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Toxins Apr 2023In Southeast Asia, the Malayan Pit Viper () is a venomous snake species of medical importance and bioprospecting potential. To unveil the diversity of its toxin genes,...
Venom Gland Transcriptome Assembly and Characterization for (Kuhl, 1824), the Malayan Pit Viper from Malaysia: Unravelling Toxin Gene Diversity in a Medically Important Basal Crotaline.
In Southeast Asia, the Malayan Pit Viper () is a venomous snake species of medical importance and bioprospecting potential. To unveil the diversity of its toxin genes, this study assembled and analyzed the venom gland transcriptome of from Malaysia. The expression of toxin genes dominates the gland transcriptome by 53.78% of total transcript abundance (based on overall FPKM, Fragments Per Kilobase Million), in which 92 non-redundant transcripts belonging to 16 toxin families were identified. Snake venom metalloproteinase (SVMP, PI > PII > PIII) is the most dominant family (37.84% of all toxin FPKM), followed by phospholipase A (29.02%), bradykinin/angiotensin-converting enzyme inhibitor-C-type natriuretic peptide (16.30%), C-type lectin (CTL, 10.01%), snake venom serine protease (SVSP, 2.81%), L-amino acid oxidase (2.25%), and others (1.78%). The expressions of SVMP, CTL, and SVSP correlate with hemorrhagic, anti-platelet, and coagulopathic effects in envenoming. The SVMP metalloproteinase domains encode hemorrhagins (kistomin and rhodostoxin), while disintegrin (rhodostomin from P-II) acts by inhibiting platelet aggregation. CTL gene homologues uncovered include rhodocytin (platelet aggregators) and rhodocetin (platelet inhibitors), which contribute to thrombocytopenia and platelet dysfunction. The major SVSP is a thrombin-like enzyme (an ancrod homolog) responsible for defibrination in consumptive coagulopathy. The findings provide insight into the venom complexity of and the pathophysiology of envenoming.
Topics: Animals; Malaysia; Transcriptome; Snake Venoms; Agkistrodon; Metalloproteases; Viper Venoms
PubMed: 37235350
DOI: 10.3390/toxins15050315 -
European Archives of... Sep 2023Disturbance of cochlear microcirculation is discussed as final common pathway of various inner ear diseases. Hyperfibrinogenemia causing increased plasma viscosity is a... (Randomized Controlled Trial)
Randomized Controlled Trial
PURPOSE
Disturbance of cochlear microcirculation is discussed as final common pathway of various inner ear diseases. Hyperfibrinogenemia causing increased plasma viscosity is a possible factor for a critical reduction of cochlear blood flow that might lead to sudden sensorineural hearing loss (SSHL). The aim was to determine the efficacy and safety of drug-induced defibrinogenation by ancrod for SSHL.
METHODS
Double-blind, randomized, placebo-controlled, multicenter, parallel group, phase II (proof-of-concept) study (planned enrollment: 99 patients). Patients received an infusion of ancrod or placebo (day 1) followed by subcutaneous administrations (day 2, 4, 6). Primary outcome was the change in pure tone audiogram air conduction average until day 8.
RESULTS
The study was terminated early due to slow recruiting (31 enrolled patients: 22 ancrod, 9 placebo). A significant improvement of hearing loss was registered in both groups (ancrod: - 14.3 dB ± 20.4 dB, - 39.9% ± 50.4%; placebo: - 22.3 dB ± 13.7 dB, - 59.1% ± 38.0%). A statistically significant group-difference was not detected (p = 0.374). Placebo response of 33.3% complete and 85.7% at least partial recovery was observed. Plasma fibrinogen levels were reduced significantly by ancrod (baseline: 325.2 mg/dL, day 2: 107.2 mg/dL). Ancrod was tolerated well, no adverse drug reaction was of severe intensity, no serious adverse events occurred.
CONCLUSION
Ancrod reduced fibrinogen levels that support its mechanism of action. The safety profile can be rated positively. Since the planned number of patients could not be enrolled, no efficacy conclusion can be drawn. The high rate of placebo response challenges clinical trials for SSHL and needs to be considered in future investigations. Trial registrations This study was registered in the EU Clinical Trials Register, EudraCT-No. 2012-000066-37 at 2012-07-02.
