RESUMEN

Vipegrin is a 6.8 kDa Kunitz-type serine proteinase inhibitor purified from Russell's viper (Vipera russelii russelii) venom. Kunitz-type serine proteinase inhibitors are non-enzymatic proteins and are ubiquitous constituents of viper venoms. Vipegrin could significantly inhibit the catalytic activity of trypsin. It also posseses disintegrin-like properties and could inhibit collagen and ADP-induced platelet aggregation in a dose-dependent manner. Vipegrin is cytotoxic to MCF7 human breast cancer cells and restricts its invasive property. Confocal microscopic analysis revealed that Vipegrin could induce apoptosis in MCF7 cells. Vipegrin disrupts cell to cell adhesion of MCF7 cells through its disintegrin-like activity. It also causes disruption of attachment of MCF7 cells to synthetic (poly L-lysine) and natural (fibronectin, laminin) matrices. Vipegrin did not cause cytotoxicity on non-cancerous HaCaT, human keratinocytes. The observed properties indicate that Vipegrin may help the development of a potent anti-cancer drug in future.

Asunto(s)

Daboia , Inhibidores de Serina Proteinasa , Animales , Humanos , Inhibidores de Serina Proteinasa/farmacología , Venenos de Víboras , Desintegrinas , Agregación Plaquetaria , Daboia/metabolismo

RESUMEN

Longitudinal metabolomics and lipidomics analyses were carried out on the blood plasma of mice injected intramuscularly with venoms of the viperid species Bothrops asper or Daboia russelii. Blood samples were collected 1, 3, 6, and 24 h after venom injection, and a control group of non-envenomed mice was included. Significant perturbations in metabolomics and lipidomics were observed at 1, 3, and 6 h, while values returned close to those of control mice by 24 h, hence reflecting a transient pattern of metabolic disturbance. Both venoms induced significant changes in amino acids, as well as in several purines and pyrimidines, and in some metabolites of the tricarboxylic acid cycle. KEGG analysis of metabolic pathways that showed those with the greatest change included aminoacyl tRNA synthesis and amino acid biosynthesis and metabolism pathways. With regard to lipid metabolism, there was an increase in triglycerides and some acyl carnitines and a concomitant drop in the levels of some phospholipids. In addition, envenomed mice had higher levels of cortisol, heme, and some oxidative stress markers. The overall pattern of metabolic changes in envenomed mice bears similarities with the patterns described in several traumatic injuries, thus underscoring a metabolic response/adaptation to the injurious action of the venoms.

Asunto(s)

Bothrops , Venenos de Crotálidos , Daboia , Ratones , Animales , Bothrops/metabolismo , Lipidómica , Hidrocortisona , Modelos Animales de Enfermedad , Daboia/metabolismo , Ponzoñas/metabolismo , Aminoácidos/metabolismo , Purinas/metabolismo , Hemo/metabolismo , Triglicéridos/metabolismo , Pirimidinas/metabolismo , ARN de Transferencia/metabolismo , Venenos de Crotálidos/toxicidad , Venenos de Crotálidos/metabolismo , Antivenenos/farmacología

RESUMEN

Snakebite is a neglected tropical disease with significant morbidity and mortality. Thrombotic microangiopathy (TMA) is an important but poorly understood complication of snakebite associated with acute kidney injury (AKI). Numerous treatments have been attempted based on limited evidence. We conducted a systematic review of TMA following snakebite using a pre-determined case definition of blood film red cell schistocytes or histologically diagnosed TMA. The search strategy included major electronic databases and grey literature. We present a descriptive synthesis for the outcomes of AKI, dialysis free survival (DFS), other end-organ damage, overall survival, and interventions with antivenom and therapeutic plasmapheresis (TPE). This study was prospectively registered with PROSPERO (CRD42019121436). Seventy-two studies reporting 351 cases were included, predominantly small observational studies. Heterogeneity for study selection, design, reporting and outcomes were observed. The commonest envenoming species were hump-nosed vipers (Hypnale spp.), Russell's viper (Daboia russelii) and Australian brown snakes (Pseudechis spp.). The prevalence of TMA was at least 5.4% in proven and probable Hypnale bites, and 10-15% of Australian elapid envenomings, AKI occurred in 94% (293/312) of TMA cases, excluding case reports. The majority of cases with AKI required dialysis. Included prospective and retrospective cohort studies reporting interventions and renal outcomes showed no evidence for benefit from antivenom or TPE with respect to DFS in dialysis dependant AKI. The Grading of Recommendations, Assessment, Development and Evaluations (GRADE) assessment for quality of accumulated evidence for interventions was low. The major complication of TMA following snakebite is AKI. AKI improves in most cases. We found no evidence to support benefit from antivenom in snakebite associated TMA, but antivenom remains the standard of care for snake envenoming. There was no evidence for benefit of TPE in snakebite associated TMA, so TPE cannot be recommended. The quality of accumulated evidence was low, highlighting a need for high quality larger studies.

