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Journal of Thrombosis and Thrombolysis Nov 2017While snake venom derived enzymes, such as the thrombin-like activity possessing ancrod, have been used to treat thrombotic disease by defibrinogenating patients, the...
While snake venom derived enzymes, such as the thrombin-like activity possessing ancrod, have been used to treat thrombotic disease by defibrinogenating patients, the therapeutic potential of fibrinogenolytic snake venom enzymes, such as those derived from Crotalus atrox, have not been fully explored. However, one of the potential risks of administering fibrinogenolytic enzymes to effect defibrinogenation is hemorrhage secondary to hypofibrinogenemia. The present investigation sought to determine if human fibrinogen modified with carbon monoxide (CO) and iron (Fe) could resist degradation by C. atrox venom as has been seen in vitro in a recently developed rabbit model of envenomation. Compared with unmodified human fibrinogen, CO/Fe modified fibrinogen administered prior to envenomation had significantly shorter onset of coagulation and greater strength; however, when administered after envenomation, there was no differences between the two types of fibrinogen. Of interest, when administered after envenomation, both types of fibrinogen delayed the onset of coagulation while increasing plasma clot strength, a mixed effect likely secondary to formation of fibrinogen degradation products. Further preclinical investigations are needed to further define the benefits and risks of the use of fibrinogenolytic enzymes as defibrinogenating agents, as well as the risks of the "biochemical brakes" used to modulate the activity or substrate of the fibrinogenolytic enzyme.
Topics: Animals; Blood Coagulation; Carbon Monoxide; Crotalid Venoms; Crotalus; Fibrinogen; Humans; Iron; Rabbits
PubMed: 28889321
DOI: 10.1007/s11239-017-1549-2 -
Otology & Neurotology : Official... Jun 2017Disturbance of cochlear microcirculation is considered to be the final common pathway of various inner ear diseases. Hyperfibrinogenemia causing increased plasma...
OBJECTIVE
Disturbance of cochlear microcirculation is considered to be the final common pathway of various inner ear diseases. Hyperfibrinogenemia causing increased plasma viscosity is a known risk factor for sudden sensorineural hearing loss and may lead to a critical reduction of cochlear blood flow. The aim of this study was to evaluate the effect of a substantial reduction of plasma fibrinogen levels by drug-induced defibrinogenation for the treatment of acute hearing loss in vivo.
METHODS
Acute hearing loss was induced by hyperfibrinogenemia (i.v. injection of 330 mg/kg BW fibrinogen), using a guinea pig animal model. Parameters of cochlear microcirculation and hearing thresholds were quantified by intravital microscopy and evoked response audiometry. After obtaining baseline values, the course of hearing loss and disturbances of microcirculation were investigated under influence of intravenous defibrinogenation therapy (ancrod), corticosteroid, or placebo treatment, using 5 animals/group.
RESULTS
Acute hyperfibrinogenemia caused hearing loss from 10 ± 7 to 26 ± 10 dB SPL at baseline. Drug-induced reduction of fibrinogen levels showed a significant increase of cochlear microcirculation (1.6-fold) and recovered hearing threshold (11 ± 6 dB SPL). Placebo or corticosteroid treatment had no effect on hearing loss (35 ± 7 dB SPL and 32 ± 18 dB SPL, respectively).
CONCLUSION
Acute hyperfibrinogenemia resulted in hearing loss. Drug-induced reduction of elevated fibrinogen levels caused an increase in cochlear blood flow and a decrease in hearing thresholds. Placebo or corticosteroid treatment had no effect. Reduction of plasma fibrinogen levels could serve as a clinical treatment option for acute hearing loss.
Topics: Ancrod; Animals; Audiometry, Evoked Response; Auditory Threshold; Cochlea; Disease Models, Animal; Fibrinogen; Fibrinolytic Agents; Guinea Pigs; Hearing Loss, Sensorineural; Male; Microcirculation
PubMed: 28369007
DOI: 10.1097/MAO.0000000000001400 -
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 -
Journal of Thrombosis and Thrombolysis Feb 2017A complication of defibrinogenation therapy with snake venom enzymes such as ancrod is hypofibrinogenemia associated bleeding secondary to no human-derived inhibitor...
