RESUMEN

Ancrod, a serine protease purified from the venom of Agkistrodon rhodostoma, is highly specific for fibrinogen. It causes anticoagulation by defibrinogenation and has been used as a therapeutic anticoagulant for the treatment of moderate to severe forms of peripheral arterial circulatory disorders in a variety of countries. The DNA of ancrod was amplified by recursive PCR with a yeast bias codon and cloned into the pGEM-T Easy vector. In order to achieve a high level secretion and a full activity expression of ancrod in Pichia pastoris (P. pastoris), the P. pastoris protein disulfide bond isomerase (PpPDI) was co-overexpressed in the strain. The secretion characteristics of ancrod with and without PpPDI were examined. With co-overexpression of PpPDI, the production of recombinant ancrod (rAncrod) was increased to 315 mg/L in the culture medium, which is twofold higher than the control strain carrying only the ancrod gene. Through purified by Ni²âº affinity chromatography and phenyl Sepharose column, the purity of rAncrod was found to be as high as 95.2%. The fibrinogenolytic and zymographic activities of the rAncrod were determined and found to be similar to that of the native protein. This improved expression system can facilitate further studies and the industrial production of ancrod.

Asunto(s)

Agkistrodon/metabolismo , Ancrod/metabolismo , Venenos de Crotálidos/enzimología , Proteínas Fúngicas/metabolismo , Pichia/enzimología , Proteína Disulfuro Isomerasas/metabolismo , Proteínas Recombinantes/metabolismo , Serina Endopeptidasas/metabolismo , Ancrod/química , Ancrod/genética , Animales , Anticoagulantes/metabolismo , Western Blotting , Cromatografía de Afinidad , Venenos de Crotálidos/química , Venenos de Crotálidos/genética , Electroforesis en Gel de Poliacrilamida , Fibrinógeno/metabolismo , Proteínas Fúngicas/genética , Expresión Génica , Vectores Genéticos , Pichia/genética , Reacción en Cadena de la Polimerasa , Proteína Disulfuro Isomerasas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Serina Endopeptidasas/química , Serina Endopeptidasas/genética

RESUMEN

The relationship between microstructural features and macroscopic mechanical properties of engineered tissues was investigated in pure and mixed composite scaffolds consisting of collagen Type I and fibrin proteins containing embedded smooth muscle cells. In order to vary the matrix microstructure, fibrin polymerization in mixed constructs was initiated using either the blood-derived enzyme thrombin or the snake venom-derived enzyme ancrod, each at low and high concentrations. Microstructural features of the matrix were quantified by analysis of high resolution scanning electron micrographs. Mechanical properties of the scaffolds were assessed by uniaxial tensile testing as well as creep testing. Viscoelastic parameters were determined by fitting creep data to Burger's four-parameter model. Oscillatory dynamic mechanical testing was used to determine the storage modulus, loss modulus, and phase shift of each matrix type. Mixed composite scaffolds exhibited improved tensile stiffness and strength, relative to pure collagen matrices, as well as decreased deformation and slower relaxation in creep tests. Storage and loss moduli were increased in mixed composites compared with pure collagen, while phase shift was reduced. A correlation analysis showed that the number of fiber bundles per unit volume was positively correlated with matrix modulus, strength, and dynamic moduli, though this parameter was negatively correlated with phase shift. Fiber diameter also was negatively correlated with scaffold strength. This study demonstrates how microstructural features can be related to the mechanical function of protein matrices and provides insight into structure-function relationships in such materials. This information can be used to identify and promote desirable microstructural features when designing biomaterials and engineered tissues.

