Snake Venom Proteomics, Immunoreactivity and Toxicity Neutralization Studies for the Asiatic Mountain Pit Vipers, Ovophis convictus, Ovophis tonkinensis, and Hime Habu, Ovophis okinavensis.

Snakebite envenomation is a serious neglected tropical disease, and its management is often complicated by the diversity of snake venoms. In Asia, pit vipers of the Ovophis species complex are medically important venomous snakes whose venom properties have not been investigated in depth. This study characterized the venom proteomes of Ovophis convictus (West Malaysia), Ovophis tonkinensis (northern Vietnam, southern China), and Ovophis okinavensis (Okinawa, Japan) by applying liquid chromatography-tandem mass spectrometry, which detected a high abundance of snake venom serine proteases (SVSP, constituting 40-60% of total venom proteins), followed by phospholipases A2, snake venom metalloproteinases of mainly P-III class, L-amino acid oxidases, and toxins from other protein families which were less abundant. The venoms exhibited different procoagulant activities in human plasma, with potency decreasing from O. tonkinensis > O. okinavensis > O. convictus. The procoagulant nature of venom confirms that consumptive coagulopathy underlies the pathophysiology of Ovophis pit viper envenomation. The hetero-specific antivenoms Gloydius brevicaudus monovalent antivenom (GbMAV) and Trimeresurus albolabris monovalent antivenom (TaMAV) were immunoreactive toward the venoms, and cross-neutralized their procoagulant activities, albeit at variably limited efficacy. In the absence of species-specific antivenom, these hetero-specific antivenoms may be useful in treating coagulotoxic envenomation caused by the different snakes in their respective regions.

Monitoring Compound-Related Effects on Coagulability in Rats and Cynomolgus and Rhesus Monkeys by Thrombin Generation Kinetic Measurement.

Thrombin generation assay (TGA) is a sensitive method for the assessment of the global clotting potential of plasma. This kinetic assay can detect both hypocoagulable and hypercoagulable conditions: delayed or reduced thrombin generation leading to a prolonged clotting time, or induced thrombin activity, shifting the coagulation cascade toward thrombosis. The purpose of this study is to qualify the TGA in nonhuman primates (NHP) and rats for its use during nonclinical in vivo and in vitro studies. Blood was drawn from nonanesthetized animals, and platelet-poor plasma was obtained after double centrifugation; coefficients of variation were <10% for all derived parameters of thrombin generation assessed with 5 pM of tissue factor. Thrombin generation was evaluated in vitro in rat and NHP plasmas with ascending doses of unfractionated heparin (UFH), recombinant tissue factor, and anticoagulant compounds. Thrombin generation was decreased with UFH and anticoagulant compounds, but was increased in the presence of tissue factor, in a dose-dependent manner. In a rat model of inflammation, animals were administered a low dose of lipopolysaccharides. Thrombin generation measurements were decreased 3 hours post-LPS administration with a nadir at 24 hours, while thrombin-antithrombin complexes reached a peak at 8 hours, supporting an earlier production of thrombin. In conclusion, these data demonstrated that TGA can be performed in vitro for screening of compounds expected to have effects on coagulation cascade, and thrombin generation can be measured at interim time points during nonclinical in vivo studies in rats and NHP.

Venomous Landmines: Clinical Implications of Extreme Coagulotoxic Diversification and Differential Neutralization by Antivenom of Venoms within the Viperid Snake Genus Bitis.

The genus Bitis comprises 17 snake species that inhabit Africa and the Arabian Peninsula. They are responsible for a significant proportion of snakebites in the region. The venoms of the two independent lineages of giant Bitis (B. arietans and again in the common ancestor of the clade consisting of B. gabonica, B. nasicornis, B. parviocula and B. rhinoceros) induce an array of debilitating effects including anticoagulation, hemorrhagic shock and cytotoxicity, whilst the dwarf species B. atropos is known to have strong neurotoxic effects. However, the venom effects of the other species within the genus have not been explored in detail. A series of coagulation assays were implemented to assess the coagulotoxic venom effects of fourteen species within the genus. This study identified procoagulant venom as the ancestral condition, retained only by the basal dwarf species B. worthingtoni, suggesting anticoagulant venom is a derived trait within the Bitis genus and has been secondarily amplified on at least four occasions. A wide range of anticoagulant mechanisms were identified, such as coagulant and destructive activities upon fibrinogen in both giant and dwarf Bitis and the action of inhibiting the prothrombinase complex, which is present in a clade of dwarf Bitis. Antivenom studies revealed that while the procoagulant effects of B. worthingtoni were poorly neutralized, and thus a cause for concern, the differential mechanisms of anticoagulation in other species were all well neutralized. Thus, this study concludes there is a wide range of coagulotoxic mechanisms which have evolved within the Bitis genus and that clinical management strategies are limited for the procoagulant effects of B. worthingtoni, but that anticoagulant effects of other species are readily treated by the South African polyvalent antivenom. These results therefore have direct, real-work implications for the treatment of envenomed patients.

