RESUMEN
Marine organisms are increasingly being investigated as sources of bioactive molecules with therapeutic applications as nutraceuticals and pharmaceuticals. In particular, nutraceuticals are gaining popularity worldwide owing to their therapeutic potential and incorporation in functional foods and dietary supplements. Abalone, a marine gastropod, contains a variety of bioactive compounds with anti-oxidant, anti-thrombotic, anti-inflammatory, anti-microbial, and anti-cancer activities. For thousands of years different cultures have used abalone as a traditional functional food believing consumption provides health benefits. Abalone meat is one of the most precious commodities in Asian markets where it is considered a culinary delicacy. Recent research has revealed that abalone is composed of many vital moieties like polysaccharides, proteins, and fatty acids that provide health benefits beyond basic nutrition. A review of past and present research is presented with relevance to the therapeutic potential of bioactive molecules from abalone.
Asunto(s)
Organismos Acuáticos/química , Gastrópodos/química , Mariscos/análisis , Animales , Antiinfecciosos/química , Antiinfecciosos/farmacología , Antiinflamatorios/química , Antiinflamatorios/farmacología , Anticoagulantes/química , Anticoagulantes/farmacología , Antineoplásicos/química , Antineoplásicos/farmacología , Antioxidantes/química , Antioxidantes/farmacología , Acuicultura , Suplementos Dietéticos , Fibrinolíticos/química , Fibrinolíticos/farmacología , Manipulación de AlimentosRESUMEN
The venoms of Australian snakes contain a myriad of pharmacologically active toxin components. This study describes the identification and comparative analysis of two distinct toxin families, the kunitztype serine protease inhibitors and waprins, and demonstrates a previously unknown evolutionary link between the two. Multiple cDNA and full-length gene isoforms were cloned and shown to be composed of three exons separated by two introns. A high degree of identity was observed solely within the first exon which coded for the propeptide sequence and its cleavage site, and indicates that each toxin family has arisen from a gene duplication event followed by diversification only within the portion of the gene coding for the functional toxin. It is proposed that while the mechanism of toxin secretion is highly conserved, diversification of mature toxin sequences allows for the existence of multiple protein isoforms in the venom to adapt to variations within the prey environment.
Asunto(s)
Venenos Elapídicos/genética , Evolución Molecular , Péptidos/genética , Inhibidores de Proteasas/química , Secuencia de Aminoácidos , Animales , Australia , Secuencia de Bases , ADN Complementario/genética , Venenos Elapídicos/química , Genoma , Immunoblotting , Datos de Secuencia Molecular , Péptidos/química , Alineación de SecuenciaRESUMEN
Envenomation from Australian elapid snakes results in a myriad of neurological effects due to post-synaptic neurotoxins that bind and inhibit nicotinic acetylcholine receptors (nAChRs) of neurons and muscle fibres. However, despite the significant physiological effects of these toxins, they have remained largely undercharacterised at the molecular level. This study describes the identification and comparative analysis of multiple neurotoxin isoforms from ten Australian snakes, including functional characterisation of two of these isoforms, Os SNTX-1 from Oxyuranus scutellatus and the more potent Pt LNTX-1 from Pseudonaja textilis. Electrophysiological recordings from adrenal chromaffin cells demonstrate that both neurotoxins act as competitive antagonists of nAChRs in a concentration-dependent manner. Their effects upon spontaneous and nerve-evoked membrane responses at the amphibian neuromuscular junction provide further evidence that both toxins bind muscle nAChRs in an irreversible manner. This study represents one of the most comprehensive descriptions to date of the sequences and activity of individual Australian elapid neurotoxins.