Asunto(s)

Ancrod/metabolismo , Colágeno Tipo I/química , Matriz Extracelular/química , Fibrina/metabolismo , Resistencia a la Tracción/fisiología , Animales , Aorta , Materiales Biocompatibles/química , Células Cultivadas , Colágeno Tipo I/fisiología , Matriz Extracelular/fisiología , Matriz Extracelular/ultraestructura , Fibrinógeno/química , Fibrinógeno/fisiología , Miocitos del Músculo Liso/fisiología , Ratas , Ingeniería de Tejidos

RESUMEN

Protein C activation initiated by the thrombin-thrombomodulin complex forms the major physiological anticoagulant pathway. Agkistrodon contortrix contortrix protein C activator, a glycosylated single-chain serine proteinase, activates protein C without relying on thrombomodulin. The crystal structures of native and inhibited Agkistrodon contortrix contortrix protein C activator determined at 1.65 and 1.54 A resolutions, respectively, indicate the pivotal roles played by the positively charged belt and the strategic positioning of the three carbohydrate moieties surrounding the catalytic site in protein C recognition, binding, and activation. Structural changes in the benzamidine-inhibited enzyme suggest a probable function in allosteric regulation for the anion-binding site located in the C-terminal extension, which is fully conserved in snake venom serine proteinases, that preferentially binds Cl(1-) instead of SO(4)(2-).

Asunto(s)

Ancrod/química , Ancrod/metabolismo , Venenos de Crotálidos/química , Venenos de Crotálidos/metabolismo , Péptidos/química , Péptidos/metabolismo , Proteína C/metabolismo , Agkistrodon/genética , Agkistrodon/metabolismo , Regulación Alostérica , Secuencia de Aminoácidos , Ancrod/antagonistas & inhibidores , Ancrod/genética , Animales , Benzamidinas/farmacología , Dominio Catalítico , Venenos de Crotálidos/antagonistas & inhibidores , Venenos de Crotálidos/genética , Cristalografía por Rayos X , Hemostasis , Técnicas In Vitro , Péptidos y Proteínas de Señalización Intercelular , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/antagonistas & inhibidores , Péptidos/genética , Conformación Proteica , Homología de Secuencia de Aminoácido , Inhibidores de Serina Proteinasa/farmacología , Electricidad Estática , Trombomodulina/metabolismo

RESUMEN

In brain physiology, cerebrovascular interactions regulate both, vascular functions, such as blood vessel branching and endothelial cell homeostasis, as well as neuronal functions, such as local synaptic activity and adult neurogenesis. In brain pathology, including stroke, HIV encephalitis, Alzheimer Disease, multiple sclerosis, bacterial meningitis, and glioblastomas, rupture of the vasculature allows the entry of blood proteins into the brain with subsequent edema formation and neuronal damage. Fibrin is a blood-derived protein that is not produced by cells of the nervous system, but accumulates only after disease associated with vasculature rupture. This review presents evidence from human disease and animal models that highlight the role of fibrin in nervous system pathology. Our review presents novel experimental data that extend the role of fibrin, from that of a blood-clotting protein in cerebrovascular pathologies, to a component of the perivascular extracellular matrix that regulates inflammatory and regenerative cellular responses in neurodegenerative diseases.

Asunto(s)

Barrera Hematoencefálica , Fibrina/metabolismo , Sistema Nervioso , Secuencia de Aminoácidos , Ancrod/genética , Ancrod/metabolismo , Animales , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/patología , Barrera Hematoencefálica/ultraestructura , Fibrinógeno/metabolismo , Fibrinolíticos/metabolismo , Homeostasis , Humanos , Inflamación/metabolismo , Inflamación/patología , Macrófagos/metabolismo , Datos de Secuencia Molecular , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/patología , Vaina de Mielina/metabolismo , Regeneración Nerviosa/fisiología , Sistema Nervioso/irrigación sanguínea , Sistema Nervioso/metabolismo , Sistema Nervioso/patología , Neuroglía/citología , Neuroglía/metabolismo , Neuronas/citología , Neuronas/metabolismo , Transducción de Señal/fisiología