Proteomic and functional variation within black snake venoms (Elapidae: Pseudechis).

Pseudechis (black snakes) is an Australasian elapid snake genus that inhabits much of mainland Australia, with two representatives confined to Papua New Guinea. The present study is the first to analyse the venom of all 9 described Pseudechis species (plus one undescribed species) to investigate the evolution of venom composition and functional activity. Proteomic results demonstrated that the typical Pseudechis venom profile is dominated by phospholipase A2 toxins. Strong cytotoxicity was the dominant function for most species. P. porphyriacus, the most basal member of the genus, also exhibited the most divergent venom composition, being the only species with appreciable amounts of procoagulant toxins. The relatively high presence of factor Xa recovered in P. porphyriacus venom may be related to a predominantly amphibian diet. Results of this study provide important insights to guide future ecological and toxinological investigations.

Biological and Enzymatic Characterization of Proteases from Crude Venom of the Ant Odontomachus bauri.

Hymenoptera venoms constitute an interesting source of natural toxins that may lead to the development of novel therapeutic agents. The present study investigated the enzymatic and biological characteristics of the crude venom of the ant Odontomachus bauri. Its crude venom presents several protein bands, with higher staining for six proteins with gelatinolytic activity (17, 20, 26, 29, 43 and 48 kDa). The crude venom showed high proteolytic activity on azocasein at optimal pH 8.0 and 37 °C. In the presence of protease inhibitors as aprotinin, leupeptin and EDTA, the azocaseinolytic activity was reduced by 45%, 29% and 9%, respectively, suggesting that the enzymes present in the crude venom belong to the three classes of proteases, with the serine proteases in greater intensity. The crude venom degraded the fibrinogen α-chain faster than the ß-chain, while the fibrinogen γ-chain remained unchanged. In biological assays, O. bauri venom showed hemolytic and coagulant activity in vitro, and defibrinating activity in vivo. In addition, the venom showed antimicrobial activity against Staphylococcus aureus and Escherichia coli as well as antiparasitic activity on Toxoplasma gondii infection in vitro. In that sense, this study sheds perspectives for pharmacological applications of O. bauri venom enzymes.

Label-Free (XIC) Quantification of Venom Procoagulant and Neurotoxin Expression in Related Australian Elapid Snakes Gives Insight into Venom Toxicity Evolution.

This study demonstrates a direct role of venom protein expression alteration in the evolution of snake venom toxicity. Avian skeletal muscle contractile response to exogenously administered acetylcholine is completely inhibited upon exposure to South Australian and largely preserved following exposure to Queensland eastern brown snake Pseudonaja textilis venom, indicating potent postsynaptic neurotoxicity of the former and lack thereof of the latter venom. Label-free quantitative proteomics reveals extremely large differences in the expression of postsynaptic three-finger α-neurotoxins in these venoms, explaining the difference in the muscle contractile response and suggesting that the type of toxicity induced by venom can be modified by altered expression of venom proteins. Furthermore, the onset of neuromuscular paralysis in the rat phrenic nerve-diaphragm preparation occurs sooner upon exposure to the venom (10 µg/mL) with high expression of α-neurotoxins than the venoms containing predominately presynaptic ß-neurotoxins. The study also finds that the onset of rat plasma coagulation is faster following exposure to the venoms with higher expression of venom prothrombin activator subunits. This is the first quantitative proteomic study that uses extracted ion chromatogram peak areas (MS1 XIC) of distinct homologous tryptic peptides to directly show the differences in the expression of venom proteins.

Elucidation of procoagulant mechanism and pathophysiological significance of a new prothrombin activating metalloprotease purified from Daboia russelii russelii venom.