Asunto(s)

Lesión Renal Aguda/etiología , Antivenenos/uso terapéutico , Daboia/metabolismo , Plasmaféresis , Mordeduras de Serpientes/complicaciones , Microangiopatías Trombóticas/etiología , Venenos de Víboras/efectos adversos , Lesión Renal Aguda/sangre , Lesión Renal Aguda/terapia , Animales , Australia/epidemiología , Femenino , Humanos , Masculino , Prevalencia , Estudios Prospectivos , Estudios Retrospectivos , Mordeduras de Serpientes/sangre , Mordeduras de Serpientes/terapia , Microangiopatías Trombóticas/sangre , Microangiopatías Trombóticas/terapia

RESUMEN

For most antivenoms there is little information from clinical studies to infer the relationship between dose and efficacy or dose and toxicity. Antivenom dose-finding studies usually recruit too few patients (e.g. fewer than 20) relative to clinically significant event rates (e.g. 5%). Model based adaptive dose-finding studies make efficient use of accrued patient data by using information across dosing levels, and converge rapidly to the contextually defined 'optimal dose'. Adequate sample sizes for adaptive dose-finding trials can be determined by simulation. We propose a model based, Bayesian phase 2 type, adaptive clinical trial design for the characterisation of optimal initial antivenom doses in contexts where both efficacy and toxicity are measured as binary endpoints. This design is illustrated in the context of dose-finding for Daboia siamensis (Eastern Russell's viper) envenoming in Myanmar. The design formalises the optimal initial dose of antivenom as the dose closest to that giving a pre-specified desired efficacy, but resulting in less than a pre-specified maximum toxicity. For Daboia siamensis envenoming, efficacy is defined as the restoration of blood coagulability within six hours, and toxicity is defined as anaphylaxis. Comprehensive simulation studies compared the expected behaviour of the model based design to a simpler rule based design (a modified '3+3' design). The model based design can identify an optimal dose after fewer patients relative to the rule based design. Open source code for the simulations is made available in order to determine adequate sample sizes for future adaptive snakebite trials. Antivenom dose-finding trials would benefit from using standard model based adaptive designs. Dose-finding trials where rare events (e.g. 5% occurrence) are of clinical importance necessitate larger sample sizes than current practice. We will apply the model based design to determine a safe and efficacious dose for a novel lyophilised antivenom to treat Daboia siamensis envenoming in Myanmar.

Asunto(s)

Antivenenos/administración & dosificación , Antivenenos/efectos adversos , Mordeduras de Serpientes/terapia , Venenos de Víboras/antagonistas & inhibidores , Anafilaxia/inducido químicamente , Animales , Antivenenos/uso terapéutico , Teorema de Bayes , Coagulación Sanguínea/efectos de los fármacos , Ensayos Clínicos Fase II como Asunto/métodos , Simulación por Computador , Relación Dosis-Respuesta a Droga , Humanos , Modelos Estadísticos , Mianmar , Daboia/metabolismo

RESUMEN

Russell's viper (Daboia russelii) is, together with Naja naja, Bungarus caeruleus and Echis carinatus, a member of the medically important 'Big Four' species responsible for causing a large number of morbidity and mortality cases across the Indian subcontinent. Despite the wide distribution of Russell's viper and the well-documented ubiquity of the phenomenon of geographic variability of intraspecific snake venom composition, Indian polyvalent antivenoms against the "Big Four" venoms are raised against venoms sourced mainly from Chennai in the southeastern Indian state of Tamil Nadu. Biochemical and venomics investigations have consistently revealed notable compositional, functional, and immunological differences among geographic variants of Russell's viper venoms across the Indian subcontinent. However, these studies, carried out by different laboratories using different protocols and involving venoms from a single geographical region, make the comparison of the different venoms difficult. To bridge this gap, we have conducted bioactivities and proteomic analyses of D. russelii venoms from the three corners of the Indian subcontinent, Pakistan, Bangladesh, and Tamil Nandu (India) and Sri Lanka, along with comparative in vivo neutralization and in vitro third-generation antivenomics of antivenoms used in India, Bangladesh and Sri Lanka. These analyses let us to propose two alternative routes of radiation for Russell's viper in the Indian subcontinent. Both radiations, towards the northeast of India and Bangladesh and towards south India and Sri Lanka, have a common origin in Pakistan, and provide a phylovenomics ground for rationalizing the geographic variability in venom composition and their distinct immunoreactivity against available antivenoms. BIOLOGICAL SIGNIFICANCE: Russell's viper (Daboia russelii), the Indian cobra (Naja naja), the common krait (Bungarus caeruleus), and the saw-scaled viper (Echis carinatus) constitute the 'Big Four' snake species responsible for most snakebite envenomings and deaths in the Indian subcontinent. Despite the medical relevance of Daboia russelii, and the well documented variations in the clinical manifestations of envenomings by this wide distributed species, which are doubtless functionally related to differences in venom composition of its geographic variants, antivenoms for the clinical treatment of envenomings by D. russelii across the Indian subcontinent are invariably raised using venom sourced mainly from the southeastern Indian state of Tamil Nadu. We have applied a phylovenomics approach to compare the venom proteomes of Russell's vipers from the three corners of the Indian subcontinent, Pakistan, Bangladesh, and South India/Sri Lanka, and have assessed the in vitro (third-generation antivenomics) and in vivo preclinical efficacy of a panel of homologous antivenoms. The identification of two dispersal routes of ancestral D. russelii into the Indian subcontinent provides the ground for rationalizing the variability in composition and immunoreactivity of the venoms of extant geographic variants of Russell's viper. Such knowledge is relevant for envisioning strategies to improve the clinical coverage of anti- D. russelii antivenoms.