RESUMEN

The thrombin-like serine protease ancrod from the Malayan pit viper Agkistrodon rhodostoma was expressed in mouse epithelial cells (C127). Oligosaccharide constituents were liberated from tryptic glycopeptides by treatment with peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase F. Neutral oligosaccharide alditols obtained after reduction and enzymic desialylation were separated by two-dimensional HPLC and characterized by methylation analysis, liquid secondary-ion mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and sequential degradation with exoglycosidases. In contrast to natural ancrod, the recombinant glycoprotein carries exclusively diantennary, triantennary and tetraantennary N-glycans with Gal beta 4 GlcNAc beta (type-2) antennae which were, in part, further substituted by host-cell-specific structural elements such as Gal alpha 3 residues or N-acetyllactosamine repeats. As a characteristic feature, a substantial proportion of the oligosaccharides bears a GalNAc beta 4Glc-NAc antenna. Studies at the level of individual N-glycosylation sites demonstrated that glycans with N, N'-diacetyllactosediamine units are not specifically attached but occur at all sites in varying amounts. Hence, the putative recognition signal (Pro70-Lys-Lys) for glycoprotein hormone N-acetylgalactosaminyltransferase, present in this glycoprotein in close proximity to Asn79, does not convey site-specific transfer of GalNAc residues in these cells.

Asunto(s)

Agkistrodon/metabolismo , Ancrod/metabolismo , Ancrod/química , Ancrod/genética , Animales , Secuencia de Carbohidratos , Carbohidratos/análisis , Línea Celular , Clonación Molecular , Glicosilación , Ratones , Datos de Secuencia Molecular , Polisacáridos/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción

RESUMEN

The mechanism of morphologic change of human cultured umbilical vein endothelial cells (HUVECs) caused by fibrin was investigated. Ancrod, a thrombin-like enzyme, did not cause morphologic alteration of HUVEC by itself at concentrations ranging from 0.01 to 10 U/ml. However, when 0.02 U/ml of ancrod was added to cultured HUVEC monolayers in the presence of citrated plasma, it caused pronounced morphologic change of HUVEC after 6-10 h incubation period. Gly-Pro-Arg-Pro (4 mg/ml), an inhibitor of fibrin polymerization, prevented the morphologic alteration, indicating that the morphologic alteration was caused by the polymerized fibrin. The morphologic change of HUVEC caused by ancrod-generated fibrin was not observed in the presence of an intracellular calcium mobilization inhibitor TMB-8 (50 microM), and the morphologic alteration was also less pronounced with BAPTA(15 microM)-loaded HUVECs and HUVECs pretreated with EGTA (1.2 mM). Ancrod (in Medium 199) itself did not stimulate phosphoinositide breakdown of HUVEC. However, when ancrod was present in plasma, it caused an increase of [3H]IP1 of HUVECs preloaded with [3H]myoinositol. This IP1 increment was inhibited by Gly-Pro-Arg-Pro. The increase of IP1 was significantly inhibited by the pretreatment of monoclonal antibodies 23C6 and 7E3 directed against alpha v beta 3 integrin. Neomycin (1 mM) and pertussis toxin (100 ng/ml), but not aspirin or mepacrine, blocked this enhanced phosphoinositide breakdown. The morphologic change was also prevented by the monoclonal antibodies, 23C6 and 7E3. These results suggest that both intra- and extra-cellular calcium participate in the event of morphologic change of HUVEC caused by ancrod-generated fibrin, and the morphologic change is mediated, at least in part, by fibrin binding to integrin alpha v beta 3 on HUVECs, causing the subsequent activation of the endogenous G-protein coupled phospholipase C.

