Search

Background: The Western diamondback rattlesnake ( Crotalus atrox ) is a medically important venomous snake in the Southwestern United States, injuring humans, and their companion animals. The goals of this investigation were to utilize a rabbit model of subcutaneous envenomation to assess Crotalus atrox venom coagulopathy and determine the efficacy of a ruthenium-containing antivenom (RA) in attenuating it., Methods: Sedated New Zealand White rabbits had viscoelastic measurements of whole blood coagulation kinetics obtained from ear artery samples. Crotalus atrox venom (4 mg/kg) was injected subcutaneously and changes in coagulation determined over three hours and compared to samples obtained prior to envenomation. Other rabbits had site-directed RA injected 5 min after venom injection., Results: A significant decrease in the velocity of clot growth and thrombus strength was observed in animals injected with venom alone. Site-directed administration of RA resulted in no change in coagulation over the 3 h following venom injection. The interaction of antivenom administration and time was significantly different in the cases of clot growth velocity and strength., Conclusions: A novel rabbit model was used to define the toxicodynamic profile of coagulopathy of Crotalus atrox venom and demonstrate the efficacy of RA. Future investigation is planned involving other medically important venoms and RA administration., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Ruthenium chloride (RuCl 3 ) is widely utilized for synthesis and catalysis of numerous compounds in academia and industry and is utilized as a key molecule in a variety of compounds with medical applications. Interestingly, RuCl 3 has been demonstrated to modulate human plasmatic coagulation and serves as a constituent of a compounded inorganic antivenom that neutralizes the coagulopathic effects of snake venom in vitro and in vivo. Using thrombelastography, this investigation sought to determine if RuCl 3 inhibition of the fibrinogenolytic effects of Crotalus atrox venom could be modulated by vehicle composition in human plasma. Venom was exposed to RuCl 3 in 0.9% NaCl, phosphate-buffered saline (PBS), or 0.9% NaCl containing 1% dimethyl sulfoxide (DMSO). RuCl 3 inhibited venom-mediated delay in the onset of thrombus formation, decreased clot growth velocity, and decreased clot strength. PBS and DMSO enhanced the effects of RuCl 3 . It is concluded that while a Ru-based cation is responsible for significant inhibition of venom activity, a combination of Ru-based ions containing phosphate and DMSO enhances RuCl 3 -mediated venom inhibition. Additional investigation is indicated to determine what specific Ru-containing molecules cause venom inhibition and what other combinations of inorganic/organic compounds may enhance the antivenom effects of RuCl 3 .

Eastern Diamondback Rattlesnake ( Crotalus adamanteus ) envenomation is a medical emergency encountered in the Southeastern United States. The venom contains a snake venom thrombin-like enzyme (SVTLE) that is defibrinogenating, causing coagulopathy without effects on platelets in humans. This investigation utilized thrombelastographic methods to document this coagulopathy kinetically on the molecular level in a rabbit model of envenomation via the analyses of whole blood samples without and with platelet inhibition. Subsequently, the administration of a novel ruthenium compound containing site-directed antivenom abrogated the coagulopathic effects of envenomation in whole blood without platelet inhibition and significantly diminished loss of coagulation in platelet-inhibited samples. This investigation provides coagulation kinetic insights into the molecular interactions and results of SVTLE on fibrinogen-dependent coagulation and confirmation of the efficacy of a ruthenium antivenom. These results serve as a rationale to investigate the coagulopathic effects of other venoms with this model and assess the efficacy of this site-directed antivenom.

Venomous snake bite adversely affects millions of people yearly, but few animal models allow for the determination of toxicodynamic timelines with hemotoxic venoms to characterize the onset and severity of coagulopathy or assess novel, site-directed antivenom strategies. Thus, the goals of this investigation were to create a rabbit model of subcutaneous envenomation to assess venom toxicodynamics and efficacy of ruthenium-based antivenom administration. New Zealand White rabbits were sedated with midazolam via the ear vein and had viscoelastic measurements of whole blood and/or plasmatic coagulation kinetics obtained from ear artery samples. Venoms derived from Crotalus scutulatus scutulatus , Bothrops moojeni , or Calloselasma rhodostoma were injected subcutaneously, and changes in coagulation were determined over three hours and compared to samples obtained prior to envenomation. Other rabbits had ruthenium-based antivenoms injected five minutes after venom injection. Viscoelastic analyses demonstrated diverse toxicodynamic patterns of coagulopathy consistent with the molecular composition of the proteomes of the venoms tested. The antivenoms tested attenuated venom-mediated coagulopathy. A novel rabbit model can be used to characterize the onset and severity of envenomation by diverse proteomes and to assess site-directed antivenoms. Future investigation is planned involving other medically important venoms and antivenom development.

In recent years a variety of metals (cadmium, chromium, copper, iron) have been demonstrated to modulate coagulation in vitro and in vivo. One group of metals, the platinoids, have not been assessed, and such investigation is justified given the thromboembolic phenomena associated with platinum-based chemotherapy. Thus, the goal of the present investigation was to assess the effects of carboplatin, cisplatin (platinum compounds), NAMI-A, and ruthenium chloride (ruthenium compounds) on human plasmatic coagulation. Human plasma was exposed to clinically relevant, equimolar concentrations of the aforementioned platinum and ruthenium compounds, with changes in plasmatic coagulation assessed via thrombelastography. The first series of experiments demonstrated no significant modulation of coagulation by the platinum compounds, while NAMI-A demonstrated mild hypercoagulability and ruthenium chloride exerted marked hypercoagulability. A second series of experiments utilizing a variety of specialized modifications of thrombelastography focused on ruthenium chloride revealed that this compound enhances prothrombin activation. While the hypercoagulability associated with platinum compounds in vivo do not appear to have a basis in plasmatic biochemistry, it appears that ruthenium compounds can exert procoagulant properties by enhancing the common pathway of human plasmatic coagulation. Future investigation of Ru based chemotherapeutic agents in development to assess procoagulant activity as part of evaluating their potential clinical safety is warranted.