Asunto(s)
Venenos Elapídicos/toxicidad , Neurotoxinas/toxicidad , Receptores Nicotínicos/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Australia , Secuencia de Bases , Bufo marinus , Cartilla de ADN/genética , ADN Complementario/genética , Venenos Elapídicos/genética , Venenos Elapídicos/aislamiento & purificación , Elapidae/genética , Electrofisiología , Técnicas In Vitro , Datos de Secuencia Molecular , Neurotoxinas/genética , Neurotoxinas/aislamiento & purificación , Antagonistas Nicotínicos/aislamiento & purificación , Antagonistas Nicotínicos/toxicidad , Receptores Nicotínicos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/toxicidad , Homología de Secuencia de AminoácidoRESUMEN
Australian terrestrial elapid snakes contain amongst the most potently toxic venoms known. However, despite the well-documented clinical effects of snake bite, little research has focussed on individual venom components at the molecular level. To further characterise the components of Australian elapid venoms, a complementary (cDNA) microarray was produced from the venom gland of the coastal taipan (Oxyuranus scutellatus) and subsequently screened for venom gland-specific transcripts. A number of putative toxin genes were identified, including neurotoxins, phospholipases, a pseudechetoxin-like gene, a venom natriuretic peptide and a nerve growth factor together with other genes involved in cellular maintenance. Venom gland-specific components also included a calglandulin-like protein implicated in the secretion of toxins from the gland into the venom. These toxin transcripts were subsequently identified in seven other related snake species, producing a detailed comparative analysis at the cDNA and protein levels. This study represents the most detailed description to date of the cloning and characterisation of different genes associated with envenomation from Australian snakes.
Asunto(s)
Venenos Elapídicos/genética , Elapidae/genética , Aminoácido Oxidorreductasas/genética , Secuencia de Aminoácidos , Animales , Venenos de Crotálidos/genética , ADN Complementario , Venenos Elapídicos/metabolismo , Elapidae/metabolismo , Perfilación de la Expresión Génica , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosfolipasas A/genética , Fosfolipasas A/metabolismo , Filogenia , ARN Mensajero/metabolismo , Alineación de Secuencia , Venenos de Serpiente/genéticaRESUMEN
The incidence of vein-graft occlusion associated with myocardial infarction and thrombosis following the use of the plasmin inhibitor, aprotinin, to reduce blood loss during vascular surgery has prompted the isolation of an alternative kinetically distinct inhibitor of plasmin from the venom of Pseudonaja textilis. This inhibitor has been called textilinin (Txln) and two distinct forms have been isolated from the Brown-snake venom (molecular weight, 6688 and 6692). A comparison of plasmin inhibitor constants for aprotinin and the Txlns 1 and 2 indicated that the former bound very tightly (inhibitor constant, Ki approximately 10(-11) mol/l), while both of the latter bound less tightly (Ki approximately 10(-9) mol/l). Homogeneity of Txlns 1 and 2 was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and mass spectrometry. A sequence difference of six amino acids was observed between the two forms of Txln. Txln 1 and 2 showed, respectively, 45 and 43% homology with aprotinin, while there was 58 and 55% homology, respectively, with a plasmin inhibitor from the venom of eastern Taipan, Oxyuranus scutellatus. Both Txlns have six cysteines, like other inhibitors of this group, and homology was determined by alignment of these cysteines. Both have been shown to reduce blood loss by about 60% in a murine tail vein bleeding model. It is proposed that the kinetic profiles of Txln 1 and 2 for plasmin allow the arrest of haemorrhage without the possible threat of thrombosis.
Asunto(s)
Venenos Elapídicos , Venenos Elapídicos/aislamiento & purificación , Venenos Elapídicos/farmacología , Fibrinolisina/antagonistas & inhibidores , Hemorragia/tratamiento farmacológico , Inhibidores de Serina Proteinasa/aislamiento & purificación , Inhibidores de Serina Proteinasa/farmacología , Secuencia de Aminoácidos , Animales , Aprotinina/farmacología , Pérdida de Sangre Quirúrgica/prevención & control , Venenos Elapídicos/genética , Ratones , Datos de Secuencia Molecular , Inhibidores de Serina Proteinasa/genéticaRESUMEN
The use of aspirin as an anti-platelet drug is limited by its propensity to induce gastric injury and by its adverse effect on vascular prostacyclin formation. Two phenolic non-steroidal anti-inflammatory drugs (salicylic acid and diflunisal) were modified by esterification with a series of O-acyl moieties. The short-term ulcerogenic in vitro and in vivo anti-platelet properties, pharmacodynamic profiles, and extent of hepatic extraction of these phenolic esters were compared with aspirin (acetylsalicylic acid). The more lipophilic esters (longer carbon chain length in O-acyl group) show significantly less gastrotoxicity in stressed rats than does aspirin after a single oral dose. The in vitro and in vivo anti-platelet studies show that these phenolic esters inhibited (1) arachidonate-triggered human platelet aggregation and (2) thrombin-stimulated rat serum thromboxane A2 production by platelets in the clotting process almost as effectively as aspirin. The hepatic extractions of these O-acyl derivatives are significantly higher than those of aspirin. The pharmacodynamic studies show that these O-acyl derivatives of salicylic acid and diflunisal probably bind to, or combine with, the same site on the platelet cyclooxygenase as aspirin. Replacing the O-acetyl group with longer chain O-acyl moiety in this series of phenolic esters markedly reduced the potential of these agents to induce short-term gastric injury but did not lessen their activity as inhibitors of platelet aggregation. These non-acetyl salicylates may therefore represent a novel class of anti-platelet drugs with less ulcerogenic potential.
