hours. So batroxobin is mainly excreted through the kidneys in its degraded form. So it isn’t detectable with an immunoassay.

Metabolism

All the species responded differently to batroxobin exposure. This means that their ability to metabolize this protease is not the same. They all have their own half-lifes. The half-life in dogs are the highest 3.9 h and 5.8 h. In rabbit and mouse the half-life values were very low, 0.3 h and 0.4 h respectively. Because batroxobin is an enzyme, it is degraded by a protease, and cleaved in smaller unfunctional parts.

Markers for batroxobin

To look at the integrity of batroxobin you can use SDS-PAGE gel analysis on 4 to 15% polyacrylamide gradient gels in a non-reducing environment. The protein was seen on the gel as a single band with molecular weight of 32,000 Da. To be sure this was batroxobin, the amino acid sequence was analyzed afterwards. The protein suspected to be batroxobin was resolved by SDS- PAGE and the band was moved to a PVDF membrane, where it was identified by Ponceau Red staining. The band was then submitted for amino acid sequence analysis. Also a SDS-PAGE analysis was done with batroxobin-treated fibrinogen. This showed bands on the gel that referred to Aα-, Bβ-, and γ-chains, indicating that there were no contaminating proteases in the batroxobin preparation (data not shown). To know for sure the fibrinogen was cleaved by batroxobin in Aα-, Bβ-, and γ-chains, these bands were compared with fibrinogen cleaved by thrombin which produces FpA and or FpB. The quantity FpA and FpB is measured by HPLC using synthetic FpA (Bachem) and FpB (LifeTein) as internal standards. To be sure FpA and FpB are measured, mass spectrometry was used. As expected, batroxobin only released FpA from fibrinogen, whereas thrombin released both FpA and FpB.

Toxicity

An overdose of batroxobin will eventually lead to death, due to hemostatic effects. No lethal- or safe dose has been determined in humans yet. The safe dose for rats is 3.0 KU/kg and for Macaca mulatta 1.5 KU/kg. The lethal dose has only been studied in mice and is 712.5548 ± 191.4479 KU/kg.

Efficacy

Batroxobin could be used as a medical adhesive against bleeding injuries. A study was done, where researchers aimed to examine the effects of batroxobin-containing adhesive for hemostasis. The fibrinogen conversion ability of batroxobin was evaluated by a fibrinogen clotting assay and a whole blood clotting assay. Both results proved that batroxobin containing adhesive forms blood clots. Higher concentrations of batroxobin more rapidly induced hemostasis. Another study has tested the effects of 2 units of batroxobin on perioperative blood loss and coagulation in patients with low molecular weight heparin (LMWH), while undergoing a hip replacement. This is done by giving 240 patients LMWH, 12 hours previously to the hip operation. These patients were randomly divided into a testing group (120 patients) and a control group (120 patients). The testing group and the control group received 2 units of batroxobin and 50 mg mannitol respectively 10 minutes before incision. There is tested on perioperative blood loss, postoperative 24 drainage and blood routine test, prothrombin time, activated partial thromboplastin time and fibrinogen. The study showed that the perioperative blood loss in the test group was 422,64 mL, which is less than the 667,67 mL blood loss of the control group. Red blood cell, hemoglobin and red blood cell volume and platelet were decreasing after the operation in both groups, but not significant different between the two groups. Also were there no drug-related adverse effects found in both groups, nor were there differences found in hospitalization between the two groups. The study concludes that 2 units of batroxobin could reduce the perioperative blood loss in patients getting a hip replacement operation with LMWH. There has also been studies done to examine the effects of batroxobin with continuous transcranial doppler monitoring in patients with acute cerebral stroke. The aim was to find out whether the effect of continuous transcranial Doppler (TCD) monitoring could be increased by the use of batroxobin on patients suffering from an acute cerebral stroke beyond the thrombolytic time window. In this study 90 patients were analyzed within twelve hours of the beginning of the symptoms. The patient were divided into a target and a control group which respectively got batroxobin with and without one hour of continuous TCD monitoring. Clinical sereneness of the strokes were based on the National Institutes of Health Stroke Scale (NIHSS) score, Barthel index (BI), Thrombolysis in Brain Ischemia score (TIBI), the incidence of advancing stroke, and the recurrence of cerebral infarction. The target group showed a significant better NIHSS score 57 days after the treatment, when compared with the control group. Also the incidence of advancing stroke one week afterwards and the incidence of stroke iteration after three months were significantly reduced in the target group. Safety of TCD monitoring in the clinical use for the groups was confirmed because no significant difference between the groups of the incidence of intracranial hemorrhage had shown one week after the use of TCD monitoring. The study concludes that the use of batroxobin combined with continuous TCD monitoring reduced the chance of advancing stroke and stroke recurrence after the treatment.

Adverse effects

The effects of batroxobin from different species are slightly different. There will be an overview of two different snakes, Bothrops atrox and Bothrops moojeni. When injected with batroxobin there will be major symptoms affecting the hemostatic system. Batroxobin from Bothrops atrox mediates the formation of fibrin I. This then renders the blood more prone to coagulation. An overdose will lead to excessive blood-clotting, which can result in bleeding due to damage to blood vessels or even thrombosis and pulmonary embolism. The clots can also cause secondary organ damage, such as renal damage or intracerebral hemorrhage. If the dose is high enough this will eventually lead to death. In the case of batroxobin from the Bothrops moojeni also formation of fibrin I is mediated, however there is also a release of tissue plasminogen activator (t-PA). This results in a rapid degradation of fibrin I and a lowered fibrinogen concentration. Which leads to a reduction of blood viscosity and blood coagulability. The blood is thinner and there is an increased tendency to bleed and to leak blood. Hypovolemic shock could occur. This shock can cause organs to stop working and eventually lead to death.

Effects on animals

There have been studies on a variety of animals. These animals include rats, mice, Macaca mulatta and rabbits.

Single-dose effects

At low dose the batroxobin significantly shortened bleeding times and clotting times in mice and rats. So a low dose of batroxobin acts as a coagulant. Furthermore, batroxobin didn’t affect the prothrombin time, thrombin time or activated partial thromboplastin time, but did lower the fibrinogen levels and released fibrinopeptide A in blood. In rabbits injection of batroxobin lead to a decrease in blood loss in liver wounds and shortened the bleeding time, coagulation time, activated partial thromboplastin time, prothrombin time and thrombin time.

Long-term effects

The long-term effects of batroxobin are tested on rats and Macaca mulatta. The subjects received an injection of batroxobin once a day for 30 consecutive days. In rats the batroxobin had pharmacological and toxicological effects on the hematology system and liver. There was a decrease of fibrinogen. The effects were reversible. In Macaca mulatta, the fibrinogen concentration was significantly decreased and the decrease was dose-dependent. There was a local inflammatory reaction in the injection sites of the animals. After 15 days there were antibodies to batroxobin detected. The conclusion was that batroxobin has pharmacological and toxicological effects on the hematology system of Macaca mulatta and the effects are reversible.

Clinical use

The medicine Defibrase ©is the trade name given to batroxobin and is isolated from the venom of Bothrops moojeni. It functions as an defibrinogenating agent and is used for patients with thrombosis. The batroxobin from the snake Bothrops atrox is patented as Reptilase and used as a hemostatic drug.