The processes of blood coagulation and fibrinolysis that in part maintain the physical integrity of the circulatory system and fluidity of its contents are complex as they are critical for life. While the roles played by cellular components and circulating proteins in coagulation and fibrinolysis are widely acknowledged, the impact of metals on these processes is at best underappreciated. In this narrative review we identify twenty-five metals that can modulate the activity of platelets, plasmatic coagulation, and fibrinolysis as determined by in vitro and in vivo investigations involving several species besides human beings. When possible, the molecular interactions of the various metals with key cells and proteins of the hemostatic system were identified and displayed in detail. It is our intention that this work serve not as an ending point, but rather as a fair evaluation of what mechanisms concerning metal interactions with the hemostatic system have been elucidated, and as a beacon to guide future investigation.

Carbon monoxide releasing molecule-2 (CORM-2), an emerging therapeutic in human medicine, enhances plasmatic coagulation and attenuates fibrinolysis in vitro in human, rabbit and horse plasma and ameliorates hypocoagulation and hyperfibrinolysis secondary to venom exposure in human plasma in vitro. Fibrinogenases in rattlesnake venom cause decreased clot strength, and in the presence of tissue plasminogen activator (tPA) in vitro, a markedly increased rate of clot lysis. CO interacts with a haem group on fibrinogen, changing its configuration so that the fibrin clot is strengthened and more resistant to fibrinolysis. We hypothesized that CORM-2 enhances coagulation and attenuates fibrinolysis in canine plasma exposed to C viridis venom. We measured the effects of C viridis venom on clot strength, rates of coagulation and fibrinolysis in both pooled canine plasma and plasma from individual naturally envenomed dogs, with and without CORM-2, using thromboelastography (TEG). We tested venom effects on coagulation using tissue factor (TF) activated TEG and on both coagulation and fibrinolysis using TF-activated TEG with added tPA. We found that 17.9 µg/mL of venom causes a mean 26.4% decrease in clot strength, a 61.8% decrease in maximum rate of thrombus generation, 75% faster clot lysis, a 226% increase in maximum rate of lysis and a 92% decrease in total clot life span (CLS). CORM-2 ameliorated these effects, increasing CLS in the presence of venom by 603%. Additionally, we showed that CORM-2 has similar effects in vitro on plasma from naturally envenomed dogs, showing promise as an adjunct therapy for snake envenomation., (© 2019 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).)

The Mojave rattlesnake is a unique species of pit viper that expresses either a highly potent phospholipase A 2 (PLA 2 )-dependent neurotoxin containing venom nearly devoid of fibrinogenolytic metalloproteinases (venom type A) or a hemotoxic venom with a high percentage of metalloproteinases and PLA 2 without any neurotoxin present (venom type B) depending on its geographical location in the Southwestern United States and Mexico. Given that PLA 2 have been demonstrated to affect coagulation, it was hypothesized that the anticoagulant effects of both type A and B venoms could be assessed by thrombelastography, and determination made if these venoms were heme modulated. Both venom types were exposed to carbon monoxide releasing molecule-2 or its inactivated molecule (0 or 100 µM) in isolation and then placed in human plasma with consequent coagulation kinetics assessed by thrombelastography. It was determined that type A venom was twice as potent as an anticoagulant compared to type B venom, and that both venoms were inhibited by carbon monoxide releasing molecule-2 but not its inactivated molecule. Given the lack of proteolytic activity of type A venom and the dependence of neurotoxicity on PLA 2 activity, it may be possible that carbon monoxide could inhibit neurotoxicity based on inhibition of PLA 2 anticoagulant activity. These data may serve as the rationale for extension of these observations into animal models to determine if CO may inhibit not just hemotoxic venom, but also PLA 2 -dependent neurotoxic venom.

Pain-acute, chronic and debilitating-is the most feared neurotoxicity resulting from a survivable venomous snake bite. The purpose of this review is to present in a novel paradigm what we know about the molecular mechanisms responsible for pain after envenomation. Progressing from known pain modulating peptides and enzymes, to tissue level interactions with venom resulting in pain, to organ system level pain syndromes, to geographical level distribution of pain syndromes, the present work demonstrates that understanding the mechanisms responsible for pain is dependent on "location, location, location". It is our hope that this work can serve to inspire the molecular and epidemiologic investigations needed to better understand the neurotoxic mechanisms responsible for these snake venom mediated diverse pain syndromes and ultimately lead to agent specific treatments beyond anti-venom alone.

Thrombocytosis has been feared as a source of thrombotic complications during the conduct of cardiopulmonary bypass (CPB) for patients undergoing cardiac procedures. We present a patient urgently requiring repair/replacement of three heart valves that had preexisting myelofibrosis with thrombocytosis (platelet count of 800,000 per µl) and neutrophilia (40,000 per µl). Despite achieving an activated clotting time > 500 s with heparin and antithrombin concentrate administration prior to CPB, the pump oxygenator and reservoir demonstrated significant clot just prior to restoration of the patient's circulation. The patient subsequently suffered a severe protamine reaction that was successfully managed. A review of the literature of similar patients and the relevant cellular and biochemical mechanisms in this setting are presented, with potential therapeutic approaches to prevent such complications noted., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Procoagulant snake venoms have been inhibited by the ruthenium containing compounds CORM-2 and RuCl 3 separately, presumably by interacting with critical histidine or other sulfur-containing amino acids on key venom enzymes. However, combinations of these and other platinoid containing compounds could potentially increase, decrease or not affect the procoagulant enzyme function of venom. Thus, the purpose of this investigation was to determine if formulations of platinoid compounds could inhibit venom procoagulant activity and if the formulated compounds interacted to enhance inhibition. Using a human plasma coagulation kinetic model to assess venom activity, six diverse venoms were exposed to various combinations and concentrations of CORM-2, CORM-3, RuCl 3 and carboplatin (a platinum containing compound), with changes in venom activity determined with thrombelastography. The combinations of CORM-2 or CORM-3 with RuCl 3 were found to enhance inhibition significantly, but not in all venoms nor to the same extent. In sharp contrast, carboplatin-antagonized CORM-2 mediated the inhibition of venom activity. These preliminary results support the concept that platinoid compounds may inhibit venom enzymatic activity at the same or different molecular sites and may antagonize inhibition at the same or different sites. Further investigation is warranted to determine if platinoid formulations may serve as potential antivenoms.