Asunto(s)
Aspirina/farmacología , Diflunisal/farmacología , Fibrinolíticos/farmacología , Mucosa Gástrica/efectos de los fármacos , Hígado/metabolismo , Inhibidores de Agregación Plaquetaria/farmacología , Úlcera Gástrica/prevención & control , Animales , Antiinflamatorios/farmacología , Artritis Experimental/inducido químicamente , Artritis Experimental/patología , Aspirina/análogos & derivados , Aspirina/farmacocinética , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Diflunisal/análogos & derivados , Diflunisal/farmacocinética , Relación Dosis-Respuesta a Droga , Femenino , Mucosa Gástrica/patología , Técnicas In Vitro , Inactivación Metabólica , Agregación Plaquetaria/efectos de los fármacos , Ratas , Ratas Wistar , Esteroides , Tromboxano A2/metabolismoRESUMEN
The recent high prevalence of fatal bites by Brown snakes (Pseudonaja genus) has led to this study of venom yields from 66 brown snake milkings over 15 months. The amount of venom obtained from all species was higher than reported previously. Electrophoretic and Western blotting analyses of their venoms showed significantly lower avidity of Brown snake antivenom (BS-AV) for the prothrombin activator (PA) component (190 kD) than for other venom components, including the neurotoxins. The LD50 of P. inframacula has been determined for the first time. SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) and Western blotting studies have shown that the Pseudonaja venoms contained proportionately more PA component than venoms of the Taipan (Oxyuranus scutellatus) or the Fierce snake (O. microlepidotus). Neutralization of the prothrombin activator of the Common Brown snake (P. textilis) (Pt-PA) by BS-AV was found to be time dependent and 40% remained unneutralized after 30 minutes incubation. Adult rats administered quantities of Pt-PA (IV) died with acute disseminated intravascular coagulation. Rats were made resistant to Pt-PA by preheparinization or by induction of tolerance to increasing quantities of Pt-PA. There is no evidence that Pt-PA has intrinsic toxicity apart from being a procoagulant. The improvement of BS-AV by addressing its deficiencies should be canvassed.
Asunto(s)
Antivenenos/uso terapéutico , Venenos de Serpiente/aislamiento & purificación , Venenos de Serpiente/toxicidad , Animales , Antivenenos/inmunología , Electroforesis en Gel de Poliacrilamida , Dosificación Letal Mediana , Masculino , Ratones , Ratones Endogámicos BALB C , Pruebas de Neutralización , Protrombina/aislamiento & purificación , Protrombina/metabolismo , Ratas , Ratas Sprague-Dawley , Venenos de Serpiente/inmunología , Especificidad de la EspecieRESUMEN
The cardiovascular and haematological effects of purified prothrombin activator derived from the venom of the Australian Common Brown Snake (Pseudonaja textilis) were studied in anaesthetised, mechanically ventilated dogs. Severe depression of systemic blood pressure and cardiac output and a rise in central venous pressure were observed. Thrombocytopenia, prolongation of both prothrombin time and activated partial thromboplastin time and a reduction in serum fibrinogen were also observed. All of these observed effects were prevented by the prior administration of heparin--a naturally occurring anticoagulant. We conclude that the prothrombin activator in Pseudonaja textilis venom may cause cardiovascular depression due to myocardial dysfunction secondary to disseminated intravascular coagulation.