Asunto(s)

Antivenenos/farmacología , Daboia , Mordeduras de Serpientes , Venenos de Víboras/antagonistas & inhibidores , Animales , Asia Occidental , Ratones , Proteómica , Daboia/clasificación , Daboia/metabolismo , Mordeduras de Serpientes/tratamiento farmacológico , Mordeduras de Serpientes/metabolismo , Mordeduras de Serpientes/patología , Especificidad de la Especie , Venenos de Víboras/toxicidad

RESUMEN

Envenomings by some populations of the Russell's viper (Daboia russelii) are characterized by a systemic capillary leak syndrome (CLS) which causes hemoconcentration, and is associated with the severity of envenoming. We adapted a model of CLS in mice by assessing hemoconcentration. The venom of D. russelii from Pakistan, but not that of another viperid, Bothrops asper, induced hemoconcentration and an increment in vascular permeability, being devoid of hemorrhagic activity at the doses tested. These findings reveal a dichotomous pattern of vasculotoxicity in viperid snake venoms. This difference might depend on variations in venom composition, especially regarding metalloproteinases (SVMPs), which are low in Pakistani D. russelii and high in B. asper. Inhibition of SVMPs and phospholipases A2 in D. russelii venom did not abrogate hemoconcentration. An hemoconcentration-inducing fraction was obtained by chromatography, which contains vascular endothelial growth factor (VEGF), a known potent inducer of increment in vascular permeability. Exudates collected from tissue injected with venom also induced hemoconcentration, and the effect was inhibited by antivenom. However, the amount of venom in exudate required to induce the effect is low, as compared with venom dissolved in saline solution, hence suggesting that endogenous proteins present in the exudate, probably inflammatory mediators, potentiate the effect.

Asunto(s)

Vasos Sanguíneos/patología , Daboia/metabolismo , Venenos de Víboras/toxicidad , Secuencia de Aminoácidos , Animales , Vasos Sanguíneos/efectos de los fármacos , Permeabilidad Capilar/efectos de los fármacos , Fraccionamiento Químico , Quimiocinas/sangre , Exudados y Transudados , Hematócrito , Hemorragia/sangre , Hemorragia/patología , Hipoalbuminemia/sangre , Hipoalbuminemia/complicaciones , Hipoalbuminemia/patología , Ratones , Pakistán , Mordeduras de Serpientes/sangre , Mordeduras de Serpientes/patología , Venenos de Víboras/química

RESUMEN

INTRODUCTION: The Russell's Viper (RV) (Daboia russelii), a category I medically important snake, is responsible for a significant level of morbidity and mortality in the Indian sub-continent. Areas covered: The current review highlights the variation in RV venom (RVV) composition from different geographical locales on the Indian sub-continent, as revealed by biochemical and proteomic analyses. A comparison of these RVV proteomes revealed significant differences in the number of toxin isoforms and relative toxin abundances, highlighting the impact of geographic location on RVV composition. Antivenom efficacy studies have shown differential neutralization of toxicity and enzymatic activity of different RVV samples from the Indian sub-continent by commercial polyvalent antivenom (PAV). The proteome analysis has provided deeper insights into the variation of RVV composition leading to differences in antivenom efficacy and severity of clinical manifestations post RV-envenomation across the Indian sub-continent. Expert commentary: Variation in RVV antigenicity due to geographical differences and poor recognition of low molecular mass (<20 kDa) RVV toxins by PAV are serious concerns for effective antivenom treatment against RV envenomation. Improvements in immunization protocols that take into account the poorly immunogenic components and geographic variation in RVV composition, can lead to better hospital management of RV bite patients.

Asunto(s)

Antivenenos/uso terapéutico , Variación Biológica Poblacional , Daboia/genética , Mordeduras de Serpientes/terapia , Venenos de Víboras/química , Animales , Antivenenos/inmunología , Humanos , India , Filogeografía , Proteómica/métodos , Daboia/metabolismo , Mordeduras de Serpientes/inmunología , Venenos de Víboras/genética , Venenos de Víboras/inmunología