Topics: Humans; Ancrod; Fibrinogen; Glucocorticoids; Hearing Loss, Sensorineural; Hearing Loss, Sudden; Treatment Outcome; Proof of Concept Study
PubMed: 36881166
DOI: 10.1007/s00405-023-07896-z -
ASN Neuro 2022Diabetic human and murine retinas revealed pronounced microglial morphological activation and vascular abnormalities associated with inflammation. Pharmacological...
Diabetic human and murine retinas revealed pronounced microglial morphological activation and vascular abnormalities associated with inflammation. Pharmacological fibrinogen depletion using ancrod dampened microglial morphology alterations, resolved fibrinogen accumulation, rescued axonal integrity, and reduced inflammation in the diabetic murine retina.
Topics: Ancrod; Animals; CX3C Chemokine Receptor 1; Fibrinogen; Humans; Inflammation; Mice; Microglia; Retina
PubMed: 36221892
DOI: 10.1177/17590914221131446 -
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 -
Toxins Jun 2022Snakebite is a neglected tropical disease that causes high rates of global mortality and morbidity. Although snakebite can cause a variety of pathologies in victims,...
Snakebite is a neglected tropical disease that causes high rates of global mortality and morbidity. 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. Despite polyclonal antibody-based antivenoms being the mainstay life-saving therapy for snakebite, they are associated with limited cross-snake species efficacy, as there is often extensive toxin variation between snake venoms, including those used as immunogens for antivenom production. This restricts the therapeutic utility of any antivenom to certain geographical regions. In this study, we explored the feasibility of using recombinantly expressed toxins as immunogens to stimulate focused, pathology-specific, antibodies in order to broadly counteract specific toxins associated with snakebite envenoming. Three snake venom serine proteases (SVSP) toxins, sourced from geographically diverse and medically important viper snake venoms, were successfully expressed in HEK293F mammalian cells and used for murine immunisation. Analyses of the resulting antibody responses revealed that ancrod and RVV-V stimulated the strongest immune responses, and that experimental antivenoms directed against these recombinant SVSP toxins, and a mixture of the three different immunogens, extensively recognised and exhibited immunological binding towards a variety of native snake venoms. While the experimental antivenoms showed some reduction in abnormal clotting parameters stimulated by the toxin immunogens and crude venom, specifically reducing the depletion of fibrinogen levels and prolongation of prothrombin times, fibrinogen degradation experiments revealed that they broadly protected against venom- and toxin-induced fibrinogenolytic functional activities. Overall, our findings further strengthen the case for the use of recombinant venom toxins as supplemental immunogens to stimulate focused and desirable antibody responses capable of neutralising venom-induced pathological effects, and therefore potentially circumventing some of the limitations associated with current snakebite therapies.
Topics: Animals; Antivenins; Fibrinogen; Mammals; Mice; Serine Proteases; Snake Bites; Snake Venoms; Snakes; Viper Venoms
PubMed: 35878181
DOI: 10.3390/toxins14070443 -
Immunologic Research Apr 2022CD4FoxP3 regulatory T cells (CD4 Tregs) are known to dampen inflammation following severe trauma. Platelets were shown to augment their posttraumatic activation in burn...
CD4FoxP3 regulatory T cells (CD4 Tregs) are known to dampen inflammation following severe trauma. Platelets were shown to augment their posttraumatic activation in burn injury, but the exact mechanisms remain unclear. We hypothesized that platelet activation mechanisms via GPIIb/IIIa, fibrinogen, and PAR4 have an immunological effect and modulate CD4 Treg activation early after trauma. Therefore, C57Bl/6 N mice were injected with tirofiban (GPIIb/IIIa inhibition), ancrod (fibrinogen splitting enzyme), or tcY-NH (selective PAR4 antagonist peptide) before inducing a third-degree burn injury of 25% of the total body surface area. Changes in coagulation, and local and systemic CD4 Treg activity were assessed via rotational thromboelastometry (ROTEM®) and phospho-flow cytometry 1 h post intervention. The inhibition of GPIIb/IIIa and fibrinogen locally led to a higher basic activity of CD4 Tregs compared to non-inhibited animals. In contrast, PAR4 disruption on platelets locally led to an increased posttraumatic activation of CD4 Tregs. Fibrinogen led to complete elimination of coagulation, whereas GPIIb/IIIa or PAR4 inhibition did not. GPIIb/IIIa receptor and fibrinogen inhibition increase CD4 Tregs activity independently of trauma. Both are crucial for thrombus formation. We suggest platelets trapped in thrombi are unable to interact with CD4 Tregs but augment their activity when circulating freely. In contrast, PAR4 seems to reduce CD4 Treg activation following trauma. In summary, GPIIb/IIIa-, PAR4-, and fibrinogen-dependent pathways in platelets modulate CD4 Treg baseline activity, independently from their hemostatic functionality. PAR4-dependent pathways modulate the posttraumatic interplay of platelets and CD4 Tregs.