Asunto(s)
Venenos Elapídicos/análisis , Venenos Elapídicos/farmacología , Fibrinolíticos/farmacología , Corazón/efectos de los fármacos , Heparina/farmacología , Animales , Coagulación Sanguínea/efectos de los fármacos , Presión Sanguínea/efectos de los fármacos , Gasto Cardíaco/efectos de los fármacos , Presión Venosa Central/efectos de los fármacos , Perros , Venenos Elapídicos/aislamiento & purificación , Electrocardiografía/efectos de los fármacos , Productos de Degradación de Fibrina-Fibrinógeno/análisis , Fibrinógeno/análisis , Fibrinolíticos/aislamiento & purificación , Bloqueo Cardíaco/inducido químicamente , Frecuencia Cardíaca/efectos de los fármacos , Hemodinámica/efectos de los fármacos , Volumen Sistólico/efectos de los fármacos , Taquicardia/inducido químicamente , Trombocitopenia/inducido químicamenteRESUMEN
Assumptions regarding the elaboration of plasma cross-linked fibrin degradation products (XLFbDPs) in vivo were tested using an experimental model in which particulate human fibrin was infused into rabbits and the products of lysis monitored with an immunoassay utilizing DD-3B6/22, a monoclonal antibody to human cross-linked derivatives. XLFbDPs were generated following the infusion of a suspension of cross-linked fibrin, attaining a peak between 40 and 60 min, then falling at a rate approximating a plasma half-life of 2 h. The major in vivo products of lysis of cross-linked fibrin, identified by SDS-PAGE of immunoextracted plasma, were D-dimer and high-molecular-weight moieties. Peak levels of XLFbDPs achieved correlated with the amount of fibrin administered. Since XLFbDP levels were no higher when fibrin infusion was followed by infusions of streptokinase and human plasminogen, it is concluded that endogenous mechanisms of lysis were already maximally stimulated. Infusions of non-cross-linked (NXL) fibrin or of fibrinogen led to much smaller, but measurable, rises in XLFbDP. In the latter group, XLFbDP levels rose further following fibrinolytic therapy. Treatment with epsilon aminocaproic acid (EACA) caused partial (greater than 50%) inhibition of lysis while pre-treatment with nitrogen mustard, inducing leucopenia, virtually abolished the appearance of XLFbDPs in the circulation. This implies that fibrinolytic responses are substantially dependent upon cellular functions sensitive to nitrogen mustard.
Asunto(s)
Productos de Degradación de Fibrina-Fibrinógeno/análisis , Fibrina/metabolismo , Fibrinólisis , Ácido Aminocaproico/farmacología , Animales , Anticuerpos Monoclonales/inmunología , Productos de Degradación de Fibrina-Fibrinógeno/inmunología , Humanos , Inmunoensayo , Leucocitos/fisiología , Leucopenia/sangre , Leucopenia/inducido químicamente , Pulmón/química , Masculino , Mecloretamina/farmacología , Mecloretamina/toxicidad , Modelos Biológicos , Plasminógeno/farmacología , Conejos , Estreptoquinasa/farmacologíaRESUMEN
Blood was obtained from four patients envenomated by the Australian common brown snake, Pseudonaja textilis textilis. This elapid snake has one of the most toxic venoms in the world, containing extremely potent neurotoxic and coagulant components. The latter is a potent complete prothrombinase, converting prothrombin to alpha-thrombin, and comprises more than 30% of the total venom protein. The four envenomated patients developed a typical consumption coagulopathy. Serial serum and plasma samples from patients were studied by immunoaffinity adsorption, 2-alanine precipitation of fibrinogen and fibrinogen-related products and 2-dimensional immunoelectrophoresis, and assayed for crosslinked fibrin degradation products as D dimer, using the monoclonal antibody, DD-3B6/22. These procedures showed the virtually complete disappearance of fibrinogen, accompanied by the appearance of large quantities of fibrinogen and fibrin degradation products consisting of both crosslinked and noncrosslinked species. With recovery, a homogeneous high molecular weight fibrinogen was observed. The data suggest that the prothrombin activator of this venom causes the generation of thrombin which subsequently converts fibrinogen to fibrin and stimulates partial crosslinking of both alpha and gamma-chains. The resultant disseminated intravascular coagulation is accompanied by very active secondary fibrinolysis which apparently limits the extent of any microvascular thrombosis but which may contribute to a bleeding tendency.