The demonstration that carbon monoxide releasing molecules (CORMs) affect experimental systems by the release of carbon monoxide, and not via the interaction of the inactivated CORM, has been an accepted paradigm for decades. However, it has recently been documented that a radical intermediate formed during carbon monoxide release from ruthenium (Ru)-based CORM (CORM-2) interacts with histidine and can inactivate bee phospholipase A 2 activity. Using a thrombelastographic based paradigm to assess procoagulant activity in human plasma, this study tested the hypothesis that a Ru-based radical and not carbon monoxide was responsible for CORM-2 mediated inhibition of Atheris, Echis , and Pseudonaja species snake venoms. Assessment of the inhibitory effects of ruthenium chloride (RuCl 3 ) on snake venom activity was also determined. CORM-2 mediated inhibition of the three venoms was found to be independent of carbon monoxide release, as the presence of histidine-rich albumin abrogated CORM-2 inhibition. Exposure to RuCl 3 had little effect on Atheris venom activity, but Echis and Pseudonaja venom had procoagulant activity significantly reduced. In conclusion, a Ru-based radical and ion inhibited procoagulant snake venoms, not carbon monoxide. These data continue to add to our mechanistic understanding of how Ru-based molecules can modulate hemotoxic venoms, and these results can serve as a rationale to focus on perhaps other, complementary compounds containing Ru as antivenom agents in vitro and, ultimately, in vivo.

Using thrombelastography to gain mechanistic insights, recent investigations have identified enzymes and compounds in Naja and Crotalus species' neurotoxic venoms that are anticoagulant in nature. The neurotoxic venoms of the four extant species of Dendroaspis (the Black and green mambas) were noted to be anticoagulant in nature in human blood, but the mechanisms underlying these observations have never been explored. The venom proteomes of these venoms are unique, primarily composed of three finger toxins (3-FTx), Kunitz-type serine protease inhibitors (Kunitz-type SPI) and <7% metalloproteinases. The anticoagulant potency of the four mamba venoms available were determined in human plasma via thrombelastography; vulnerability to inhibition of anticoagulant activity to ethylenediaminetetraacetic acid (EDTA) was assessed, and inhibition of anticoagulant activity after exposure to a ruthenium (Ru)-based carbon monoxide releasing molecule (CORM-2) was quantified. Black mamba venom was the least potent by more than two orders of magnitude compared to the green mamba venoms tested; further, Black Mamba venom anticoagulant activity was not inhibited by either EDTA or CORM-2. In contrast, the anticoagulant activities of the green mamba venoms were all inhibited by EDTA to a greater or lesser extent, and all had anticoagulation inhibited with CORM-2. Critically, CORM-2-mediated inhibition was independent of carbon monoxide release, but was dependent on a putative Ru-based species formed from CORM-2. In conclusion, there was great species-specific variation in potency and mechanism(s) responsible for the anticoagulant activity of Dendroaspis venom, with perhaps all three protein classes-3-FTx, Kunitz-type SPI and metalloproteinases-playing a role in the venoms characterized.

Bee venom phospholipase A 2 (PLA 2 ) has potential for significant morbidity. Ruthenium (Ru)-based carbon monoxide releasing molecules (CORM) inhibit snake venoms that are anticoagulant and contain PLA 2 . In addition to modulating heme-bearing proteins with carbon monoxide, these CORM generate reactive Ru species that form adducts with histamine residues resulting in changes in protein function. This study sought to identify anticoagulant properties of bee venom PLA 2 via catalysis of plasma phospholipids required for thrombin generation. Another goal was to determine if Ru-based CORM inhibit bee venom PLA 2 via carbon monoxide release or via potential binding of reactive Ru species to a key histidine residue in the catalytic site of the enzyme. Anticoagulant activity of bee venom PLA 2 was assessed via thrombelastography with normal plasma. Bee venom PLA 2 was then exposed to different CORM and a metheme forming agent and anticoagulant activity was reassessed. Using Ru, boron and manganese-based CORM and a metheme forming agent, it was demonstrated that it was unlikely that carbon monoxide interaction with a heme group attached to PLA 2 was responsible for inhibition of anticoagulant activity by Ru-based CORM. Exposure of PLA 2 to a Ru-based CORM in the presence of histidine-rich human albumin resulted in loss of inhibition of PLA 2 . Ru-based CORM likely inhibit bee venom PLA 2 anticoagulant activity via formation of reactive Ru species that bind to histidine residues of the enzyme.

Background: A phenomena of interest is the in vitro anticoagulant effects of neurotoxins found in elapid venoms that kill by paralysis. These enzymes include phospholipase A2 (PLA2), and it has recently been demonstrated that carbon monoxide inhibits the PLA2-dependent neurotoxin contained in Mojave rattlesnake type A venom. The purpose of this investigation was to assess if the anticoagulant activity of elapid venoms containing PLA2 and/or three finger toxins could be inhibited by carbon monoxide., Methods: Venoms collected from Bungarus multicinctus, Micrurus fulvius, and five Naja species were exposed to carbon monoxide via carbon monoxide releasing molecule-2 prior to placement into human plasma. Coagulation kinetics were assessed via thrombelastography., Results: Compared with plasma without venom addition, all venoms had significant anticoagulant effects, with a 160-fold range of concentrations having similar anticoagulant effects in a species-specific manner. Carbon monoxide significantly inhibited the anticoagulant effect of all venoms tested, but inhibition was not complete in all cases., Conclusion: Given that individual neurotoxin activity often depends on intact activity that includes anticoagulant action, it may be possible that carbon monoxide inhibits neurotoxicity. Future investigation is justified to assess such carbon monoxide mediated inhibition with purified neurotoxins in vitro and in vivo.