Topics: Animals; Blood Platelets; Burns; Fibrinogen; Hemostatics; Mice; Mice, Inbred C57BL; Platelet Glycoprotein GPIIb-IIIa Complex; T-Lymphocytes, Regulatory; Thrombosis
PubMed: 34932195
DOI: 10.1007/s12026-021-09258-5 -
Blood Mar 2019Platelets play a pivotal role in stimulating liver regeneration after partial hepatectomy in rodents and humans. Liver regeneration in rodents is delayed when platelets...
Platelets play a pivotal role in stimulating liver regeneration after partial hepatectomy in rodents and humans. Liver regeneration in rodents is delayed when platelets are inhibited. However, the exact mechanisms whereby platelets accumulate and promote liver regeneration remain uncertain. Thrombin-dependent intrahepatic fibrin(ogen) deposition was recently reported after partial hepatectomy (PHx) in mice, but the role of fibrin(ogen) deposits in liver regeneration has not been investigated. We tested the hypothesis that fibrin(ogen) contributes to liver regeneration by promoting intrahepatic platelet accumulation and identified the trigger of rapid intrahepatic coagulation after PHx. PHx in wild-type mice triggered rapid intrahepatic coagulation, evidenced by intrahepatic fibrin(ogen) deposition. Intrahepatic fibrin(ogen) deposition was abolished in mice with liver-specific tissue factor deficiency, pinpointing the trigger of coagulation after PHx. Direct thrombin activation of platelets through protease-activated receptor-4 did not contribute to hepatocyte proliferation after PHx, indicating that thrombin contributes to liver regeneration primarily by driving intrahepatic fibrin(ogen) deposition. Fibrinogen depletion with ancrod reduced both intrahepatic platelet accumulation and hepatocyte proliferation after PHx, indicating that fibrin(ogen) contributes to liver regeneration after PHx by promoting intrahepatic platelet accumulation. Consistent with the protective function of fibrin(ogen) in mice, low postoperative plasma fibrinogen levels were associated with liver dysfunction and mortality in patients undergoing liver resection. Moreover, increased intrahepatic fibrin(ogen) deposition was evident in livers of patients after liver resection but was remarkably absent in patients displaying hepatic dysfunction postresection. The results suggest a novel mechanism whereby coagulation-dependent intrahepatic fibrin(ogen) deposition drives platelet accumulation and liver regeneration after PHx.
Topics: Adult; Aged; Aged, 80 and over; Animals; Blood Coagulation; Female; Fibrinogen; Follow-Up Studies; Hepatectomy; Humans; Liver Diseases; Liver Regeneration; Male; Mice; Mice, Inbred C57BL; Middle Aged; Platelet Activation; Prognosis; Prospective Studies; Receptors, Thrombin; Thrombin; Thromboplastin
PubMed: 30655274
DOI: 10.1182/blood-2018-08-869057 -
Journal of Thrombosis and Haemostasis :... May 2018
An international collaborative study to calibrate the WHO 2nd International Standard for Ancrod (15/106) and the WHO Reference Reagent for Batroxobin (15/140): communication from the SSC of the ISTH.
Topics: Ancrod; Batroxobin; Calibration; Fibrinolysis; Fibrinolytic Agents; Humans; International Cooperation; Observer Variation; Predictive Value of Tests; Reference Standards; Reproducibility of Results; Thrombin Time; World Health Organization
PubMed: 29607604
DOI: 10.1111/jth.13996 -
Neuron Dec 2017Blood-brain barrier (BBB) disruption alters the composition of the brain microenvironment by allowing blood proteins into the CNS. However, whether blood-derived...