The procoagulant proteases present in Russell's Viper venom (RVV) are responsible for promoting consumption coagulopathy in victims. In this study, a procoagulant metalloprotease (Rusviprotease) possessing prothrombin activating and α-fibrinogenase properties has been purified and characterized from RVV. Rusviprotease is a 26.8 kDa glycoprotein which also exists in other multimeric forms. The peptide mass fingerprinting and secondary structure analyses of Rusviprotease revealed its similarity with snake venom prothrombin activators and metalloproteases. Similar to group A prothrombin activators, Rusviprotease cleaved prothrombin independent of any co-factor requirement generating meizothrombin which is further cleaved to form thrombin. The Km and Vmax values of Rusviprotease towards prothrombin were determined to be 1.73 µM, and 153.5 nM thrombin generated/min/µmoles of Rusviprotease, respectively. The Km and Vmax values of Rusviprotease towards fibrinogen were calculated to be 3.14 µM and 78.7 nmol/min, respectively. Spectrofluorometric study provided the evidence of interaction between Rusviprotease and factor Xa with a Kd value of 6.64 nM. This interaction augmented the prothrombin activating property of the factor Xa-prothrombinase-Rusviprotease complex by 2.5 fold. Intravenous injection of Rusviprotease to BALB/c mice (0.1 mg/kg) resulted in in vivo defibrinogenation rendering the blood incoagulable. In conclusion, Rusviprotease is the first example of a prothrombin activator with fibrinogenolytic property purified from Daboia russelii russelii venom.

Pulmonary and hemostatic toxicity of multi-walled carbon nanotubes and zinc oxide nanoparticles after pulmonary exposure in Bmal1 knockout mice.

BACKGROUND: Pulmonary exposure to nanoparticles (NPs) may affect, in addition to pulmonary toxicity, the cardiovascular system such as procoagulant effects, vascular dysfunction and progression of atherosclerosis. However, only few studies have investigated hemostatic effects after pulmonary exposure. METHODS: We used Bmal1 (brain and muscle ARNT-like protein-1) knockout (Bmal1(-/-)) mice which have a disturbed circadian rhythm and procoagulant phenotype, to study the pulmonary and hemostatic toxicity of multi-walled carbon nanotubes (MWCNTs) and zinc oxide (ZnO) NPs after subacute pulmonary exposure. Bmal1(-/-) and wild-type (Bmal1(+/+)) mice were exposed via oropharyngeal aspiration, once a week, during 5 consecutive weeks, to a cumulative dose of 32 or 128 µg MWCNTs or 32 or 64 µg ZnO NPs. RESULTS: MWCNTs caused a pronounced inflammatory response in the lung with increased cell counts in the broncho-alveolar lavage and increased secretion of interleukin-1ß and cytokine-induced neutrophil chemo-attractant (KC), oxidative stress (increased ratio of oxidized versus reduced glutathione and decreased total glutathione) as well as anemic and procoagulant effects as evidenced by a decreased prothrombin time with increased fibrinogen concentrations and coagulation factor (F)VII. In contrast, the ZnO NPs seemed to suppress the inflammatory (decreased neutrophils in Bmal1(-/-) mice) and oxidative response (increased total glutathione in Bmal1(-/-) mice), but were also procoagulant with a significant increase of FVIII. The procoagulant effects, as well as the significant correlations between the pulmonary endpoints (inflammation and oxidative stress) and hemostasis parameters were more pronounced in Bmal1(-/-) mice than in Bmal1(+/+) mice. CONCLUSIONS: The Bmal1(-/-) mouse is a sensitive animal model to study the procoagulant effects of engineered NPs. The MWCNTs and ZnO NPs showed different pulmonary toxicity but both NPs induced procoagulant effects, suggesting different mechanisms of affecting hemostasis. However, the correlation analysis suggests a causal association between the observed pulmonary and procoagulant effects.

An alternative micromethod to access the procoagulant activity of Bothrops jararaca venom and the efficacy of antivenom.