RESUMEN

Inflammation-mediated disorders are on the rise and hence, there is an urgent need for the design and synthesis of new anti-inflammatory drugs with higher affinity and specificity for their potential targets. The current study presents an effective and new protocol for the synthesis of thienyl-pyrazoles through 3 + 2 annulations using a recyclable heterogeneous catalyst Amberlyst-15. Chalcones 3(a-g) prepared from 3-methylthiophene-2-carbaldehyde and acetophenones by Claisen-Schmidt approach reacted with semicarbazide hydrochloride 4 in the presence of Amberlyst-15 in acetonitrile at room temperature producing thienyl-pyrazole carboxamides 5(a-h) in good yields. Alternatively, the compounds 5(a-h) were prepared by conventional method using acetic acid (30%) medium. Structures of synthesized new pyrazoles were confirmed by spectral and crystallographic studies. All the new compounds were evaluated for their in vitro inhibition of Phospholipase A2 from Vipera russelli and preliminary studies revealed that, amongst the designed series, compounds 5b, 5c and 5h showed promising inhibition. Further, the compounds exhibited linear mixed-type inhibition behavior for the sPLA2 enzyme indicating that they bind to an allosteric site distinct from either the calcium or substrate binding site on the enzyme. These kinetic conclusions were further validated by macromolecular rigid-body docking whereby compounds 5c and 5h showed binding to distinct pockets on the protein. These findings present a promising series of lead molecules that can serve as prototypes for the treatment of inflammatory related disorders.

Asunto(s)

Sitio Alostérico/efectos de los fármacos , Daboia/metabolismo , Fosfolipasas A2 Grupo II/metabolismo , Inhibidores de Fosfolipasa A2/química , Inhibidores de Fosfolipasa A2/farmacología , Pirazoles/química , Pirazoles/farmacología , Animales , Antiinflamatorios no Esteroideos/síntesis química , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/farmacología , Catálisis , Diseño de Fármacos , Fosfolipasas A2 Grupo II/química , Simulación del Acoplamiento Molecular , Inhibidores de Fosfolipasa A2/síntesis química , Pirazoles/síntesis química , Estirenos/química

RESUMEN

Snake venom Kunitz-type serine protease inhibitors (KSPIs) exhibit various biological functions including anticoagulant activity. This study elucidates the occurrence and subunit stoichiometry of a putative complex formed between two KSPIs (Rusvikunin and Rusvikunin-II) purified from the native Rusvikunin complex of Pakistan Russell's Viper (Daboia russelii russelii) venom (RVV). The protein components of the Rusvikunin complex were identified by LC-MS/MS analysis. The non-covalent interaction between two major components of the complex (Rusvikunin and Rusvikunin-II) at 1:2 stoichiometric ratio to form a stable complex was demonstrated by biophysical techniques such as spectrofluorometric, classical gel-filtration, equilibrium gel-filtration, circular dichroism (CD), dynamic light scattering (DLS), RP-HPLC and SDS-PAGE analyses. CD measurement showed that interaction between Rusvikunin and Rusvikunin-II did not change their overall secondary structure; however, the protein complex exhibited enhanced hydrodynamic diameter and anticoagulant activity as compared to the individual components of the complex. This study may lay the foundation for understanding the basis of protein complexes in snake venoms and their role in pathophysiology of snakebite.

Asunto(s)

Daboia/metabolismo , Complejos Multiproteicos/metabolismo , Inhibidores de Serina Proteinasa/metabolismo , Venenos de Víboras/metabolismo , Secuencia de Aminoácidos , Animales , Aprotinina/química , Aprotinina/metabolismo , Bovinos , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Electroforesis en Gel de Poliacrilamida , Fibrina/metabolismo , Fibrinógeno/metabolismo , Humanos , Complejos Multiproteicos/química , Unión Proteica , Proteolisis , Inhibidores de Serina Proteinasa/química , Albúmina Sérica Bovina/metabolismo , Espectrofotometría , Espectrometría de Masas en Tándem , Venenos de Víboras/química

RESUMEN

To evaluate the characteristic features of the dilute Russell's viper venom time (DRVVT) titer in the antiphospholipid syndrome (APS). The medical record of 3660 consecutive patients with DRVVT orders between 2006 and 2015 were examined for criteria satisfying the diagnosis of APS. DRVVT titer was studied as a function of titer distribution, titer stability, and clinicopathologic features. Twenty-six patients were diagnosed with APS based on a persistently positive DRVVT and a history of arterial or venous thrombosis. DRVVT titer was mostly of low magnitude (65-77% of patients), was of similar value between initial and repeat testing (mean DRVVT titer 1.40 vs. 1.38; P = 0.858; mean time interval 216 days), and was positively associated with anticardiolipin antibodies (IgG and IgM) and antibeta-2-glycoprotein I antibodies (IgG and IgM) (P < 0.020). Low titer and moderate/high titer DRVVT were associated with a triple positive antiphospholipid antibody profile in 0 and 62% of patients, respectively (P < 0.020). DRVVT titer in APS was predominantly low in magnitude, stable over time, and associated with specific antiphospholipid antibody profiles.

