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Toxins Jul 2022Snakebite is a neglected tropical disease that causes considerable death and disability in the tropical world. Although snakebite can cause a variety of pathologies in...
Snakebite is a neglected tropical disease that causes considerable death and disability in the tropical world. Although snakebite can cause a variety of pathologies in victims, haemotoxic effects are particularly common and are typically characterised by haemorrhage and/or venom-induced consumption coagulopathy. Antivenoms are the mainstay therapy for treating the toxic effects of snakebite, but despite saving thousands of lives annually, these therapies are associated with limited cross-snake species efficacy due to venom variation, which ultimately restricts their therapeutic utility to particular geographical regions. In this study, we sought to explore the potential of ssDNA aptamers as toxin-specific inhibitory alternatives to antibodies. As a proof of principle model, we selected snake venom serine protease toxins, which are responsible for contributing to venom-induced coagulopathy following snakebite envenoming, as our target. Using SELEX technology, we selected ssDNA aptamers against recombinantly expressed versions of the fibrinogenolytic SVSPs ancrod from the venom of and batroxobin from . From the resulting pool of specific ssDNA aptamers directed against each target, we identified candidates that exhibited low nanomolar binding affinities to their targets. Downstream aptamer-linked immobilised sorbent assay, fibrinogenolysis, and coagulation profiling experiments demonstrated that the candidate aptamers were able to recognise native and recombinant SVSP toxins and inhibit the toxin- and venom-induced prolongation of plasma clotting times and the consumption of fibrinogen, with inhibitory potencies highly comparable to commercial polyvalent antivenoms. Our findings demonstrate that rationally selected toxin-specific aptamers can exhibit broad in vitro cross-reactivity against toxin isoforms found in different snake venoms and are capable of inhibiting toxins in pathologically relevant in vitro and ex vivo models of venom activity. These data highlight the potential utility of ssDNA aptamers as novel toxin-inhibiting therapeutics of value for tackling snakebite envenoming.
Topics: Antivenins; Disseminated Intravascular Coagulation; Hemorrhage; Humans; Snake Bites; Snake Venoms
PubMed: 35878207
DOI: 10.3390/toxins14070469 -
Journal of Thrombosis and Haemostasis :... Feb 2003Thrombin causes platelet activation via multiple pathways, and deficient thrombin generation reduces platelet contractile force (PCF) during clot retraction. We...
Thrombin causes platelet activation via multiple pathways, and deficient thrombin generation reduces platelet contractile force (PCF) during clot retraction. We hypothesized that PCF in blood samples from clotting factor-deficient patients would be diminished due to delayed or deficient thrombin generation. Blood samples from patients with fibrinogen, and factor V, VII, VIII, IX, X, XI and XIII deficiencies were compared to samples from normal controls. PCF in patient blood clotted with thrombin (1 NIH UmL(-1)) was compared to PCF in clots formed with batroxobin (0.25 micro g mL(-1)). PCF in the former should be normal, but PCF in the latter is dependent on thrombin generation within the sample and might be deficient. In factor VII-(n = 2, P < 0.05), factor VIII-(n = 6, P < 0.005) and factor XI-(n = 2, P < 0.05) deficient platelet-rich plasmas, PCF in batroxobin-induced clots was significantly lower than in thrombin-induced clots. In factor IX deficiency (n = 2), one patient had a dramatic reduction in PCF while a second patient had increased PCF. PCF was insignificantly (P = 0.346) reduced in two patients with factor X deficiency, and was normal in one patient with factor V deficiency. The factor X result is consistent with work in model systems, which indicates that as little as 1-3% factor X activity is sufficient to restore thrombin generation to normal. The factor V result probably indicates that the deficiency is incomplete. PCF in thrombin-induced clots was normal in all of these patients. Low fibrinogen and factor XIII deficiency reduced PCF in both thrombin- and batroxobin-induced clots. These results indicate that PCF is reduced, probably due to delayed thrombin generation, in some factor-deficient platelet-rich plasma samples.
Topics: Batroxobin; Blood Platelets; Case-Control Studies; Clot Retraction; Coagulation Protein Disorders; Elasticity; Humans; In Vitro Techniques; Platelet Activation; Thrombin; Time Factors
PubMed: 12871496
DOI: 10.1046/j.1538-7836.2003.00021.x -
Acta Pharmacologica Sinica Feb 2000To study the effects of batroxobin (Bat) on neurons survival, neurobehavioral test, ATP levels and hydroxyl radical outputs in hippocampus during forebrain...