Blood-brain barrier (BBB) disruption alters the composition of the brain microenvironment by allowing blood proteins into the CNS. However, whether blood-derived molecules serve as extrinsic inhibitors of remyelination is unknown. Here we show that the coagulation factor fibrinogen activates the bone morphogenetic protein (BMP) signaling pathway in oligodendrocyte progenitor cells (OPCs) and suppresses remyelination. Fibrinogen induces phosphorylation of Smad 1/5/8 and inhibits OPC differentiation into myelinating oligodendrocytes (OLs) while promoting an astrocytic fate in vitro. Fibrinogen effects are rescued by BMP type I receptor inhibition using dorsomorphin homolog 1 (DMH1) or CRISPR/Cas9 activin A receptor type I (ACVR1) knockout in OPCs. Fibrinogen and the BMP target Id2 are increased in demyelinated multiple sclerosis (MS) lesions. Therapeutic depletion of fibrinogen decreases BMP signaling and enhances remyelination in vivo. Targeting fibrinogen may be an upstream therapeutic strategy to promote the regenerative potential of CNS progenitors in diseases with remyelination failure.
Topics: Activin Receptors, Type I; Animals; Blood Vessels; Bone Morphogenetic Proteins; Fibrinogen; Lysophosphatidylcholines; Mice; Mice, Inbred C57BL; Mice, Knockout; Microarray Analysis; Myelin Sheath; Oligodendrocyte Precursor Cells; Plasmids; Remyelination; Signal Transduction
PubMed: 29103804
DOI: 10.1016/j.neuron.2017.10.008 -
Journal of Thrombosis and Haemostasis :... Apr 2017Essentials Immunity and coagulation are linked during sepsis but the role of thrombin is not fully elucidated. We investigated the effect of thrombin inhibition on...
UNLABELLED
Essentials Immunity and coagulation are linked during sepsis but the role of thrombin is not fully elucidated. We investigated the effect of thrombin inhibition on murine Klebsiella pneumosepsis outcome. Thrombin is crucial for survival and limiting bacterial growth in pneumonia derived sepsis. Thrombin improves host defense via fibrin and enhancement of platelet-neutrophil interactions.
SUMMARY
Background Innate immunity and coagulation are closely linked during sepsis. Their interaction can be detrimental to the outcome because of microvascular failure but can also enhance host defense. The role of thrombin therein has not been fully elucidated. Objective We aimed to investigate the contribution of thrombin to the host response during pneumonia-derived sepsis. Methods Mice treated with the specific thrombin inhibitor dabigatran or control chow were infected with the common human sepsis pathogen Klebsiella (K.) pneumoniae via the airways. In subsequent infection experiments, mice were additionally treated with ancrod to deplete fibrinogen. Ex vivo Klebsiella growth was assessed by incubating human whole blood or specific blood components in various conditions with Klebsiella. Results Thrombin inhibition by dabigatran enhanced bacterial outgrowth and spreading, and accelerated mortality. Thrombin inhibition did not influence neutrophil recruitment to the lung or activation or neutrophil extracellular trap formation. Dabigatran reduced D-dimer formation and fibrin deposition in the lung. Fibrin depletion also enhanced bacterial outgrowth and spreading, and thrombin inhibition had no additional effect. Both thrombin and fibrin polymerization inhibited ex vivo Klebsiella outgrowth in human whole blood, which was neutrophil dependent, and the effect of thrombin required the presence of platelets and platelet protease activated receptor-1. In vivo thrombin inhibition reduced platelet-neutrophil complex formation and endothelial cell activation, but did not prevent sepsis-induced thrombocytopenia or organ damage. Conclusions These results suggest that thrombin plays an important role in protective immunity during pneumonia-derived sepsis by fibrin polymerization and enhancement of platelet-neutrophil interactions.
Topics: Animals; Blood Coagulation; Blood Platelets; Cell Communication; Dabigatran; Extracellular Traps; Female; Fibrin; Fibrinogen; Flow Cytometry; Humans; Immune System; Immunity, Innate; Klebsiella Infections; Klebsiella pneumoniae; Lung; Mice; Mice, Inbred C57BL; Microcirculation; Neutrophils; Pneumonia, Bacterial; Sepsis; Thrombin
PubMed: 28092405
DOI: 10.1111/jth.13625