Asunto(s)

Síndrome Antifosfolípido/sangre , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Inhibidor de Coagulación del Lupus/sangre , Trombosis/sangre , Venenos de Víboras/química , Adulto , Anciano , Animales , Síndrome Antifosfolípido/diagnóstico , Biomarcadores/sangre , Femenino , Humanos , Masculino , Persona de Mediana Edad , Tiempo de Tromboplastina Parcial , Daboia/metabolismo , Trombosis/diagnóstico , beta 2 Glicoproteína I/antagonistas & inhibidores , beta 2 Glicoproteína I/sangre , beta 2 Glicoproteína I/inmunología

RESUMEN

Dilute Russell's viper venom (DRVV) testing and activated partial thromboplastin time (APTT) have been effectively used in combination for lupus anticoagulant testing. The purpose of our study was to evaluate the role of mixing in activated partial thromboplastin and dilute Russell's viper venom testing for evaluation of lupus anticoagulants. Citrated blood from patients who were not on oral anticoagulant therapy was studied. Mixing study with 1 : 1 normal plasma for elevated APTT and also few samples with elevated screen time was carried out. Elevated APTT was seen in only 48.1% of patients with lupus anticoagulant. Correction of APTT was seen in 27.8% of lupus anticoagulant-positive patients. DRVV test on mixing resulted in 83.8% false-negative values. Integrated DRVV test could be a standalone test for testing lupus anticoagulant. Mixing study may be restricted for patients on oral anticoagulants or patients with strong lupus anticoagulant.

Asunto(s)

Aborto Espontáneo/sangre , Recolección de Muestras de Sangre/normas , Inhibidor de Coagulación del Lupus/sangre , Trombosis/sangre , Venenos de Víboras/química , Aborto Espontáneo/diagnóstico , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Biomarcadores/sangre , Niño , Reacciones Falso Negativas , Femenino , Humanos , Masculino , Persona de Mediana Edad , Tiempo de Tromboplastina Parcial , Embarazo , Tiempo de Protrombina , Daboia/metabolismo , Trombosis/diagnóstico

RESUMEN

The pathophysiological significance of a toxic fraction (GF-VI DEAE-II) isolated from Russell's viper venom (RVV) is characterized. GF-VI DEAE-II represents 1.6% of the total RVV protein and it comprises of a 27.6kDa minor component (RP-I) (0.04%, w/w) and a major 6.6kDa non-enzymatic peptide (1.11%, w/w), named Rusvitoxin. The LC-MS/MS analysis of RP-I showed its identity to snake venom serine proteases, whereas Rusvitoxin demonstrated its close identity with snake venom three finger toxins, cytotoxins and cardiotoxins particularly from Naja sp. GF-VI DEAE-II was found to be non-cytotoxic to the tested mammalian cancer cells and non-hemolytic; nevertheless, it demonstrated α-fibrin(ogen)ase activity and in vivo toxicity in BALB/c mice with an LD50 (i.p.) of 2.3mg/kg. GF-VI DEAE-II induced lethargy and hind-leg paralysis in mice within 10min of i.p. injection. GF-VI DEAE-II induced hyperfibrinogenomia, and significantly altered (p<0.05) the plasma levels of factor X, pro- and anti-inflammatory cytokines viz. TNF-α, IL-6 and IL-10 in treated mice. Histological observations of tissues and biochemical properties of serum from GF-VI DEAE-II-treated mice suggested multiple organ dysfunctions. Conversely, Rusvitoxin at a dose of 5mg/kg did not induce toxicity in BALB/c mice. At 1:15 (antigen: antivenom, w/w) ratio, commercially polyvalent and monovalent antivenoms neutralized more than 80% of the fibrinolytic and anticoagulant activities of GF-VI DEAE-II. The present study suggests the significant role of GF-VI DEAE-II in RVV-induced pathogenesis in victim/prey.

Asunto(s)

Citotoxinas/efectos adversos , Citotoxinas/farmacología , Daboia/metabolismo , Venenos de Moluscos/efectos adversos , Venenos de Moluscos/farmacología , Venenos de Víboras/efectos adversos , Venenos de Víboras/farmacología , Animales , Anticoagulantes/metabolismo , Antivenenos/farmacología , Fibrinolíticos/metabolismo , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Ratones , Ratones Endogámicos BALB C , Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

This is the first report on the characterization of a snaclec (RVsnaclec) purified from Daboia russelii russelii venom. The RVsnaclec is a heterodimer of two subunits, α (15.1 kDa) and ß (9 kDa). These subunits are covalently linked to form multimeric (αß)2 and (αß)4 structures. Peptide mass fingerprinting analysis of RVsnaclec via LC-MS/MS demonstrated its similarity to snaclecs purified from other viperid snake venoms. Two tryptic peptide sequences of RVsnaclec revealed the putative conserved domains of C-type lectin (CTL). RVsnaclec dose-dependently increased the Ca-clotting time and prothrombin time of platelet-poor plasma (PPP); however, it did not affect the partial thromboplastin time (APTT) or thrombin time of PPP. The in vitro and in vivo anticoagulant activity of RVsnaclec is correlated to its binding and subsequent uncompetitive inhibition of FXa (Ki = 0.52 µmole) in a Ca(2+)-independent manner; however, supplementation with 0.25 mM Ca(2+) enhanced the Xa binding potency of RVsnaclec. Monovalent or polyvalent antivenom failed to neutralize its anticoagulant potency, and RVsnaclec did not inhibit trypsin, chymotrypsin, thrombin or plasmin. RVsnaclec was devoid of hemolytic activity or cytotoxicity against several human cancer cell lines, demonstrated concentration-dependent aggregation and deaggregation of human platelets, and inhibited the ADP-induced aggregation of platelet. RVsnaclec (5.0 mg/kg body weight) was non-lethal to mice and showed no adverse pharmacological effects, suggesting that it has potential as a lead compound for future therapeutic applications in cardiovascular disorders.