AIM
To study the effects of batroxobin (Bat) on neurons survival, neurobehavioral test, ATP levels and hydroxyl radical outputs in hippocampus during forebrain ischemia-reperfusion in gerbils.
METHODS
The forebrain ischemia was induced by occluding the bilateral common carotid arteries for 10 min in gerbils, and ATP levels and 2, 3-dihydroxybenzoic acid (DHBA) outputs were assayed by HPLC. The neurons survival were assessed by histology, and behavioral tests of gerbils were assessed by open field test.
RESULTS
The number of neurons survival in Ir at d 7 postischemic insult were (7 +/- 4)% of sham-operated gerbils, much less than that in Bat (45 +/- 16)%. The levels of explore activities of ischemic gerbils was 175% and 159% of sham-operated gerbils at d 3 and d 6 postischemic insult, much more than that in Bat (120% d 3 and 140% d 6). Hippocampal ATP levels in Ir were 64% of sham-operated gerbils at reperfusion 60 min, much less than that in Bat I and II (82% and 89% respectively). The hippocampal 2,3-DHBA outputs in Ir increased by 4.5 folds of sham-operated gerbils at reperfusion 60 min, but the 2,3-DHBA outputs in Bat I and Bat II were only 2.6 and 2.4 folds respectively.
CONCLUSION
Bat possesses the inhibitory effects on DND and OH. production following cerebral ischemia-reperfusion in gerbils.
Topics: Adenosine Triphosphate; Animals; Batroxobin; Brain Ischemia; Cell Survival; Fibrinolytic Agents; Gerbillinae; Hippocampus; Hydroxybenzoates; Male; Maze Learning; Neurons; Reperfusion Injury
PubMed: 11263264
DOI: No ID Found -
Research and Practice in Thrombosis and... Jul 2023Variants of fibrinogen sequences that bind to thrombin's catalytic sites are mostly associated with bleeding phenotypes, while variants with fibrinogen...
BACKGROUND
Variants of fibrinogen sequences that bind to thrombin's catalytic sites are mostly associated with bleeding phenotypes, while variants with fibrinogen nonsubstrate-thrombin-binding sites are commonly believed to cause thrombosis. AαGlu39 and BβAla68 play important roles in fibrin(ogen)-thrombin-nonsubstrate binding. The BβAla68Thr variant has been described in several unrelated families with apparent thrombotic phenotypes.
OBJECTIVES
Homozygous AαGlu39Lys variant (fibrinogen BOE II) was identified in a boy with dysfibrinogenemia who had multiple cerebral hemorrhages. A series of analyses were performed to assess the variant's functions and elucidate underlying bleeding mechanisms.
METHODS
Abnormal fibrinogen was purified from plasma and subjected to Western blot, fibrinogen and fibrin monomer polymerization, clottability, fibrinopeptides release, activated factor (F)XIII (FXIIIa) cross-linking, fibrinolysis, and scanning electron microscopy analyses.
RESULTS
Fibrinogen BOE II weakened the binding capacity of thrombin to fibrinogen and delayed the formation of fibrin clots. The release of fibrinopeptides, polymerization of fibrinogen catalyzed by thrombin, and cross-linking of FXIIIa of fibrinogen BOE II were impaired. In contrast, batroxobin-catalyzed fibrinogen polymerization and desA/desAB fibrin monomer polymerization did not differ from those in normal controls. Fibrin clots formed by fibrinogen BOE II were composed of thicker fibrin fibers and showed a faster fibrinolysis rate.
CONCLUSION
Defective fibrin(ogen)-thrombin-nonsubstrate binding is not necessarily associated with thrombotic disorders. When the hypercoagulable state created by increased circulating free thrombin is insufficient to compensate for defective hemostasis caused by slowly formed but rapidly lysed clots, the primary concern of thrombin-binding deficiency dysfibrinogenemia appears to be hemorrhage rather than thrombosis.