Asunto(s)
Coagulación Intravascular Diseminada/etiología , Fibrinólisis , Mordeduras de Serpientes/complicaciones , Adulto , Anciano , Coagulación Sanguínea/efectos de los fármacos , Coagulación Intravascular Diseminada/sangre , Venenos Elapídicos/toxicidad , Femenino , Fibrinólisis/efectos de los fármacos , Humanos , Masculino , Persona de Mediana Edad , Mordeduras de Serpientes/sangreRESUMEN
A polypeptide inhibitor of osteoblast proliferation is described which occurs in synovial effusions of patients with rheumatoid arthritis. Partial purification of the inhibitor showed a molecular weight of approximately 81,000 by gel electrophoresis. This polypeptide seems to be unique as no inhibitor of osteoblasts of similar molecular weight has been previously described in rheumatoid synovial effusions.
Asunto(s)
Artritis Reumatoide/fisiopatología , Osteoblastos/fisiología , Péptidos/aislamiento & purificación , Líquido Sinovial/fisiología , Animales , Artritis Reumatoide/metabolismo , División Celular , Células Cultivadas , Cromatografía de Afinidad , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Electroforesis en Gel de Poliacrilamida , Ratones , Peso Molecular , Péptidos/metabolismo , Líquido Sinovial/análisis , Líquido Sinovial/metabolismoRESUMEN
A simple procedure, involving chromatography on concanavalin A-Sepharose and gel filtration, has been developed for the purification of a prothrombin activator from the venom of the Australian brown snake Pseudonaja textilis textilis. The prothrombin activator, which is a major venom component, is a high molecular weight protein (Mr greater than or equal to 200,000) which yields a number of subunits when examined by SDS-PAGE. It is related antigenically to the venom prothrombin activator of the taipan Oxyuranus scutellatus. P. textilis prothrombin activator is able to coagulate citrated plasma, warfarin plasma, and Factor V- and Factor X-deficient plasmas; to convert purified human prothrombin to thrombin; and to hydrolyse the peptide p-nitroanilide substrate S-2222. Calcium ions and phospholipids had little if any effect on the rates of coagulation of citrated plasma or S-2222 hydrolysis catalysed by this enzyme.
Asunto(s)
Péptido Hidrolasas/aislamiento & purificación , Protrombina/metabolismo , Venenos de Serpiente/análisis , Animales , Australia , Pruebas de Coagulación Sanguínea , Catálisis , Cromatografía en Gel , Concanavalina A , Electroforesis en Gel de Poliacrilamida , Concentración de Iones de Hidrógeno , Peso Molecular , SefarosaRESUMEN
The measurement of crosslinked fibrin derivatives in plasma has received evaluation as a screening test in the diagnosis of venous thrombosis. Plasma samples were taken from 104 patients undergoing venography because of clinical suspicion of lower limb venous thrombosis. The samples were assayed using a monoclonal antibody identifying an epitope on D dimer and larger crosslinked fibrin derivatives in an enzyme immunoassay. 100% of patients with positive venograms had elevated levels of these molecules. While a percentage of patients with negative venograms also had increased levels, alternative clinical explanations were apparent in most. A normal D dimer value excludes the diagnosis of venous thrombosis, while an increased value supports it. The measurement of crosslinked fibrin derivatives in plasma may play a role in the selection of patients for venography.
Asunto(s)
Productos de Degradación de Fibrina-Fibrinógeno/análisis , Fibrina/metabolismo , Tromboflebitis/diagnóstico , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Monoclonales , Especificidad de Anticuerpos , Estudios de Evaluación como Asunto , Femenino , Productos de Degradación de Fibrina-Fibrinógeno/inmunología , Humanos , Masculino , Persona de Mediana Edad , Conformación ProteicaRESUMEN
Conformational and structural changes on conversion of fibrinogen to fibrin and its cross-linking by Factor XIIIa lead to the development of new antigenic determinants that permit differentiation between their plasminolytic cleavage products. A monoclonal antibody (DD-3B6/22) that is specific for cross-linked fibrin derivatives containing the D dimer configuration has been used in developing a latex agglutination procedure that can detect fibrin degradation products in either plasma or serum. Fibrinogen or its degradation products do not cross-react with this antibody. Results were calibrated with an enzyme immunoassay, which used a purified D dimer standard. Plasmas from 40 normal subjects, all having D dimer levels below 250 ng/mL measured by enzyme immunoassay, were all negative by latex assay. In contrast, positive latex agglutination titers were obtained with 87 of 88 patients with demonstrated deep venous thrombosis, pulmonary embolism, or disseminated intravascular coagulation. Compared to enzyme immunoassay, latex agglutination assay is less sensitive, but this latex procedure provides a rapid and less elaborate test for elevated levels of cross-linked fibrin degradation products in patients with thrombosis. Plasma assays for fibrin degradation products are preferable to those using serum.