The assessment of the capacity of antivenoms to neutralize the lethal activity of snake venoms still relies largely on traditional rodent lethality assay (LD50). However, adequately validated in vitro tests should be introduced for assessing antivenom neutralizing capacity in plasma of immunized horses as well as for in-process quality control. The dynamic of fibrin formation in recalcified avian plasma samples is extremely slow, when compared to that presented by mammalian plasmas. In this study, we present one new coagulant assay, by performing dose-response curve after plotting the clotting time (CT) parameter of the ROTEM profile of recalcified chicken plasma samples (target) against semi-logarithmic doses of Bothrops jararaca venom (agonist), either in absence or in presence of the semi-logarithmic doses of anti-bothropic serum (ABS) (antagonist). The mean coagulant dose 50% (CD50) was defined as the quantity of venom (in µg) which reduces CT to 900 s, between minimum and maximum responses. The CT induced by 5CD50 of the venom was used as the control for calculating the effective dose (ED) of each batch of ABS. ED was defined as the ABS dose (nanoliters, nL) at which CT induced by one amount of venom corresponding to 5CD50 is displaced to the maximum threshold (1800 s). Five batches of the ABS, previously assayed for their lethality neutralizing activity (ED50) were assayed. The correlation coefficient (r) between both in vitro (ED) and in vivo (ED50) values was 0.87 (p value < 0.05). We propose this micro method as highly sensitive for characterization and quantification of possible procoagulant activity of small doses of snake venoms (nanograms) and for detecting small doses (nanoliters) of specific antibodies against this effect in little volume samples of biological fluids.

Systemic effects induced by the venom of the snake Bothrops caribbaeus in a murine model.

Snakebite envenoming by Bothrops caribbaeus, an endemic viperid from the Lesser Antillean island of Saint Lucia, is clinically characterized by local tissue damage and systemic thrombosis that can lead to cerebral, myocardial or pulmonary infarctions and venous thromboses. Systemic effects (lethality, pulmonary hemorrhage, thrombocytopenia and coagulopathy) induced by intravenous (i.v.) administration of B. caribbaeus venom were studied in mice. The role of snake venom metalloproteinases (SVMPs) in these systemic alterations was assessed by inhibition with the chelating agent calcium disodium ethylenediaminetetraacetic acid (CaNa(2)EDTA). A snake C-type lectin-like (snaclec) and a type P-III hemorrhagic SVMP were isolated and characterized from this venom, and the effect of venom and the isolated snaclec on human platelet aggregation was studied in vitro. Results indicate that SVMPs play an important role in the overall toxicity of B. caribbaeus venom, being responsible for systemic hemorrhage and lethality, but not thrombocytopenia, whereas the isolated snaclec is involved in the thrombocytopenic effect. Both venom and snaclec induce platelet aggregation/agglutination. Moreover, the snaclec binds directly to glycoprotein Ib (GPIb) and induces agglutination in washed fixed platelets. On the other hand, B. caribbaeus venom hydrolyzed fibrinogen in vitro and induced a partial drop of fibrinogen levels with an increase in fibrin/fibrinogen degradation products (FDP) levels in vivo. The negative result for D-dimer (DD) in plasma is consistent with the lack of microscopic evidence of pulmonary thrombosis and endothelial cell damage. Likewise, no increments in plasma sE-selectin levels were detected. The absence of thrombosis in this murine model suggests that this effect may be species-specific.

Biochemical and biological characterization of two serine proteinases from Colombian Crotalus durissus cumanensis snake venom.

Two clotting serine proteinases, named Cdc SI and Cdc SII, were isolated and characterized for the first time from Colombian Crotalus durissus cumanensis snake venom. The enzymes were purified using two chromatographic steps: molecular exclusion on Sephacryl S-200 and RP-HPLC on C8 Column. The molecular masses of the proteins, determined by MALDI-TOF mass spectrometry, were 28,561.4 and 28,799.2 Da for Cdc SI and Cdc SII, respectively. The aim of the present study was to evaluate enzymatic, coagulant and toxic properties of the two enzymes. The serine proteinases hydrolyzed specific chromogenic substrate (BaPNA) and exhibited a Michaelis-Menten behavior. Cdc SI had V(max) of 0.038 ± 0.003 nmol/min and K(M) of 0.034 ± 0.017 mM, while Cdc SII displayed values of V(max) of 0.267 ± 0.011 nmol/min and K(M) of 0.145 ± 0.023 mM. N-terminal sequences were VIGGDEXNIN and VIGGDICNINEHNFLVALYE for Cdc SI and Cdc SII, respectively. Molecular masses, N-terminal sequences, inhibition assays, and enzymatic profile suggest that Cdc SI and Cdc SII belong to the family of snake venom thrombin-like enzymes. These serine proteinases differed in their clotting activity on human plasma, showing a minimum coagulant dose of 25 µg and 0.571 µg for Cdc SI and Cdc SII, respectively. Enzymes also showed coagulant activity on bovine fibrinogen and degraded chain α of this protein. Toxins lack hemorrhagic and myotoxic activities, but are capable to induce defibrin(ogen)ation, moderate edema, and an increase in vascular permeability. These serine proteinases may contribute indirectly to the local hemorrhage induced by metalloproteinases, by causing blood clotting disturbances, and might also contribute to cardiovascular alterations characteristic of patients envenomed by C. d. cumanensis in Colombia.