Snake venom enzymes of the L-amino acid oxidase (LAAO) class are responsible for tissue hemorrhage, edema, and derangement of platelet function. However, what role, if any, these flavoenzymes play in altering plasmatic coagulation have not been well defined. Using coagulation kinetomic analyses (thrombelastograph-based), it was determined that the LAAO derived from Crotalus adamanteus venom displayed a procoagulant activity associated with weak clot strength (no factor XIII activation) similar to thrombin-like enzymes. The procoagulant activity was not modified in the presence of reduced glutathione, demonstrating that the procoagulant activity was likely due to deamination, and not hydrogen peroxide generation by the LAAO. Further, unlike the raw venom of the same species, the purified LAAO was not inhibited by carbon monoxide releasing molecule-2 (CORM-2). Lastly, exposure of the enzyme to phenylmethylsulfonyl fluoride (PMSF) resulted in the LAAO expressing anticoagulant activity, preventing contact activation generated thrombin from forming a clot. In sum, this investigation for the first time characterized the LAAO of a snake venom as both a fibrinogen polymerizing and an anticoagulant enzyme acting via oxidative deamination and not proteolysis as is the case with thrombin-like enzymes (e.g., serine proteases). Using this thrombelastographic approach, future investigation of purified enzymes can define their biochemical nature.

Lizards in the genus Heloderma are the most ancient venomous reptiles, with a traceable lineage nearly 100 million years old. The proteome of the venom of three of the remaining species (Heloderma suspectum, H. exasperatum, H. horridum) are very conserved, with kallikrein-like activity present to cause critical hypotension to immobilize and outright kill prey. Kallikrein-like activity would be expected to activate the contact protein pathway of coagulation, which would be detectable with thrombelastography in human plasma. Thus, it was proposed to determine if kallikrein-like activity could be detected with thrombelastography, and if this activity could be inhibited by carbon monoxide (CO) via a putative heme-based mechanism. Procoagulant activity of each venom was assessed via thrombelastography with normal plasma, and kallikrein-like activity confirmed with FX-depleted plasma. Venom was then exposed to carbon monoxide releasing molecule-2 (CORM-2) or its inactive releasing molecule to assess CO inhibition. All three venoms demonstrated kallikrein-like activity with the same potency and inhibition of activity by CO. In conclusion, the present work documented that procoagulant, kallikrein-like activity containing venoms of the oldest species of venomous reptiles was inhibited by CO, potentially via heme modulation. This is also the first identification and characterization of a kallikrein-like enzyme utilizing coagulation factor-depleted plasma to assess venom that inflicts hypotension. Future investigations will continue to define the vulnerability of venom enzymatic activities to CO.

Snakebite with hemotoxic venom continues to be a major source of morbidity and mortality worldwide. Our laboratory has characterized the coagulopathy that occurs in vitro in human plasma via specialized thrombelastographic methods to determine if venoms are predominantly anticoagulant or procoagulant in nature. Further, the exposure of venoms to carbon monoxide (CO) or O -phenylhydroxylamine (PHA) modulate putative heme groups attached to key enzymes has also provided mechanistic insight into the multiple different activities contained in one venom. The present investigation used these techniques to characterize fourteen different venoms obtained from snakes from North, Central, and South America. Further, we review and present previous thrombelastographic-based analyses of eighteen other species from the Americas. Venoms were found to be anticoagulant and procoagulant (thrombin-like activity, thrombin-generating activity). All prospectively assessed venom activities were determined to be heme-modulated except two, wherein both CO and its carrier molecule were found to inhibit activity, while PHA did not affect activity ( Bothriechis schlegelii and Crotalus organus abyssus ). When divided by continent, North and Central America contained venoms with mostly anticoagulant activities, several thrombin-like activities, with only two thrombin-generating activity containing venoms. In contrast, most venoms with thrombin-generating activity were located in South America, derived from Bothrops species. In conclusion, the kinetomic profiles of venoms obtained from thirty-two Pan-American Pit Viper species are presented. It is anticipated that this approach will be utilized to identify clinically relevant hemotoxic venom enzymatic activity and assess the efficacy of locally delivered CO or systemically administered antivenoms., Competing Interests: Dr. Vance G. Nielsen and Dr. Sam Afshar declare that they have no conflicts of interest. Mr. Nathaniel Frank has no conflict of interest except that he is the owner of Mtoxins.

Venomous snake bite and subsequent coagulopathy is a significant source of morbidity and mortality worldwide. The gold standard to treat coagulopathy caused by these venoms is the administration of antivenom; however, despite this therapy, coagulopathy still occurs and recurs. Of interest, our laboratory has demonstrated in vitro and in vivo that coagulopathy-inducing venom exposed to carbon monoxide (CO) is inhibited, potentially by an attached heme. The present investigation sought to determine if venoms derived from snakes of the African genera Atheris, Atractaspis, Causus, Cerastes, Echis, and Macrovipera that have no or limited antivenoms available could be inhibited with CO or with the metheme-inducing agent, O-phenylhydroxylamine (PHA). Assessing changes in coagulation kinetics of human plasma with thrombelastography, venoms were exposed in isolation to CO or PHA. Eight species were found to have procoagulant activity consistent with the generation of human thrombin, while one was likely fibrinogenolytic. All venoms were significantly inhibited by CO/PHA with species-specific variation noted. These data demonstrate indirectly that the heme is likely bound to these disparate venoms as an intermediary modulatory molecule. In conclusion, future investigation is warranted to determine if heme could serve as a potential therapeutic target to be modulated during treatment of envenomation by hemotoxic enzymes.