Asunto(s)

Anticoagulantes/farmacología , Daboia , Lectinas Tipo C/química , Venenos de Víboras/farmacología , Animales , Anticoagulantes/administración & dosificación , Anticoagulantes/química , Anticoagulantes/toxicidad , Coagulación Sanguínea/efectos de los fármacos , Plaquetas/citología , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Calcio/metabolismo , Factor Xa/metabolismo , Inhibidores del Factor Xa/administración & dosificación , Inhibidores del Factor Xa/química , Inhibidores del Factor Xa/farmacología , Inhibidores del Factor Xa/toxicidad , Cabras , Humanos , Lectinas Tipo C/administración & dosificación , Lectinas Tipo C/aislamiento & purificación , Ratones , Agregación Plaquetaria/efectos de los fármacos , Daboia/metabolismo , Venenos de Víboras/administración & dosificación , Venenos de Víboras/química , Venenos de Víboras/toxicidad

RESUMEN

Proteases from Russell's viper venom (RVV) induce a variety of toxic effects in victim. Therefore, four new RVV protease isoenzymes of molecular mass 32901.044 Da, 333631.179 Da, 333571.472 Da, and 34594.776 Da, were characterized in this study. The first 10 N-terminal residues of these serine protease isoenzymes showed significant sequence homology with N-terminal sequences of snake venom thrombin-like and factor V-activating serine proteases, which was reconfirmed by peptide mass fingerprinting analysis. These proteases were found to be different from previously reported factor V activators isolated from snake venoms. These proteases showed significantly different fibrinogenolytic, BAEE-esterase and plasma clotting activities but no fibrinolytic, TAME-esterase or amidolytic activity against the chromogenic substrate for trypsin, thrombin, plasmin and factor Xa. Their Km and Vmax values towards fibrinogen were determined in the range of 6.6 to 10.5 µM and 111.0 to 125.5 units/mg protein, respectively. On the basis of fibrinogen degradation pattern, they may be classified as A/B serine proteases isolated from snake venom. These proteases contain ∼ 42% to 44% of N-linked carbohydrates by mass whereas partially deglycosylated enzymes showed significantly less catalytic activity as compared to native enzymes. In vitro these protease isoenzymes induce blood coagulation through factor V activation, whereas in vivo they provoke dose-dependent defibrinogenation and anticoagulant activity in the mouse model. At a dose of 5 mg/kg, none of these protease isoenzymes were found to be lethal in mice or house geckos, suggesting therapeutic application of these anticoagulant peptides for the prevention of thrombosis.

Asunto(s)

Coagulación Sanguínea/efectos de los fármacos , Coagulantes/farmacología , Daboia/metabolismo , Factor Va/metabolismo , Fibrinógeno/metabolismo , Serina Proteasas/aislamiento & purificación , Venenos de Víboras/enzimología , Amidas/metabolismo , Secuencia de Aminoácidos , Animales , Hidrolasas de Éster Carboxílico/metabolismo , Bovinos , Compuestos Cromogénicos/metabolismo , Electroforesis en Gel de Poliacrilamida , Glicosilación , Isoenzimas/química , Isoenzimas/aislamiento & purificación , Isoenzimas/metabolismo , Ratones , Modelos Animales , Datos de Secuencia Molecular , Alineación de Secuencia , Serina Proteasas/química , Serina Proteasas/metabolismo

RESUMEN

Present report shows for the first time on the induction of in vitro angiogenesis by a 3.9 kDa novel peptide (RVVAP) purified from Russell's viper venom. Secondary structure of RVVAP is made up of 36.8% α-helix, 33.3% ß pleated sheets and 29.9% turns. Optimum angiogenesis and significant elevation in endothelial migration were observed at 50 ng/ml of RVVAP treatment; above this concentration, progressive decrease in wound healing was noted. RVVAP (1.0 µg/ml) was non-cytotoxic to U87-MG, HeLa and HT-29 cells; however, increasing the RVVAP concentration above 500 ng/ml resulted in induction of chromosomal aberrations and delay in cell cycle kinetics of Chinese hamster ovary cells.