Procoagulant and prothrombotic effects of the herbal medicine, Dipsacus asper and its active ingredient, dipsacus saponin C, on human platelets.

BACKGROUND: In spite of the growing popularity of herbal medicines and natural food supplements, their effects on cardiovascular homeostasis remain largely unknown, especially regarding pro-thrombotic risks. OBJECTIVE: In the present study, 21 herbal tea extracts were screened for the procoagulant activities on platelets, an important promoter of thrombosis to examine if herbal medicines or natural products may have prothrombotic risks. We discovered that Dipsacus asper (DA), known to have analgesic and anti-inflammatory effects, potently induced procoagulant activities in platelets. We tried to identify the active ingredient and elucidate the underlying mechanism. RESULTS: Among 10 major ingredients of DA, dipsacus saponin C (DSC) was identified as a key active ingredient in DA-induced procoagulant activities. DSC-induced procoagulant activities were achieved by the exposure of phosphatidylserine (PS) and PS-bearing microparticle generation that were caused by the alteration in the activities of phospholipid translocases: scramblase and flippase. These events were initiated by increased intracellular calcium and ATP depletion. Notably, DSC induced a series of apoptotic events including the disruption of mitochondrial membrane potential, translocation of Bax and Bak, cytochrome c release and caspase-3 activation. The key roles of apoptotic pathway and caspase activation were demonstrated by the reversal of DSC-induced PS exposure and procoagulant activities with the pretreatment of caspase inhibitors. Interestingly, EGTA reversed DSC-induced procoagulant activities and apoptotic events suggesting that an intracellular calcium increase may play a central role. These results were also confirmed in vivo where platelets of the rats exposed to DSC or DA exhibited PS exposure. Most importantly, DSC or DA administration led to increased thrombus formation. CONCLUSION: These results demonstrate that herbal medicines or natural products such as DA or DSC might have prothrombotic risks through procoagulant activation of platelets.

Validation of the calibrated thrombin generation test (cTGT) as the reference assay to evaluate the procoagulant activity of nanomaterials.

We validated a preclinical toxicological screening assay and provided guidelines to evaluate the potential impact of nanoparticles (NPs) on blood coagulation. Five NPs with various physicochemical properties were studied using several existing methods of clotting times and thrombin generation assays in human normal pool plasma. In both recalcification clotting time (RCT) and calibrated thrombin generation test (cTGT), the NPs exhibited procoagulant activity (SiO2 ≥ SiC ≥ TiC > CuO > CB) but cTGT was more sensitive and relevant than RCT. Thus, the cTGT appears as a reference assay to investigate the nanoparticle (NP) procoagulant activity in human plasma. It should be used as the reference toxicity test for evaluating the effects of nanomaterials on coagulation cascade. In addition, we also showed that the use of the Pluronic F-108 dispersant and/or the sonication for the NP suspension preparation may mask their procoagulant activity and thus should be avoided.

Vascular effects of ultrafine particles in persons with type 2 diabetes.

BACKGROUND: Diabetes confers an increased risk for cardiovascular effects of airborne particles. OBJECTIVE: We hypothesized that inhalation of elemental carbon ultrafine particles (UFP) would activate blood platelets and vascular endothelium in people with type 2 diabetes. METHODS: In a randomized, double-blind, crossover trial, 19 subjects with type 2 diabetes inhaled filtered air or 50 µg/m³ elemental carbon UFP (count median diameter, 32 nm) by mouthpiece for 2 hr at rest. We repeatedly measured markers of vascular activation, coagulation, and systemic inflammation before and after exposure. RESULTS: Compared with air, particle exposure increased platelet expression of CD40 ligand (CD40L) and the number of platelet-leukocyte conjugates 3.5 hr after exposure. Soluble CD40L decreased with UFP exposure. Plasma von Willebrand factor increased immediately after exposure. There were no effects of particles on plasma tissue factor, coagulation factors VII or IX, or D-dimer. CONCLUSIONS: Inhalation of elemental carbon UFP for 2-hr transiently activated platelets, and possibly the vascular endothelium, in people with type 2 diabetes.