Snake venom contains a myriad of classes of enzyme which have been investigated for medicinal and toxinological purposes, including phospholipase A 2 (PLA 2 ), which is responsible for anticoagulant, myotoxic and neurotoxic effects. Given the importance of PLA 2 , the purposes of the present investigation were to characterize the coagulation kinetic behavior of a PLA 2 purified from Crotalus adamanteus venom (Ca-PLA 2 ) in human plasma with thrombelastography and determine if carbon monoxide could inhibit its activity. Coagulation kinetics were determined in human plasma with a range of Ca-PLA 2 activity (0-2 U/ml) via thrombelastography. Then, using carbon monoxide releasing molecule-2 or its inactivated molecule (0 or 100 µM), the vulnerability of Ca-PLA 2 activity to carbon monoxide mediated inhibition was assessed. Lastly, the inhibitory response of Ca-PLA 2 activity to exposure to carbon monoxide releasing molecule-2 (0-100 µM) was determined. Ca-PLA 2 activity degraded the velocity of clot growth and clot strength in an activity dependent, exponential manner. Carbon monoxide inhibited Ca-PLA 2 activity in a concentration dependent fashion, with loss of detectable activity at 100 µM of carbon monoxide releasing molecule-2. These findings, while preliminary, open the possibility that other PLA 2 contained in snake venom with multiple toxicities (e.g., myotoxin, neurotoxin) may be heme bearing and CO-inhibitable, which have profound potential basic and clinical science implications.

Envenomation by vipers with hemotoxic enzymes continues to be a worldwide source of morbidity and mortality. The present work examined the effects of exposure of venom enzymes to carbon monoxide and O-phenylhydroxylamine, agents that modulate the biometal heme, by forming carboxyheme and metheme, respectively. Four venoms obtained from medically important, diverse snake venom found in Africa, Asia and Australia were analyzed. The species that had venom tested in human plasma with thrombelastography and heme modulating agents were Deinagkistrodon acutus, Protobothrops mucrosquamatus, Dispholidus typus and Pseudonaja textilis. These venoms varied four hundred-fold in potency (ng-µg/ml) to exert procoagulant effects on human plasma; further, there was species specific variability in venom inhibition after exposure to carboxyheme or metheme agents. Lastly, using a wide range of carbon monoxide concentrations, it was determined that the factor V component of P. textilis venom was likely inhibited before the factor X component. Further investigation using this thrombelastograph-based, venom "kinetomic" methodology involving heme modulation will demonstrate in time its laboratory and clinical utility.

Aminocaproic acid (EACA) availability has recently been decreased whereas tranexamic acid (TXA) is still available as an antifibrinolytic agent to decrease blood loss associated with procedures involving cardiopulmonary bypass (CPB) by inhibiting plasmin mediated platelet activation. Given that the clinical inclination is to substitute TXA for EACA, we sought to compare the antifibrinolytic efficacy of the two agents using the clinically accepted molar ratio of EACA:TXA (7.9:1) that prevents platelet activation in a viscoelastic based system under a variety of conditions in human plasma; 25-50% therapeutic concentration (EACA 32.5-65 µg/ml, TXA 5-10 µg/ml) in the presence of 1500-3000 IU tissue-type plasminogen activator, with 0-50% dilution of plasma with buffer. In all equipotent concentrations, TXA provided superior antifibrinolytic action compared to EACA. It is hoped that this work will serve as a rationale to further investigate these and other similar agents, especially now in a time of unpredictable unavailability of key medications needed to optimize patient care. It is also our wish that these data assist perfusionists, anesthesiologists and cardiothoracic surgeons with their consideration of using an antifibrinolytic agent when managing complex patients with hypercoagulable states (e.g., ventricular assist device explant, infective endocarditis) undergoing CPB.

Introduction: Copper poisoning is associated with severe multiorgan injury and potentially death if chelation therapy is not administered. Of interest, while important gastrointestinal and urinary tract hemorrhage is associated with copper poisoning, very little is known concerning the nature of copper induced coagulopathy., Methods: Using thrombelastography, we assessed changes in coagulation kinetics in human plasma following exposure to copper concentrations encountered during poisoning., Results: While time to commence coagulation was not compromised, both velocity of thrombus growth and final strength were diminished. This result was duplicated with one concentration of copper in factor XIII deficient plasma. This pattern of coagulation kinetic response was interpreted as copper mediated fibrinogen dysfunction, perhaps via oxidation of key fibrinogen disulfide bridges. Lastly, experiments wherein glutathione was added implicated copper generated radical oxygen species as one of the mechanisms responsible for compromised coagulation kinetics., Conclusions: While chelation therapy is the key to survival following copper poisoning, perhaps this and future investigations of how copper affects coagulation may provide insight into effective supportive therapy for patients with active bleeding.

Geographic isolation and other factors result in evolution-driven diversity of the enzymatic composition of venom of pit vipers in the same genus. The present investigation sought to characterize venoms obtained from such genetically diverse Ovophis and Trimeresurus pit vipers utilizing thrombelastographic coagulation kinetic analyses. The coagulation kinetics of human plasma were assessed after exposure to venom obtained from two Ovophis and three Trimeresurus species. The potency of each venom was defined (µg/mL required to equivalently change coagulation); additionally, venoms were exposed to carbon monoxide (CO) or a metheme-inducing agent to modulate any enzyme-associated heme. All venoms had fibrinogenolytic activity, with four being CO-inhibitable. While Ovophis venoms had similar potency, one demonstrated the presence of a thrombin-like activity, whereas the other demonstrated a thrombin-generating activity. There was a 10-fold difference in potency and 10-fold different vulnerability to CO inhibition between the Trimeresurus species. Metheme formation enhanced fibrinogenolytic-like activity in both Ovophis species venoms, whereas the three Trimeresurus species venoms had fibrinogenolytic-like activity enhanced, inhibited, or not changed. This novel "venom kinetomic" approach has potential to identify clinically relevant enzymatic activity and assess efficacy of antivenoms between genetically and geographically diverse species.

It has been recently demonstrated that the hemotoxic venom activity of several species of snakes can be inhibited by carbon monoxide (CO) or a metheme forming agent. These and other data suggest that the biometal, heme, may be attached to venom enzymes and may be modulated by CO. A novel fibrinogenolytic metalloproteinase, named CatroxMP-II, was isolated and purified from the venom of a Crotalus atrox viper, and subjected to proteolysis and mass spectroscopy. An ion similar to the predicted singly charged m/z of heme at 617.18 was identified. Lastly, CORM-2 (tricarbonyldichlororuthenium (II) dimer, a CO releasing molecule) inhibited the fibrinogenolytic effects of CatroxMP-II on coagulation kinetics in human plasma. In conclusion, we present the first example of a snake venom metalloproteinase that is heme-bound and CO-inhibited.