Asunto(s)

Proteínas Angiogénicas/análisis , Proteínas Angiogénicas/farmacología , Ciclo Celular/efectos de los fármacos , Aberraciones Cromosómicas/efectos de los fármacos , Daboia/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Venenos de Víboras/química , Animales , Células CHO , Movimiento Celular/efectos de los fármacos , Pollos , Cricetinae , Cricetulus , Relación Dosis-Respuesta a Droga , Células Endoteliales/efectos de los fármacos , Óvulo/efectos de los fármacos , Conformación Proteica , Cicatrización de Heridas/efectos de los fármacos

RESUMEN

Snake venoms are complex mixture of enzymatic and non-enzymatic proteins. Non-covalent protein-protein interaction leads to protein complexes, which bring about enhanced pharmacological injuries by their synergistic action. Here we report identification and characterization of a new Daboia russelii hemorrhagic complex I (DR-HC-I) containing phospholipase A2 (PLA2) and non-enzymatic peptide. DR-HC-I was isolated from the venom of D. russelii by CM-Shepadex-C25 and gel permeation chromatography. Individual components were purified and identified by RP-HPL chromatography, mass spectrometry and N-terminal amino acid sequencing. DR-HC-I complex was lethal to mice with the LD50 dose of 0.7 mg/kg body weight with hemorrhagic and neurotoxic properties. DR-HC-I complex consists of non-hemorrhagic PLA2 and neurotoxic non-enzymatic peptide. The non-enzymatic peptide quenched the intrinsic fluorescence of PLA2 in a dose dependent manner, signifying the synergistic interaction between two proteins. PLA2 and peptide toxin in a 5:2 M ratio induced skin hemorrhage in mice with MHD 20 µg. However, addition of ANS (1-Anilino-8-naphthalene sulfonate) to DR-HC-I complex inhibited skin hemorrhagic effect and also synergic interaction. But there was no impact on PLA2 due to this synergistic interaction, and indirect hemolytic or plasma re-calcification activity. However, the synergistic interaction of PLA2 and non-enzymatic peptide contributes to the enhanced venom-induced hemorrhage and toxicity of Daboia russellii venom.

Asunto(s)

Daboia/metabolismo , Hemorragia/inducido químicamente , Complejos Multiproteicos/metabolismo , Fosfolipasas A2/metabolismo , Enfermedades de la Piel/inducido químicamente , Venenos de Víboras/enzimología , Secuencia de Aminoácidos , Naftalenosulfonatos de Anilina/metabolismo , Animales , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Fluorescencia , Hemorragia/tratamiento farmacológico , Dosificación Letal Mediana , Espectrometría de Masas , Ratones , Datos de Secuencia Molecular , Complejos Multiproteicos/toxicidad , Péptidos/metabolismo , Péptidos/farmacología , Análisis de Secuencia de ADN , Enfermedades de la Piel/tratamiento farmacológico , Venenos de Víboras/toxicidad

RESUMEN

Two trypsin inhibitors and one chymotrypsin inhibitor from Chinese Daboia russellii siamensis venom, denoted as CBPTI-1, CBPTI-2 and CBPTI-3 were purified, characterized and cloned from lyophilized venom-derived cDNA libraries. The N-terminus of CBPTI-1 was modified and not amenable to Edman degradation sequencing, however an internal partial sequence was found to be SGRCRGHLRRIYYNPDSNKCE. The N-termini of CBPTI-2 and CBPTI-3 were unmodified and their partial sequences were established as HDRPTFCNLAPESGRCRAH and HDRPKFCYLPADPGECMAYIRSFYYDS respectively. From cloning studies CBPTI-1 was found to consist of 66 amino acid residues, while CBPTI-2 and CBPTI-3 precursors consist of 60 amino acid residues, including 6 cysteine residues. Another cDNA sequence (CBPTI-4) was also obtained. Alignment of cDNA sequences showed that CBPTI-3 exhibited similar sequence homology to CBPTI-4 cDNA except for an 8 nucleotide deletion in the open-reading frame. CBPTI-1 and CBPTI-2 were demonstrated to be potent trypsin inhibitors, but were also shown to be effectively potent in chymotrypsin inhibition. The K(i) values of CBPTI-1 and CBPTI-2 for trypsin inhibition were 4.07 × 10(-7) M and 6.66 × 10(-7) M, respectively, and they were non-competitive in their activity. CBPTI-3 showed chymotrypsin inhibition activity with a K(i) value of 2.55 × 10(-9) M, but did not show trypsin inhibitor activity.