A complication of defibrinogenation therapy with snake venom enzymes such as ancrod is hypofibrinogenemia associated bleeding secondary to no human-derived inhibitor being available to inactivate or diminish the activity of such enzymes. Of interest, ancrod contains a critical histidine residue without which enzymatic activity is inhibited, and carbon monoxide has been demonstrated to inhibit biomolecular function by interacting with histidine moieties in ion channels. We tested the hypothesis that exposure of three different snake venoms containing serine proteases with thrombin-like activity (which included ancrod) to carbon monoxide derived from carbon monoxide releasing molecule-2 would diminish their effects on plasmatic coagulation as assessed by thrombelastography. In the case of the Malayan pit viper and Eastern diamondback rattlesnake venoms, carbon monoxide diminished the effects of thrombin-like activity. In contrast, timber rattlesnake venom demonstrated enhancement of "thrombin-generating" activity with simultaneous loss of thrombin-like activity in response to carbon monoxide exposure. These findings may serve as the rational basis for not just continuing to investigate the potential of snake venom enzymes as clinical defibrinogenating agents, but to also to assess the potential to stop such agents from becoming a catalytic "runaway train" by judicious application of a biochemical "brake" such as carbon monoxide.
Topics: Ancrod; Animals; Blood Coagulation; Carbon Monoxide; Crotalid Venoms; Fibrinolytic Agents; Humans; Organometallic Compounds; Serine Proteases; Serine Proteinase Inhibitors; Thrombelastography; Thrombin
PubMed: 27787696
DOI: 10.1007/s11239-016-1442-4 -
Journal of Proteomics Oct 2016The venom of Malayan pit viper (Calloselasma rhodostoma) is highly toxic but also valuable in drug discovery. However, a comprehensive proteome of the venom that details...
UNLABELLED
The venom of Malayan pit viper (Calloselasma rhodostoma) is highly toxic but also valuable in drug discovery. However, a comprehensive proteome of the venom that details its toxin composition and abundance is lacking. This study aimed to unravel the venom complexity through a multi-step venomic approach. At least 96 distinct proteins (29 basic, 67 acidic) in 11 families were identified from the venom. The venom consists of mainly snake venom metalloproteinases (SVMP, 41.17% of total venom proteins), within which the P-I (kistomin, 20.4%) and P-II (rhodostoxin, 19.8%) classes predominate. This is followed by C-type lectins (snaclec, 26.3%), snake venom serine protease (SVSP, 14.9%), L-amino acid oxidase (7.0%), phospholipase A2 (4.4%), cysteine-rich secretory protein (2.5%), and five minor toxins (nerve growth factor, neurotrophin, phospholipase B, 5' nucleotidase and phosphodiesterase, totaling 2.6%) not reported in the proteome hitherto. Importantly, all principal hemotoxins unveiled correlate with the syndrome: SVSP ancrod causes venom-induced consumptive coagulopathy, aggravated by thrombocytopenia caused by snaclec rhodocytin, a platelet aggregation inducer, while P-II rhodostoxin mediates hemorrhage, exacerbated by P-I kistomin and snaclec rhodocetin that inhibit platelet plug formation. These toxins exist in multiple isoforms and/or complex subunits, deserving further characterization for the development of an effective, polyspecific regional antivenom.
BIOLOGICAL SIGNIFICANCE
Advents in proteomics and bioinformatics have vigorously propelled the scientific discoveries of toxins from various lineages of venomous snakes. The Malayan pit viper, Calloselasma rhodostoma, is a medically important species in Southeast Asia as its bite can cause envenomation, while the venom is also a source of bioactive compounds for drug discovery. Detailed profiling of the venom, however, is inadequate possibly due to the complex nature of the venom and technical limitation in separating the constituents into details. Integrating a multi-step fractionation method, this study successfully revealed a comprehensive and quantitative profile of the composition of the venom of this medically important venomous snake. The relative abundance of the various venom proteins is determined in a global profile, providing useful information for understanding the pathogenic roles of the different toxins in C. rhodostoma envenomation. Notably, the principal hemotoxins were identified in great details, including the variety of toxin subunits and isoforms. The findings indicate that these toxins are the principal targets for effective antivenom neutralization, and should be addressed in the production of a pan-regional polyspecific antivenom. In addition, minor toxin components not reported previously in the venom were also detected in this study, enriching the current toxin database for the venomous snakes.