PubMed: 37601017
DOI: 10.1016/j.rpth.2023.102145 -
The Journal of Biological Chemistry Mar 1987Determination of the nucleotide sequence of a cDNA for batroxobin, a thrombin-like enzyme from Bothrops atrox, moojeni venom, allowed elucidation of the complete amino... (Comparative Study)
Comparative Study
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.
Topics: Amino Acid Sequence; Animals; Base Sequence; Batroxobin; Cloning, Molecular; DNA; Disulfides; Endopeptidases; Molecular Weight; Peptide Fragments; Peptide Hydrolases; Serine Endopeptidases
PubMed: 3546302
DOI: No ID Found -
FEBS Letters Jul 2004A thrombin-like enzyme of Bothrops atrox moojeni venom, batroxobin, specifically cleaves fibrinogen alpha chain, resulting in the formation of non-crosslinked fibrin... (Comparative Study)
Comparative Study
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.
Topics: Amino Acid Sequence; Batroxobin; Cloning, Molecular; DNA, Complementary; Molecular Sequence Data; Pichia; Recombinant Proteins; Sequence Alignment; Sequence Homology, Amino Acid; Thrombin; Trypsin
PubMed: 15280019
DOI: 10.1016/j.febslet.2004.06.060 -
Acta Pharmacologica Sinica Jan 2000To study the effect of batroxobin(Bat) on dog heart ischemia/reperfusion (I/R) injury.
AIM
To study the effect of batroxobin(Bat) on dog heart ischemia/reperfusion (I/R) injury.
METHODS
Dog heart I/R injury was induced by occluding the left anterior descending coronary artery for 30 min and restoring blood perfusion for 90 min. Bat was intravenously injected before heart ischemia and 15 min before reperfusion. Plasma creatine kinase (CK), lactate dehydrogenase (LDH), and myocardial malondiaedehyde (MDA) concentrations were measured. The pathologic changes of I/R myocardium were observed.
RESULTS
Bat reduced the mortality rate of I/R dog (I/R group 65.0% vs Bat-I group 30.0% and Bat-II group 28.6%, P < 0.05). Myocytes of I/R heart showed intracellular edema, damaged mitochondria, and concentrated nucleus. Bat decreased these changes. In Bat-I and Bat-II group, plasma CK and LDH level were reduced, the +dp/dtmax and -dp/dtmax at 30 min after ischemia and 90 min after reperfusion were elevated, and left ventricular end dilation pressure (LVEDP) was lowered. The myocardial MDA contents were decreased by 42.3% and 38.1% (P < 0.01) in Bat-I and Bat-II group, respectively.
CONCLUSION
Bat may exert an apparent role against dog heart ischemia/reperfusion injury and improve myocardial function.
Topics: Animals; Batroxobin; Creatine Kinase; Dogs; Fibrinolytic Agents; Heart Function Tests; L-Lactate Dehydrogenase; Male; Malondialdehyde; Myocardial Reperfusion Injury; Myocardium
PubMed: 11263251
DOI: No ID Found -
Toxins Mar 2022A percutaneous renal biopsy is an essential tool for the diagnosis of various renal diseases; however, post-biopsy bleeding is a major complication. Hemocoagulase is a...
A percutaneous renal biopsy is an essential tool for the diagnosis of various renal diseases; however, post-biopsy bleeding is a major complication. Hemocoagulase is a detoxified and purified snake venom enzyme that is widely used to prevent post-procedural bleeding. In this study, we retrospectively analyzed the effect of hemocoagulase on post-renal biopsy bleeding. We included 221 patients who underwent percutaneous renal biopsy between April 2017 and December 2020 and analyzed post-renal biopsy hemoglobin (Hb) decline in patients who were administered a periprocedural hemocoagulase injection. After the renal biopsy, the mean Hb decrease in the entire patient cohort was 0.33 ± 0.84 g/dL. Periprocedural hemocoagulase injection lowered the Hb decline post-renal biopsy (0.50 ± 0.87 vs. 0.23 ± 0.80 g/dL, = 0.0204). The propensity-matched cohort was also adjusted for factors influencing postprocedural bleeding; periprocedural hemocoagulase injection reduced the Hb decline post-renal biopsy (0.56 ± 0.89 vs. 0.17 ± 0.74 g/dL, = 0.006). There were no adverse events (e.g., thrombosis and anaphylactic shock) due to hemocoagulase. Our study demonstrated the beneficial effect of hemocoagulase on post-renal biopsy Hb decline, suggesting its clinical value in preventing post-renal biopsy bleeding.