Asunto(s)
Anticuerpos Monoclonales , Reactivos de Enlaces Cruzados , Factor XIII/análisis , Productos de Degradación de Fibrina-Fibrinógeno/análisis , Humanos , Pruebas de Fijación de Látex , TransglutaminasasRESUMEN
An improved procedure for the purification of fragment D dimer derived from crosslinked plasma fibrin is described which entails chromatofocusing chromatography using PBE 94 and polybuffer 74, and gel chromatography on Sephacryl S-300. The procedure provides a preparation of D dimer which behaves as a single macromolecular entity with molecular weight 190,000 in sedimentation equilibrium studies. Only a single protein band is observed in polyacrylamide gel electrophoresis conducted in the presence or absence of sodium dodecyl sulfate, while patterns characteristic of gamma'-gamma' chains are observed under denaturing conditions after reduction of the preparation with beta-mercaptoethanol. The D dimer contains no demonstrable E antigen by a range of electrophoretic and immunologic techniques. Advantages of this method for obtaining D dimer in high yield include the use of plasma as starting material, the use of a simple lysis regimen in the presence of Ca2+, and the use of simple chromatographic techniques performed under nondenaturing conditions.
Asunto(s)
Productos de Degradación de Fibrina-Fibrinógeno/aislamiento & purificación , Coagulación Sanguínea , Centrifugación por Gradiente de Densidad , Cromatografía/métodos , Electroforesis en Gel de Poliacrilamida , Humanos , Inmunodifusión , Técnicas para Inmunoenzimas , Focalización Isoeléctrica , Peso Molecular , Unión Proteica , Desnaturalización Proteica , SolubilidadRESUMEN
We have devised a simple enzyme immunoassay to detect and quantitate autoantibodies against derivatives of fibrinogen. This assay has been applied with a range of antigens including a fibrinogen lysate (containing X, Y, D and E), D dimer, D dimer-E and a preparation of high molecular weight complexes derived from crosslinked fibrin. We have found that autoantibodies interacting with these antigens can be detected in varying concentrations in most sera from both normal subjects and patients with a variety of diseases and are evidently of mixed Ig class. These autoantibodies are directed against at least several cryptic antigens which appear during fibrinogen/fibrin degradation and some appear to be directed specifically against cross-linked fibrin derivatives. No clear disease correlates have yet emerged but a relationship between elevated levels and prior infective, thrombotic, inflammatory or traumatic disorders is likely. It is suggested that these autoantibodies may contribute to the catabolism of fibrinogen derivatives, provide a marker of thrombosis, and sometimes produce pathologic effects.
Asunto(s)
Autoanticuerpos/análisis , Productos de Degradación de Fibrina-Fibrinógeno/inmunología , Ensayo de Inmunoadsorción Enzimática , Fibrina/inmunología , Humanos , Inmunoglobulinas/análisisRESUMEN
Fibrinogen degradation, fibrin polymerisation, and the insertion of cross links into fibrin by fibrin stabilising factor lead to the appearance of new antigenic determinants. Antibodies against these antigenic sites may react specifically with the derivatives but not with the parent molecules. We have utilised a monoclonal antibody, which interacts with the cross linked fragment D dimer and related high molecular weight fibrin derivatives, to develop an enzyme immunoassay which measures cross linked fibrin derivatives in plasma and serum using D dimer as standard. Mean concentration in plasma from normal subjects was 75 ng/ml with an upper limit of about 144 ng/ml. Concentrations in patients with pulmonary embolism, deep venous thrombosis, arterial thromboembolism, and disseminated intravascular coagulation were raised in all cases. Confirmation of the specific increase of cross linked fibrin derivatives in patients with disseminated intravascular coagulation was obtained by parallel monitoring of their fibrin degradation products in serum using affinity chromatography and sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis. In many patients the plasma concentrations greatly exceeded the serum values of cross linked fibrin degradation products, suggesting that the procedure can measure fibrin derivatives in plasma which are absent from serum.