We present a case of a patient administered parasternal transdermal lidocaine patch therapy as part of a multimodal analgesic regime designed to diminish opioid-associated delirium after coronary bypass surgery. The patient presented with delirium and severe methemoglobinemia (41%) that responded to discontinuation of lidocaine therapy, oxygen administration, and methylene blue administration. The clinical contributors and medicolegal implications of this degree of lidocaine-associated methemoglobin-mediated delirium are presented in the hope of avoiding similar complications in the postoperative setting after coronary bypass surgery.

Envenomation by hemotoxic enzymes continues to be a major cause of morbidity and mortality throughout the world. With regard to treatment, the gold standard to abrogate coagulopathy caused by these venoms is still the administration of antivenom; however, despite antivenom therapy, coagulopathy still occurs and recurs. Of interest, this laboratory has demonstrated in vitro and in vivo that coagulopathy inducing venom derived from snakes of the family Viperidae exposed to carbon monoxide (CO) is inhibited, potentially by an attached heme. The present investigation sought to determine if venoms derived from snakes of the Elapidae family (taipans and cobras) could also be inhibited with CO or with the metheme inducing agent, O-phenylhydroxylamine (PHA). Assessing changes in coagulation kinetics of human plasma with thrombelastography, venoms from Elapidae snakes were exposed in isolation to CO (five species) or PHA (one specie) and placed in human plasma to assess changes in procoagulant or anticoagulant activity. The procoagulant activity of two taipan venoms and anticoagulant activity of three cobra venoms were significantly inhibited by CO. The venom of the inland taipan was also inhibited by PHA. In sum, these data demonstrate indirectly that the biometal heme is likely bound to these disparate venoms as an intermediary modulatory molecule. In conclusion, CO may not just be a potential therapeutic agent to treat envenomation but also may be a potential modulator of heme as a protective mechanism for venomous snakes against injury from their own proteolytic venoms.

Envenomation by haemotoxic enzymes remains a significant source of human morbidity and mortality worldwide, with administration of long-acting or multiple doses of antivenom first-line therapy. However, coagulopathy can still occur and recur. Of interest, it has been recently demonstrated that direct, isolated exposure of snake venom enzymes with fibrinogenolytic activity to carbon monoxide (CO) abrogates venom-mediated loss of coagulation in human plasma. These observations of CO inhibition of venom fibrinogenolytic activity were subsequently repeated in rabbit whole blood. This study sought to translate these findings in an in vivo rabbit model of envenomation with fibrinogenolytic Crotalus atrox venom. Sedated rabbits were intravenously administered C. atrox venom (400 μg/kg) pre-exposed to 0 or 1000 μM carbon monoxide-releasing molecule-2 (CORM-2) in vitro. Arterial whole-blood samples demonstrated that compared to pre-envenomation values, the CORM-2-naïve venom significantly prolonged the onset of coagulation, decreased the velocity of clot growth and decreased clot strength as determined by thromboelastography an hour after venom injection. In contrast, CORM-2 pre-exposure prevented or attenuated C. atrox venom effects on coagulation kinetics. Future studies to determine whether rabbits injected with such venom subcutaneously/intramuscularly can have consequent coagulopathy abrogated by injection of carbon monoxide-releasing molecules into the 'bite site' are justified., (© 2017 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).)

Several in vitro investigations have demonstrated that anticoagulant effects of fibrinogenolytic snake venom metalloproteinases have been abrogated in human plasma by modifying fibrinogen with iron (Fe) and carbon monoxide (CO) to prevent catalysis or by directly inhibiting these enzymes with CO. To translate these findings, we chose to assess the rabbit as a model of envenomation with Crotalus atrox venom. It was determined with thrombelastography that 15 times the concentration of venom noted to compromise coagulation in plasma in vitro was required to cause coagulopathy in vivo, likely secondary to venom binding to blood cells and being cleared from the circulation rapidly. Unlike human plasma, rabbit plasma pre-treated with Fe/CO was not protected from fibrinogenolysis by venom. Consequently, the administration of purified human fibrinogen (with or without Fe/CO) would be required before venom administration to rabbits. Of greater interest, venom exposed to CO had complete loss of fibrinogenolytic effect in rabbit plasma and partial loss of activity in whole blood, indicative of unbinding of CO from venom and binding to haemoglobin. Thus, venom exposed to CO could remain partially or completely inhibited in whole blood long enough for clearance from the circulation, allowing rabbits to be a useful model to test the efficacy of regional CO administration to the bite site. Future investigations are planned to test these novel approaches to attenuate venom-mediated coagulopathy in the rabbit., (© 2017 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).)

: We present a case of a 26-year-old patient with severe infective endocarditis complicated with cerebral septic emboli that required essentially complete replacement of his circulating antithrombin activity to achieve an activated coagulation time near 480 s. The need for this degree of antithrombin administration may have been secondary to ongoing systemic inflammation and consequent thrombin generation despite blood culture results demonstrating no bacteremia. In sum, ongoing loss of endogenous antithrombin activity secondary to inflammation and the need for more than 80% normal activity to conduct safe cardiopulmonary bypass may require extraordinary administration of exogenous antithrombin in similar settings.