Topics: Batroxobin; Biopsy; Hemorrhage; Humans; Retrospective Studies
PubMed: 35324720
DOI: 10.3390/toxins14030223 -
Cartilage Apr 2021The study aims were to determine whether BST-CarGel, a chitosan scaffold for cartilage repair, can be mixed with bone marrow aspirate concentrate (BMAC) to create a cell...
OBJECTIVE
The study aims were to determine whether BST-CarGel, a chitosan scaffold for cartilage repair, can be mixed with bone marrow aspirate concentrate (BMAC) to create a cell seeded implant with comparative properties to standard BST-CarGel mixed with blood.
DESIGN
Whole blood and bone marrow were harvested from 12 patients who underwent cartilage repair surgery using BMAC after informed consent. A validated testing model was used to assess the following 6 conditions: (1) BST-CarGel mixed with whole blood (CG-WB), (2) BST-CarGel mixed with bone marrow (CG-BM), (3) BST-CarGel mixed with bone marrow concentrate (CG-BMAC), (4) whole blood (WB), (5) bone marrow (BM), and (6) bone marrow concentrate and batroxobin (BMAC-BTX). Cell retention and viability within the BST-CarGel/BMAC clots were investigated.
RESULTS
In our study, BM and BMAC (processed using the Harvest, SmartPrep2 system and reactivated with batroxibin) when combined with BST-CarGel produced a product that had similar clot contraction, macroscopic properties, and histological appearance to standard BSTCarGel mixed with blood. Mononucleated cells from the BMAC were retained within the scaffold and remained viable until clot dissolution .
CONCLUSIONS
By combining BST-CarGel with BMAC in the manner described, bone marrow-derived mononucleated cells can be retained within the chondral defect potentially negating the need for microfracture. Further work is required to confirm these potential benefits and determine if this combination will result in more durable cartilage repair and improved clinical outcomes.
Topics: Arthroplasty, Subchondral; Biocompatible Materials; Bone Marrow; Cartilage Diseases; Cartilage, Articular; Cell Culture Techniques; Chitosan; Feasibility Studies; Fractures, Stress; Humans; In Vitro Techniques; Prosthesis Design; Tissue Scaffolds; Treatment Outcome
PubMed: 30525942
DOI: 10.1177/1947603518812564 -
Blood Sep 2011Endothelial cells form the inner lining of vascular networks and maintain blood fluidity by inhibiting blood coagulation and promoting blood clot dissolution...
Endothelial cells form the inner lining of vascular networks and maintain blood fluidity by inhibiting blood coagulation and promoting blood clot dissolution (fibrinolysis). Plasmin, the primary fibrinolytic enzyme, is generated by the cleavage of the plasma protein, plasminogen, by its activator, tissue plasminogen activator. This reaction is regulated by plasminogen receptors at the surface of the vascular endothelial cells. Previous studies have identified the plasminogen receptor protein S100A10 as a key regulator of plasmin generation by cancer cells and macrophages. Here we examine the role of S100A10 and its annexin A2 binding partner in endothelial cell function using a homozygous S100A10-null mouse. Compared with wild-type mice, S100A10-null mice displayed increased deposition of fibrin in the vasculature and reduced clearance of batroxobin-induced vascular thrombi, suggesting a role for S100A10 in fibrinolysis in vivo. Compared with wild-type cells, endothelial cells from S100A10-null mice demonstrated a 40% reduction in plasminogen binding and plasmin generation in vitro. Furthermore, S100A10-deficient endothelial cells demonstrated impaired neovascularization of Matrigel plugs in vivo, suggesting a role for S100A10 in angiogenesis. These results establish an important role for S100A10 in the regulation of fibrinolysis and angiogenesis in vivo, suggesting S100A10 plays a critical role in endothelial cell function.
Topics: Animals; Annexin A2; Cells, Cultured; Endothelial Cells; Female; Fibrin; Fibrinolysin; Fibrinolysis; Hemorrhage; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Neovascularization, Physiologic; S100 Proteins
PubMed: 21768297
DOI: 10.1182/blood-2011-05-353482