FIX-Triple, a gain-of-function factor IX variant, improves haemostasis in mouse models without increased risk of thrombosis.

Engineered recombinant factor IX (FIX) with augmented clotting activity may prove useful for replacement therapy, but it has not been studied for risk of thrombosis. We used three mouse models to evaluate thrombosis risk associated with the FIX variant FIX-Triple, which has a 13-fold higher specific activity than wild-type FIX (FIX-WT). Protein infusion of FIX-Triple into haemophilia B mice was not thrombogenic, even at a dose of 13-fold higher than FIX-WT. Gene knock-in to generate mice that constitutively produce FIX-WT or FIX-Triple protein revealed that all mice expressed equal antigen levels. FIX-Triple knock-in mice that exhibited 10-fold higher FIX clotting activity did not show hypercoagulation. Adeno-associated viral (AAV) delivery of the FIX gene into mice was used to mimic gene therapy. Haemophilia B and inbred C57Bl/6 mice injected with different doses of virus particles carrying FIX-WT or FIX-Triple and expressing up to a nearly 13-fold excess (1289% of normal) of FIX clotting activity did not show increased risk of thrombosis compared with untreated wild-type mice in a normal haemostatic state. When challenged with ferric chloride (FeCl3), the mesenteric venules of AAV-treated C57Bl/6 mice that gave a nearly five-fold excess (474%) of FIX clotting activity were not thrombotic; however, thrombosis became obvious in FeCl3-challenged mice expressing extremely high FIX clotting activities (976-1289%) achieved by AAV delivery of FIX-Triple. These studies suggest that FIX-Triple is not thrombogenic at therapeutic levels and is a potential therapeutic substitute for FIX-WT.

Endogenous thrombin potential as a novel method for the characterization of procoagulant snake venoms and the efficacy of antivenom.

Venom-induced consumption coagulopathy occurs in snake envenoming worldwide but the interaction between procoagulant snake venoms and human coagulation remains poorly understood. We aimed to evaluate an assay using endogenous thrombin potential (ETP) to investigate the procoagulant properties of a range of Australian whole venoms in human plasma and compared this to traditional clotting and prothrombinase activity studies. We developed a novel modification of ETP using procoagulant snake venoms to trigger thrombin production. This was used to characterise the relative potency, calcium and clotting factor requirements of five important Australian snake venoms and efficacy of commercial antivenom, and compared this to prothrombinase activity and clotting assays. All five venoms initiated thrombin generation in the absence and presence of calcium. Pseudonaja textilis (Brown snake; p<0.0001), Hoplocephalus stephensii (Stephen's-banded snake; p<0.0001) and Notechis scutatus (tiger snake; p=0.0073) all had statistically significant increases in ETP with calcium. Venom potency varied between assays, with ETP ranging from least potent with Oxyuranus scutellatus (Taipan) venom to intermediate with N. scutatus and H. stephensii venoms to most potent with P. textilis and Tropidechis carinatus (Rough-scale snake) venoms. ETPs for N. scutatus, T. carinatus and H. stephensii venoms were severely reduced with factor V deficient plasma. Antivenom neutralized the thrombin generating capacity but not prothrombin substrate cleaving ability of the venoms. Contrary to previous studies using clotting tests and factor Xa substrates, these venoms differ in calcium requirement. ETP is a useful assay to investigate mechanisms of other procoagulant venoms and is a robust method of assessing antivenom efficacy.

High-mobility group box 1 protein promotes development of microvascular thrombosis in rats.