Asunto(s)

Ancrod/metabolismo , Endotelio Vascular/fisiología , Fibrina/metabolismo , Receptores de Vitronectina/metabolismo , Transducción de Señal , Calcio/farmacología , Adhesión Celular , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Proteínas de Unión al GTP/metabolismo , Humanos , Fosfatos de Inositol/análisis , L-Lactato Deshidrogenasa/análisis , Morfogénesis/efectos de los fármacos , Toxina del Pertussis , Fosfatidilinositoles/metabolismo , Fosfolipasas de Tipo C/metabolismo , Factores de Virulencia de Bordetella/farmacología

RESUMEN

Ancrod, a thrombin-like enzyme, has been used as defibrinogenating agent in prevention of venous thrombosis. This study showed ancrod in citrated plasma elevated 6-keto PGF1 alpha production of human umbilical vein endothelial cells; this increment of 6-keto PGF1 alpha was completely inhibited by Gly-Pro-Arg-Pro, an inhibitor of fibrin polymerization. The enhanced prostacyclin production, but not basal level of prostacyclin, was inhibited by actinomycin D and cycloheximide. In washed aspirin-pretreated cells, ancrod-formed fibrin induced restoration of prostacyclin production by a cycloheximide- and actinomycin D-sensitive process. Ancrod-formed fibrin stimulated synthesis of cyclooxygenase as probed by Western blotting and this enhancement was blocked by actinomycin D and cycloheximide. In conclusion, we first report that ancrod-formed fibrin stimulates prostacyclin production of human endothelial cells and this event is dependent on de novo synthesis of cyclooxygenase.

Asunto(s)

Ancrod/metabolismo , Endotelio Vascular/metabolismo , Epoprostenol/biosíntesis , Fibrina/farmacología , Prostaglandina-Endoperóxido Sintasas/biosíntesis , 6-Cetoprostaglandina F1 alfa/biosíntesis , Aspirina/farmacología , Western Blotting , Células Cultivadas , Cicloheximida/farmacología , Dactinomicina/farmacología , Endotelio Vascular/efectos de los fármacos , Fibrina/biosíntesis , Humanos , Cinética , Oligopéptidos/farmacología , Venas Umbilicales

RESUMEN

A new type of A alpha Glu-11 to Gly substitution has been identified in a congenitally abnormal fibrinogen, fibrinogen Mitaka II, derived from a 14-year-old female suffering from easy bruising since childhood. Plasma of the patient and fibrinogen purified therefrom were found to clot slowly by thrombin but in a normal fashion by ancrod, a thrombin-like snake venom enzyme. The ancrod-clotted fibrin gels were normally solid and turbid, whereas the thrombin-clotted gels were initially fragile and transparent but became gradually normalized during further incubation. On reverse-phase high-performance liquid chromatography, there was an additional peptide group eluted distinctly later than the corresponding normal fibrinopeptide A in the clot-liquor of the patient's samples. Sequence analysis of these aberrant peptides and isolated A alpha chains of the patient's fibrinogen showed that Glu at position 11 of the abnormal A alpha chain had been replaced by Gly. Studies using 125I-labeled thrombin showed that the binding with thrombin was evidently reduced for her fibrinogen and the aberrant fibrinopeptide A as compared with that for the normal controls, indicating that A alpha Glu-11 may be critical for the fibrinogen-thrombin interaction. Indeed, A alpha Glu-11 of fibrinogen has recently been proposed to stabilize the local conformation, including the beta-turn, and to form a salt bridge between its side-chain carboxyl group and the guanidino group of Arg-173 of thrombin based on crystallographic analyses using analogs of fibrinopeptide A complexed with thrombin (Stubb et al, Eur J Biochem 206:187, 1992 and Martin et al, J Biol Chem 267:7911, 1992).

Asunto(s)

Fibrinógenos Anormales/genética , Fibrinógenos Anormales/metabolismo , Glutamatos , Glicina , Mutación Puntual , Trombina/metabolismo , Adolescente , Secuencia de Aminoácidos , Ancrod/metabolismo , Femenino , Fibrinopéptido A/química , Fibrinopéptido A/aislamiento & purificación , Ácido Glutámico , Humanos , Cinética , Masculino , Datos de Secuencia Molecular

RESUMEN

A titration method for determination of trypsin-like serine proteinase concentration has been developed by using ZArgONp and ZLysONp, two specific chromogenic amino-acid derivatives which show the characteristics of optimal active-site titrants. Active proteinase concentration has been estimated from the effect of titrant concentration on the amplitude, at time zero, of the time-course for the instantaneous release of p-nitrophenol, preceding the steady-state reaction (burst phase).