Asunto(s)

Quimotripsina/antagonistas & inhibidores , Daboia/metabolismo , Venenos Elapídicos/química , Fragmentos de Péptidos/metabolismo , Inhibidores de Tripsina , Tripsina/metabolismo , Secuencia de Aminoácidos , Animales , Bovinos , Cromatografía Líquida de Alta Presión , Quimotripsina/análisis , Quimotripsina/metabolismo , Clonación Molecular , Venenos Elapídicos/genética , Venenos Elapídicos/metabolismo , Datos de Secuencia Molecular , Fragmentos de Péptidos/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Tripsina/análisis , Inhibidores de Tripsina/genética , Inhibidores de Tripsina/aislamiento & purificación , Inhibidores de Tripsina/metabolismo

RESUMEN

The activation of coagulation factors V and X by Russell's viper venom (RVV) has been implicated in the development of consumptive coagulopathies in severely envenomed patients. However, factor Va is prone to inactivation by activated protein C (APC), an important serine protease that negatively regulates blood coagulation. It is therefore hypothesized that APC may be down-regulated by some of the venom components. In this study, we managed to isolate a potent Kunitz-type APC inhibitor, named DrKIn-I. Using chromogenic substrate, DrKIn-I dose-dependently inhibited the activity of APC. Heparin potentiated the inhibition and reduced the IC(50) of DrKIn-I by 25-fold. DrKIn-I, together with heparin, also protected factor Va from APC-mediated inactivation. Using surface plasmon resonance, DrKIn-I exhibited fast binding kinetics with APC (association rate constant = 1.7 × 10(7) M(-1) s(-1)). Direct binding assays and kinetic studies revealed that this inhibition (K(i) = 53 pM) is due to the tight binding interactions of DrKIn-I with both heparin and APC. DrKIn-I also effectively reversed the anticoagulant activity of APC and completely restored the thrombin generation in APC-containing plasma. Furthermore, although the injection of either DrKIn-I or RVV-X (the venom factor X-activator) into ICR mice did not significantly deplete the plasma fibrinogen concentration, co-administration of DrKIn-I with RVV-X resulted in complete fibrinogen consumption and the deposition of fibrin thrombi in the glomerular capillaries. Our results provide new insights into the pathogenesis of RVV-induced coagulopathies and indicate that DrKIn-I is a novel APC inhibitor that is associated with potentially fatal thrombotic complications in Russell's viper envenomation.

Asunto(s)

Daboia/metabolismo , Coagulación Intravascular Diseminada/metabolismo , Heparina/farmacología , Inhibidor de Proteína C/farmacología , Proteína C/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Coagulación Sanguínea/efectos de los fármacos , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas Portadoras/farmacología , Clonación Molecular , ADN Complementario/química , ADN Complementario/genética , Coagulación Intravascular Diseminada/inducido químicamente , Sinergismo Farmacológico , Humanos , Cinética , Ratones , Ratones Endogámicos ICR , Datos de Secuencia Molecular , Unión Proteica , Proteína C/metabolismo , Inhibidor de Proteína C/genética , Inhibidor de Proteína C/metabolismo , Daboia/genética , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Venenos de Víboras/genética , Venenos de Víboras/metabolismo , Venenos de Víboras/farmacología , Venenos de Víboras/envenenamiento

RESUMEN

Serine proteases are widely found in snake venoms. They have variety of functions including contributions to hemostasis. In this study, five serine proteases were cloned and characterized from two different cDNA libraries: factor V activator (RVV-V), alpha fibrinogenase (RVAF) and beta fibrinogenase (RVBF) from Russell's viper (Daboia russelli siamensis), and plasminogen activator (APL-PA) and protein C activator (APL-C) from Agkistrodon piscivorus leucostoma. The snake venom serine proteases were clustered in phylogenetic tree according to their functions. K(A)/K(S) values suggested that accelerated evolution has occurred in the mature protein coding regions in cDNAs of snake venom serine proteases.

Asunto(s)

Agkistrodon/metabolismo , Venenos de Crotálidos/enzimología , Daboia/metabolismo , Filogenia , Proteínas de Reptiles/genética , Serina Endopeptidasas/genética , Venenos de Víboras/enzimología , Regiones no Traducidas 3' , Algoritmos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bases de Datos de Ácidos Nucleicos , Biblioteca de Genes , Datos de Secuencia Molecular , Proteínas de Reptiles/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Análisis de Secuencia de ADN , Serina Endopeptidasas/química

RESUMEN

Crystal of Russell Viper venom phospholipase A(2) complexed with an isoquinoline alkaloid, berberine from a herbaceous plant Cardiospermum halicacabum, was prepared and its structure was solved by X-ray crystallography. The crystal diffracted up to 1.93Å and the structure solution clearly located the position of berberine in the active site of the enzyme. Two hydrogen bonds, one direct and the other water mediated, were formed between berberine and the enzyme. Gly 30 and His 48 made these two hydrogen bonds. Additionally, the hydrophobic surface of berberine made a number of hydrophobic contacts with side chains of neighboring amino acids. Surface Plasmon Resonance studies revealed strong binding affinity between berberine and phospholipase A(2). Enzyme inhibition studies proved that berberine is a competitive inhibitor of phospholipase A(2). It was inferred that the isoquinoline alkaloid, berberine, is a potent natural inhibitor of phospholipaseA(2).

Asunto(s)

Berberina/química , Berberina/farmacología , Cristalografía por Rayos X/métodos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Fosfolipasas A/antagonistas & inhibidores , Fosfolipasas A/química , Sapindaceae/química , Animales , Fosfolipasas A/metabolismo , Estructura Secundaria de Proteína , Daboia/metabolismo , Resonancia por Plasmón de Superficie