Hemodilution-associated hypercoagulability has been the focus of several investigations because significant morbidity and mortality have been associated with perioperative thrombophilia. Because most investigations implicate imbalances in procoagulant/anticoagulant activity as the etiology of hemodilution-associated hypercoagulability, we determined the effects of dilution on coagulation kinetics and clot strength with thrombelastography (TEG(R)). Control plasma (+/-celite activation) and antithrombin (AT)-deficient (<10% activity) plasma were diluted 0%, 10%, 20%, and 30% with saline. TEG(R) variables measured included time to clot initiation (reaction time, R), speed of clot propagation (angle, alpha), and clot strength (amplitude, A; or shear elastic modulus, G). Dilution of control plasma (10%-30%) resulted in a significant (P < 0.05) 16% decrease in R values, no change in alpha values, and decrease in A and G values. AT-deficient plasma had significantly smaller R values compared with control, and dilution did not change R values in AT-deficient plasma. Celite activation eliminated dilution-associated changes in R values in control plasma but resulted in linear decreases (R(2) = 0.88-0.96, P < 0.0001) in alpha, A, and G in response to dilution. Thus, our data indirectly support the concept that decreases in AT activity cause dilution-mediated hypercoagulability in plasma. Finally, celite activation permits quantification of dilution with TEG.

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.

While the administration of antivenom to treat hemotoxic snake bite injury remains the gold standard of therapy, we have demonstrated that modifying human fibrinogen with iron and carbon monoxide renders it resistant to fibrinogenolytic snake venom enzymes. In order to translate these findings into a possible biometal-based therapy complementary to antivenom administration, a preclinical model that possesses fibrinogen that closely mimics the human molecule in response to iron and carbon monoxide needed to be identified. The goal of this investigation was to determine if a swine model could serve in this capacity by assessing the thrombelastographic response of porcine plasma to iron and carbon monoxide exposure, without or with further exposure to the fibrinogenolytic venom of the viper Crotalus atrox. Using plasma obtained from eight swine, it was determined that their plasma responded to iron and carbon monoxide in a manner similar to that of human plasma by displaying enhanced coagulation kinetics. However, in sharp contrast to the response seen with human plasma, only iron significantly protected porcine plasma coagulation kinetics from C. atrox venom degradation. Therefore the pig is an animal beyond humans that could derive benefit from the biometal-focused therapy of iron infusion to protect against venom mediated compromise of coagulation. Thus, future investigation to assess the effects of iron administration to attenuate the effects of fibrinogenolytic envenomation with a pig model is justified.

Hypofibrinogenemia is an important clinical consequence following envenomation by Lachesis muta muta, usually attenuated or prevented by administration of antivenom. The venom of L. m. muta contains both a metalloproteinase fibrinogenase and a serine protease thrombin-like enzyme, and exposure of fibrinogen to iron (Fe) and carbon monoxide (CO) has been demonstrated to decrease its catalysis by such enzymes. Using thrombelastographic analytical techniques, it was determined that this venom displayed weak procoagulant effects combined with fibrinogenolytic effects, and pretreatment of plasma with Fe and CO markedly attenuated venom-mediated effects. Additional experiments involving heparin exposure and varying calcium concentrations demonstrated that modification of fibrinogen with Fe and CO in human plasma rendered fibrinogen not recognizable to the fibrinogenolytic metalloproteinase but did not prevent polymerization by the thrombin-like serine protease. Lastly, when venom was exposed to CO in isolation and then placed in plasma, the fibrinogenase was inhibited but the thrombin-like enzyme was not inhibited. In sum, utilizing relatively facile modifications, we demonstrated with thrombelastography that Fe and/or CO addition can protect human plasmatic coagulation from fibrinogenase activity but not the effects of the thrombin-like activity of L. m. muta venom.

Estrone (E 1 ) and Estriol (E 3 ) are endogenous female hormones, present in increased concentrations during female specific physiological processes (menopause and pregnancy respectively) that are associated with increased venous thrombotic risk. These hormones are also used as hormone therapies that are also associated with increased thromboembolism risk. Viscoelastic analysis revealed no significant difference to clot formation after hormone addition, however morphological analysis showed that the addition of both E 1 and E 3 result in fibrin clots composed of thinner fibrin fibers arranged in dense matted networks. These changes to the fibrin network ultrastructure are indicative of a prothrombotic state but may also indicate hypofibrinolysis. We therefore conclude that the increased risk of venous thrombosis during pregnancy and menopause may originate from a combination of hypercoagulation and a possible hypofibrinolytic mechanism of these hormones. Therefore females with a hypercoagulable tendency that fall pregnant or enter menopause need to be monitored to prevent venous thrombotic events. The decision to use hormone therapies during and after menopause should not be taken lightly and the risk-reward scale should be closely examined to ensure it does not tip towards thrombosis and subsequent thrombotic events that ultimately could have been prevented., (© 2017 Wiley Periodicals, Inc.)

: Annually, thousands suffer venomous snake-bite from Crotalus simus and Bothrops asper vipers in central and South America. The goals of the present study were to generally characterize the thrombin-like effects of venom from these snakes in human plasma with viscoelastic methods. Human plasma was exposed to the venom of three different C. simus subspecies and venoms obtained from B. asper vipers located in three different locations in Mexico. To characterize the factor X-activating and thrombin-like activity of these venoms, plasma (normal or factor XIII deficient) was pretreated with a variety of additives (e.g., heparin) in the absence or presence of calcium prior to exposure to 2.0 μg/ml of each viper's venom. These profiles were compared with plasma without venom that had contact activation of coagulation. Coagulation kinetics were determined with thrombelastography. All venoms had thrombin-like activity, with C. s. simus creating a slow growing, weak clot that was likely mediated by metalloproteinases. In contrast, B. asper venoms had rapid onset of coagulation and a high velocity of thrombus growth. Further, B. asper venom activity was calcium-independent, activated prothrombin, activated factor XIII, and independently polymerized fibrinogen. The viscoelastic methods used were able to differentiate subspecies of C. simus and specimens of B. asper, and provide insight into the mechanisms by which the venoms acted on plasma. These methods may be useful in the profiling of similar venoms and perhaps can assist in the assessment of interventions designed to treat envenomation (e.g., antivenom).