Asunto(s)

Arginina/análogos & derivados , Lisina/análogos & derivados , Serina Endopeptidasas/metabolismo , Ancrod/metabolismo , Animales , Arginina/metabolismo , Sitios de Unión , Humanos , Calicreínas/metabolismo , Cinética , Lisina/metabolismo , Espectrofotometría , Trombina/metabolismo , Tripsina/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo

RESUMEN

Magnetically induced birefringence was used to monitor fibrin polymerization after the release of the small negatively charged A fibrinopeptides from human fibrinogen by the action of the snake-venom-derived enzymes reptilase and ancrod. A range of conditions was investigated. Fibrin polymerization in solutions of purified fibrinogen shows a distinct break near the gelation point. On addition of Ca2+ or albumin the lag period is shortened, fibre thickness is increased and the break in assembly almost vanishes, probably because both of these additives promote lateral aggregation. There are minor differences in the kinetics, depending on the venom enzyme used. The kinetics of fibrin assembly in model systems containing either Ca2+ or albumin and in human plasma with a largely dormant coagulation cascade are very similar. Therefore in the latter condition there is no significant alteration in the assembly process due to interaction between fibrin or the venom enzymes and any of the plasma proteins. When the cascade is activated, the polymerization progress curves have a character that resembles a combination of the reactions observed when the venom enzymes and endogenously generated thrombin separately induce coagulation, except for a region near gelation where, paradoxically, polymerization appears to be slower on activation. The low-angle neutron-diffraction patterns from oriented gels made with thrombin or reptilase are identical. Therefore at low resolution the packing of the monomers within fibres is the same when fibrinopeptide A only or both fibrinopeptides A and B are removed.

Asunto(s)

Fibrina , Fibrinógeno/metabolismo , Fibrinopéptido A/metabolismo , Ancrod/metabolismo , Batroxobina/metabolismo , Birrefringencia , Fibrinopéptido A/sangre , Humanos , Cinética , Sustancias Macromoleculares , Magnetismo , Modelos Biológicos

RESUMEN

Values of steady-state and pre-steady-state parameters for the hydrolysis of ZArgONp and ZLysONp catalysed by ancrod, the coagulating serine proteinase from the Malayan pit viper (Agkistrodon rhodostoma) venom, have been determined, between pH 2.5 and 8 (I = 0.1 M) at 21 +/- 0.5 degrees C, and analysed in parallel with those of bovine alpha-thrombin and porcine pancreatic beta-kallikrein-B. In addition to the well-known coagulating behaviour, ancrod also shows catalytic properties, in the hydrolysis of ZArgONp and ZLysONp, reminiscent of those of porcine pancreatic beta-kallikrein-B.

Asunto(s)

Ancrod/metabolismo , Venenos de Crotálidos/análisis , Arginina/análogos & derivados , Arginina/metabolismo , Concentración de Iones de Hidrógeno , Calicreínas/metabolismo , Cinética , Lisina/análogos & derivados , Lisina/metabolismo , Especificidad por Sustrato , Trombina/metabolismo