BACKGROUND: Sepsis is a life-threatening disorder resulting from systemic inflammatory and coagulatory responses to infection. High-mobility group box 1 protein (HMGB1), an abundant intranuclear protein, was recently identified as a potent lethal mediator of sepsis. However, the precise mechanisms by which HMGB1 exerts its lethal effects in sepsis have yet to be confirmed. We recently reported that plasma HMGB1 levels correlated with disseminated intravascular coagulation (DIC) score, indicating that HMGB1 might play an important role in the pathogenesis of DIC. OBJECTIVES: To investigate the mechanisms responsible for the lethal effects of HMGB1, and more specifically, to explore the effects of HMGB1 on the coagulation system. METHODS: Rats were exposed to thrombin with or without HMGB1, and a survival analysis, pathologic analyses and blood tests were conducted. The effects of HMGB1 on the coagulation cascade, anticoagulant pathways and surface expression of procoagulant or anticoagulant molecules were examined in vitro. RESULTS: Compared to thrombin alone, combined administration of thrombin and HMGB1 resulted in excessive fibrin deposition in glomeruli, prolonged plasma clotting times, and increased mortality. In vitro, HMGB1 did not affect clotting times, but inhibited the anticoagulant protein C pathway mediated by the thrombin-thrombomodulin complex, and stimulated tissue factor expression on monocytes. CONCLUSIONS: These findings demonstrate the procoagulant role of HMGB1 in vivo and in vitro. During sepsis, massive accumulation of HMGB1 in the systemic circulation would promote the development of DIC.

Coagulant toxicity and effectiveness in a slaughterhouse wastewater treatment plant.

Attempts were made in this study to examine the toxicity of polymer/alum addition to the aeration tank effluent prior to sludge flotation as practiced in a slaughterhouse wastewater treatment plant. Based on the Microtox toxicity assay, alum at concentrations 100-200 mg/L was found to slightly increase the toxicity level of slaughterhouse wastewater effluent. However, at higher concentrations (300-1000 mg/L), significant residual chronic toxicity remained in all slaughterhouse wastewater effluents, independent of the treatment process. Polymer, on the other hand, removed organics and solids, but polymer effluents are more toxic than alum at extremely low concentration. Results indicated that alum and polymer caused inhibitory effects to the system at soluble concentrations of approximately 400 and 60 mg/L and above, respectively. The data also indicated that the solids collected in both tests (polymer/alum) were much more toxic than those from the effluents. Sediment samples from the polymer tests were the most toxic. Furthermore, the effluent toxicity of the coagulants was dramatically more significant when used after the settlement of solids than when used in mixed liquor. In addition, strong correlations were observed between the observed toxicity for a series of supernatants and the coagulant concentrations of alum/polymer processes, and between supernatants and solids collected in both tests.

Trocarin, a blood coagulation factor Xa homologue from snake venom, causes inflammation and mitogenesis.

Trocarin, a Group D prothrombin activator from Tropidechis carinatus snake venom, has high sequence similarity to blood coagulation factor Xa (FXa). Both trocarin and FXa activate prothrombin to mature thrombin and have similar requirements for cofactors, such as factor Va, Ca2+ ions and phospholipids. In addition to its hemostatic functions, human FXa causes inflammation and induces mitogenesis in several cell types due to its interaction with effector protease receptor-1 (EPR-1). The inter-EGF domain region (L83FTKRL88) of FXa implicated in EPR-1-binding is distinctly different in trocarin (K83VLYQS88). Here we show that, interestingly, trocarin also causes edema in the mouse footpad; the inflammation, accompanied by a large purplish clot, is more persistent than the transient edema caused by FXa. Histological examination indicates significant differences between edema induced by FXa and trocarin. Moreover, trocarin-induced edema is not inhibited by a synthetic peptide based on the FXa-binding region of EPR-1, indicating that the inflammation is probably mediated by a mechanism independent of EPR-1-binding. Trocarin, like FXa, also has a mitogenic effect on bronchial smooth muscle cells mediated by an EPR-1-independent mechanism. Hence trocarin, being closely related to FXa, has similar non-hemostatic functions in mediating inflammation and mitogenesis, yet appears to act by distinctly different mechanisms.

Coagulant component in Cerastes cerastes (Egyptian sand viper) venom.

A coagulant component has been purified from Cerastes cerastes venom, using gel filtration on a Sephadex G 100 (fine) column followed by chromatography on a Bio-Rex 70 column. This compound had a proteolytic effect and could coagulate human plasma deficient in factor VIII or (VIII + IX) with the formation of a firm clot. It could also clot plasma deficient in factor X, but the clot formed was soft and not complete. The compound had no effect on platelet aggregation and was nontoxic. This compound is believed to be primarily a factor X activator, as it could replace factor VIII in hemophilic plasmas.