While topical thrombin application can markedly improve surgical hemostasis, rapid absorption of thrombin can result in pulmonary embolism and death. We report a case of accidental interstitial infiltration of topical thrombin after hemorrhoidectomy that was treated with administration of human antithrombin and heparin anticoagulation. Except for a marked decrease in antithrombin activity from super normal to normal values, the patient exhibited no laboratory or clinical signs of pulmonary embolism, thrombin mediated consumptive loss of procoagulants, or regional thrombosis. The patient had an uncomplicated recovery without sign of thrombotic morbidity. While it is hoped that such a medical misadventure should not occur, our case may serve as a reference to guide anticoagulant therapy if such a clinical scenario arises.

We present a case of a patient undergoing aortic valve replacement being inadvertently administered 5000 U of bovine thrombin instead of heparin for anticoagulation for cardiopulmonary bypass. The labeling error was made within the operating room pharmacy. The key to survival of this patient was a rapid diagnosis, administration of antithrombin and heparin, and removal of cardiac and great vessel thrombi. It is recommended that point of care anesthesia providers `prepare heparin for cardiopulmonary bypass anticoagulation, as thrombin is not used in anesthetic practice and is not contained within anesthesia cabinet medication drawers.

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.

Since the introduction of antivenom administration over a century ago to treat venomous snake bite, it has been the most effective therapy for saving life and limb. However, this treatment is not always effective and not without potential life-threatening side effects. We tested a new paradigm to abrogate the plasmatic anticoagulant effects of fibrinogenolytic snake venom metalloproteinases (SVMP) by inhibiting these Zn +2 -dependent enzymes directly with carbon monoxide (CO) exposure. Assessment of the fibrinogenolytic effects of venoms collected from the Arizona black rattlesnake, Northern Pacific rattlesnake, Western cottonmouth, Eastern cottonmouth, Broad-banded copperhead and Southern copperhead on human plasmatic coagulation kinetics was performed with thrombelastography in vitro. Isolated exposure of all but one venom (Southern copperhead) to CO significantly decreased the ability of the venoms to compromise coagulation. These results demonstrated that direct inhibition of transition metal-containing venom enzymes by yet to be elucidated mechanisms (e.g. CO, binding to Zn +2 or displacing Zn +2 from the catalytic site, CO binding to histidine residues) can in many instances significantly decrease fibrinogenolytic activity. This new paradigm of CO-based inhibition of the anticoagulant effects of SVMP could potentially diminish haemostatic compromise in envenomed patients until antivenom can be administered., (© 2016 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).)

Annually, thousands suffer poisonous snake bite, often from defibrinogenating species. Iron and carbon monoxide (CO) improve coagulation kinetics by modulation of fibrinogen as demonstrated in various Agkistrodon species and Crotalus atrox. Thus, we sought to determine whether pretreatment of plasma with iron and CO could attenuate venom-mediated catalysis of fibrinogen obtained from four common Crotalus species with known fibrinogenase activity. Human plasma was pretreated with ferric chloride (0-10 μmol/l) and CO-releasing molecule-2 (0-100 μmol/l) prior to exposure to venom from a Northern Pacific rattlesnake, Arizona black rattlesnake, prairie rattlesnake, or red diamond rattlesnake. The concentration of venom used decreased coagulation function of one or more kinetic parameters by at least 50% of normal values. Coagulation kinetics were determined with thrombelastography.Three snake venoms significantly degraded plasmatic coagulation kinetics, prolonging the onset to clot formation, diminishing velocity of clot growth and decreasing clot strength. However, red diamond rattlesnake venom exposure resulted in mixed coagulation kinetics, significantly decreasing the time to onset of coagulation without decreasing the velocity of clot growth. Iron and CO attenuated these coagulation kinetic changes in a species-specific manner. Further in vitro investigation of other fibrinogenolytic venoms is indicated to determine if iron and CO can attenuate venom compromised coagulation.

Background: The effect of endogenous hormone concentrations, specifically 17β-estradiol and progesterone, on fibrin network formation has not been established., Objectives: It is essential to understand natural hormone mechanisms since these hormones are still present in circulation while hormonal contraceptives, which are associated with increased risk of venous thromboembolism, are used., Methods: Due to the fact that these hormones are known to increase hypercoagulability and the prothrombotic state scanning electron microscopy (SEM), atomic force microscopy (AFM), thromboelastography (TEG) and turbidimetry were employed to investigate the morphology, surface roughness, viscoelastic properties and formation and lysis of fibrin., Results: 17β-estradiol and progesterone showed hypercoagulable viscoelastic properties and decreased the diameter and surface roughness of fibrin while increasing dense matted deposit occurrence. Our results suggest that the additional burden of hormonal load, together with the presence of endogenous estrogen and progesterone, may result in a prothrombotic and hypercoagulable state in females with an inflammatory predisposition., Conclusion: Our results are of clinical importance when considering hormones as either pathological agent or therapeutic intervention as will be assessed in future investigation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Since the introduction of antivenom administration 120 years ago to treat venomous snake bit, it has been the gold standard for saving life and limb. However, this therapeutic approach is not always effective and not without potential life-threatening side effects. We tested a new paradigm to abrogate the plasmatic anticoagulant effects of fibrinogenolytic snake venom metalloproteinases by modification of fibrinogen with iron and carbon monoxide and by inhibiting these Zn(2+) dependent metalloproteinases directly with carbon monoxide exposure. Assessment of the fibrinogenolytic effects of venoms collected from Puff adder, Gaboon viper and Indian cobra snakes on plasmatic coagulation kinetics was performed with thrombelastography. Pretreatment of plasma with iron and carbon monoxide exposure markedly attenuated the effects of all three venoms, and direct pretreatment of each venom with carbon monoxide also significantly decreased the ability to compromise coagulation. These results demonstrated that the introduction of a transition metal (e.g., modulation of the α-chain of fibrinogen with iron), modulation of transition metal in heme (e.g., carbon monoxide modulation of fibrinogen-bound heme iron), and direct inhibition of transition metal containing venom enzymes (e.g., CO binding to Zn(2+) or displacing Zn(2+) from the catalytic site) significantly decreased fibrinogenolytic activity. This biometal modulation strategy to attenuate the anticoagulant effects of snake venom metalloproteinases could potentially diminish hemostatic injury in envenomed patients until antivenom can be administered.