Asunto(s)
Reactivos de Enlaces Cruzados , Productos de Degradación de Fibrina-Fibrinógeno/análisis , Fibrina/análisis , Anticuerpos Monoclonales , Cromatografía de Afinidad , Coagulación Intravascular Diseminada/sangre , Electroforesis en Gel de Poliacrilamida , Epítopos/análisis , Fibrina/inmunología , Humanos , Embolia Pulmonar/sangre , Trombosis/sangreRESUMEN
A series of experiments to define the lethal potency (LD50) and electrophysiological properties of the venom of the Australian Rough-scaled Snake (Tropidechis carinatus) are described. Crude pooled venom contains at least five fractions which were separated using liquid chromatography and high pressure liquid chromatography techniques (Fractions I-V). LD50 studies are reported using each of these fractions, with data for both adult and neonatal mice. Fraction I (mol. wt greater than 100,000) was essentially non-toxic. Fraction IV (mol. wt less than or equal to 10,000) and Fraction V (mol. wt less than 1,000) were potent toxic components with LD50'S (s.c. injection; fraction in 0.1% bovine serum albumin and 0.85% saline; neonatal mice) of 0.04 mg/kg and 0.06 mg/kg respectively. LD50'S for the whole crude venom were similar in both adult and neonatal mice. Electrophysiological studies using a Bulbring preparation (rat isolated phrenic nerve-hemidiaphragm) indicated that Fractions I, IIa and IIb were inactive. Fraction IV (mol. wt less than or equal to 10,000) caused rapid neuromuscular blockade which appeared to be irreversible. Neurophysiological experiments with a rat isolated extensor digitorum longus muscle preparation suggested that the major toxic activity of the whole venom resides in Fractions III and IV, and that both of these fractions have presynaptic and postsynaptic action.
Asunto(s)
Unión Neuromuscular/efectos de los fármacos , Venenos de Serpiente/farmacología , Animales , Carbacol/farmacología , Cromatografía Líquida de Alta Presión , Electrofisiología , Femenino , Técnicas In Vitro , Dosificación Letal Mediana , Masculino , Ratones , Unión Neuromuscular/fisiología , Ratas , Ratas Endogámicas , Venenos de Serpiente/análisis , Venenos de Serpiente/toxicidad , Transmisión Sináptica/efectos de los fármacosRESUMEN
Monoclonal antibodies (MAb) were raised against human D dimer. The hybridomas were screened with a solid phase enzyme immunoassay against D dimer and fibrinogen degradation products. Among the panel of MAb identified, two distinct patterns emerged; the majority belonging to a panspecific class reacting against epitopes present on both D dimer and fibrinogen degradation product Dcate and a monospecific class reacting with determinants apparently present only on D dimer. A number of MAb were further characterised for their ability to specifically capture antigen in a solid phase enzyme immunoassay and assays were developed which have a sensitivity of 10 ng/ml for D dimer or crosslinked fibrin derivatives and may be suitable for detection of crosslinked derivatives in serum and plasma samples in a clinical situation.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Productos de Degradación de Fibrina-Fibrinógeno/análisis , Animales , Especificidad de Anticuerpos , Reacciones Cruzadas , Coagulación Intravascular Diseminada/diagnóstico , Productos de Degradación de Fibrina-Fibrinógeno/inmunología , Fibrinólisis , Humanos , Técnicas para Inmunoenzimas , RatonesRESUMEN
We have prepared a monoclonal antibody which recognises an antigenic determinant on D dimer, a specific fragment resulting from the degradation of crosslinked fibrin. This antibody has been used in the development of an enzyme-linked immunoassay for D dimer and related degradation products containing crosslinked gamma-gamma chains, to provide a simple assay of circulating crosslinked fibrin degradation products suitable for clinical use. Since these crosslinked fibrin degradation products are characteristic of fibrinolysis, as distinct from fibrinogenolysis, their measurement should aid in the diagnosis, evaluation and monitoring of thrombotic and thrombolytic states. In preliminary studies, low concentrations of crosslinked fibrin derivatives were detected in normal sera. High levels were found in 30/30 patients with disseminated intravascular coagulation and in the majority of patients having deep venous thrombosis or pulmonary embolism.