Topics: Animals; Crotalinae; Hemolytic Agents; L-Amino Acid Oxidase; Lectins, C-Type; Metalloproteases; Phospholipases A2; Proteome; Serine Proteases; Viper Venoms
PubMed: 27418434
DOI: 10.1016/j.jprot.2016.07.006 -
Journal of Thrombosis and Thrombolysis Aug 2016Fibrinogen depletion via catalysis by snake venom enzymes as a therapeutic strategy to prevent or treat thrombotic disorders was utilized for over four decades, with...
Fibrinogen depletion via catalysis by snake venom enzymes as a therapeutic strategy to prevent or treat thrombotic disorders was utilized for over four decades, with ancrod being the quintessential agent. However, ancrod eventually was found to not be of clinical utility in large scale stroke trial, resulting in the eventual discontinuation of the administration of the drug for any indication. It was hypothesized that ancrod, possessing thrombin-like activity, may have unappreciated robust coagulation kinetics. Using thrombelastographic methods, a comparison of equivalent tissue factor initiated thrombin generation and Calloselasma rhodostoma venom (rich in ancrod activity) on plasmatic coagulation kinetics was performed. The venom resulted in thrombi that formed nearly twice as fast compared to thrombin formed clots, and there was no difference in fibrinolytic kinetics initiated by tissue-type plasminogen activator. In plasma containing iron and carbon monoxide modified fibrinogen, which may be found in patients at risk of stroke, the coagulation kinetic differences observed with venom was still more vigorous than that seen with thrombin. These phenomena may provide insight into the clinical failure of ancrod, and may serve as an impetus to revisit the concept of fibrinogen depletion via fibrinogenolytic enzymes, not those with thrombin-like activity.
Topics: Afibrinogenemia; Ancrod; Animals; Blood Coagulation; Crotalid Venoms; Fibrinogen; Fibrinolysis; Humans; Kinetics; Thrombelastography; Thrombin; Thromboplastin
PubMed: 26905070
DOI: 10.1007/s11239-016-1343-6 -
Biometals : An International Journal on... Apr 2016Iron-bound fibrinogen has been noted to accelerate plasmatic coagulation in patients with divergent conditions involving upregulation of heme oxygenase activity,...
Iron-bound fibrinogen has been noted to accelerate plasmatic coagulation in patients with divergent conditions involving upregulation of heme oxygenase activity, including hemodialysis, Alzheimer's disease, sickle cell anemia, and chronic migraine. Our goal was to determine if a site of iron-fibrinogen interaction was on the alpha chain. Using thrombelastography, we compared the coagulation kinetic profiles of plasma exposed to 0-10 µM ferric chloride after activation of coagulation with thrombin generated by contact activation of plasma with the plastic sample cup or by exposure to 1 µg/ml of Calloselasma rhodostoma venom (rich in ancrod activity), which causes coagulation via polymerization of alpha chain monomers. Venom mediated coagulation always occurred before thrombin activated thrombus formation, and ferric chloride always diminished the time of onset of coagulation and increased the velocity of clot growth. Iron enhances plasmatic coagulation kinetics by modulating the alpha chain of fibrinogen.
Topics: Adult; Blood Coagulation; Chlorides; Female; Ferric Compounds; Fibrinogen; Humans; Kinetics; Male; Young Adult
PubMed: 26782808
DOI: 10.1007/s10534-016-9909-5 -
Canadian Journal of Anaesthesia =... May 2015Heparin anticoagulation followed by protamine reversal is commonly used in cardiopulmonary bypass (CPB) cardiac procedures, but this strategy has some limitations. The... (Review)
Review
PURPOSE
Heparin anticoagulation followed by protamine reversal is commonly used in cardiopulmonary bypass (CPB) cardiac procedures, but this strategy has some limitations. The primary objective of this study was to determine the reliable alternatives for anticoagulation during CPB for cardiac surgery. For each drug proposed, the secondary objectives were to outline the main advantages and disadvantages, to propose a therapeutic protocol, and to provide a cost-benefit analysis.
SOURCE
A systematic review of the literature was performed between September 2012 and December 2013. It was based on the protocol established by the "Cochrane collaboration Handbook". Twenty articles were analyzed. The Thériaque database from the University Hospital of Grenoble made the economic analysis possible.
PRINCIPAL FINDINGS
Seven alternative anticoagulation strategies were considered: danaparoid sodium, lepirudin, argatroban, bivalirudin, ancrod, idraparinux, and EP217609. Danaparoid sodium has issues with individual variability. Several studies (EVOLUTION-ON, CHOOSE-ON) proposed a reliable therapeutic protocol for bivalirudin. Ancrod resulted in an increase in the transfusion of blood products. Direct thrombin inhibitors offer a promising alternative. EP217609 is a synthetic anticoagulant currently undergoing Phase IIa clinical trials. It is an indirect inhibitor of factor Xa, a direct inhibitor of free and bound thrombin, and can be neutralized by avidin.
CONCLUSIONS
The ideal anticoagulation strategy for cardiac surgery with CPB does not exist. Heparin and protamine remain the gold standard for anticoagulation therapy. To date, bivalirudin is the most promising molecule despite its high cost and lack of a readily available antagonist.
Topics: Anticoagulants; Cardiopulmonary Bypass; Cost-Benefit Analysis; Drug Costs; Heparin; Hirudins; Humans; Peptide Fragments; Protamines; Recombinant Proteins
PubMed: 25697279
DOI: 10.1007/s12630-015-0339-6 -
American Journal of Physiology. Renal... Aug 2014Fibrinogen (Fg) has been implicated in the pathogenesis of several fibrotic disorders by acting as a profibrotic ligand for a variety of cellular surface receptors and...
Fibrinogen (Fg) has been implicated in the pathogenesis of several fibrotic disorders by acting as a profibrotic ligand for a variety of cellular surface receptors and by modulating the provisional fibrin matrix formed after injury. We demonstrated increased renal Fg expression after unilateral ureteral obstruction and folic acid (FA) nephropathy in mice, respectively. Urinary Fg excretion was also increased in FA nephropathy. Using in vitro and in vivo approaches, our results suggested that IL-6 mediates STAT3 activation in kidney fibrosis and that phosphorylated (p)STAT3 binds to Fgα, Fgβ, and Fgγ promoters in the kidney to regulate their transcription. Genetically modified Fg heterozygous mice (∼75% of normal plasma Fg levels) exhibited only 3% kidney interstitial fibrosis and tubular atrophy after FA nephropathy compared with 24% for wild-type mice. Fibrinogenolysis through Ancrod administration after FA reduced interstitial fibrosis more than threefold compared with vehicle-treated control mice. Mechanistically, we show that Fg acts synergistically with transforming growth factor (TGF)-β1 to induce fibroblast proliferation and activates TGF-β1/pSMAD2 signaling. This study offers increased understanding of Fg expression and molecular interactions with TGF-β1 in the progression to kidney fibrosis and, importantly, indicates that fibrinogenolytics like Ancrod present a treatment opportunity for a yet intractable disease.
Topics: Ancrod; Animals; Disease Progression; Fibrinogen; Fibrosis; Hep G2 Cells; Humans; Interleukin-6; Kidney; Kidney Diseases; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; STAT3 Transcription Factor; Smad2 Protein; Transforming Growth Factor beta1; Ureteral Obstruction
PubMed: 25007874
DOI: 10.1152/ajprenal.00189.2014