RESUMEN

Kinetics for the hydrolysis of p-nitrophenyl esters of N-alpha-carbobenzoxy-L-amino acids catalyzed by Ancrod were determined between pH 5 and 10 (I = 0.1 M) at 21 +/- 0.5 degrees C; the results are consistent with the minimum three-step mechanism: (formula: see text) For all substrates examined, the pH profiles of kcat and/or kcat/Km reflect the ionization of two groups with pKa values ranging between 6.9 and 7.2, and 9.3 and 9.6 (probably, the histidine residue involved in the catalytic triad and the N-terminus, respectively); at variance, values of Km are pH-independent. Moreover, the formation of the E X S complexes may be regarded as a pseudo-equilibrium process, and the acylation step (k + 2) is always rate-limiting in catalysis. Among p-nitrophenyl esters examined, ZArgONp shows the most favourable kinetic parameters and may be the substrate of choice for Ancrod, in that it allows the determination of the enzyme concentration as low as 1 X 10(-9) M (approximately equal to 0.1 Ancrod units/ml), at the optimum pH value (approximately equal to 8). The catalytic behaviour of Ancrod is compared to that of serine proteinases acting on cationic and non-cationic substrates; differences in kinetics, which refer to a lower enzyme:substrate affinity, may be related to a higher rigidity, lower hydrophobicity and/or adverse steric hindrance of the S1 subsite of Ancrod.

Asunto(s)

Ancrod/metabolismo , Aminoácidos/metabolismo , Animales , Compuestos de Bencilo/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Lisina/análogos & derivados , Lisina/metabolismo , Matemática , Nitrofenoles/metabolismo , Trombina/metabolismo

RESUMEN

Most plasma fibrinogen molecules, when analyzed by Na Dod SO4 polyacrylamide gel electrophoresis migrate in three major positions, designated bands I, II, and III, respectively. Band I reflects the presence of intact molecules, whereas bands II and III represent molecules containing catabolic Aalpha chain fragments. In order to establish the Aalpha chain composition of band II molecules, we analyzed plasma fraction I-6 which consists amost exclusively of material migrating in this band position. Analyses of ancrod- and of reptilase-treated samples by Na Dod SO4 polyacrylamide gel electrophoresis and gel scanning densitometry permitted assessment of the Aalpha chain composition of fraction I-6. We found that an Aalpha chain derivative, termed Aalpha/4, comprises more than 70% of the Aalpha chain population of I-6. Consistent with this finding and with studies showing that band II molecules were the most abundant species of catabolic derivatives in plasma, Aalpha/4 was shown to be the most abundant of the Aalpha chain (core) fragments in blood.

Asunto(s)

Fibrinógeno/química , Ancrod/metabolismo , Batroxobina/metabolismo , Biomarcadores/sangre , Densitometría , Electroforesis en Gel de Poliacrilamida , Fibrina/metabolismo , Fibrinógeno/aislamiento & purificación , Fibrinógeno/metabolismo , Humanos , Hidrólisis , Cinética , Peso Molecular , Multimerización de Proteína , Trombina/metabolismo

RESUMEN

A patient with a functionally defective fibrinogen (fibrinogen Chapel Hill) has been investigated. Fibrinogen Chapel Hill is characterized by hypofibrinogenemia, with a plasma concentration about one third of normal, as measured both functionally and immunochemically. Fibrinogen survival is normal; so also is fibrinopeptide release. A polymerization defect in this fibrinogen results in the delay of fibrin fibrils in solution to form a normal three-dimensional gel. This defect is not associated with end-to-end aggregation or with lateral associations in solution. Delayed gelation results from an abnormality in a tertiary contact site involved in network branching, which is associated with the hydrophilic, carboxy-terminal segment of the alpha chain. Fibrinogen Chapel Hill exhibits two additional abnormal responses, which are also associated with the same region. The early plasmin cleavages of fibrinogen and fragment X are delayed, and there is a concomitant delay in the appearance of fragments Y, D, and E. This fibrinogen also has an unusual sensitivity to Ancrod proteolysis, whereby Ancrod cleaves a large carboxy-terminal segment of the alpha chain more rapidly than in normal fibrinogen. The abnormalities in fibrinogen Chapel Hill can be explained by a structural abnormality which is functionally related to an alpha chain associated polymerization domain.

Asunto(s)

Afibrinogenemia/genética , Trastornos de la Coagulación Sanguínea/genética , Anomalías Congénitas , Fibrinógeno/genética , Adulto , Ancrod/metabolismo , Electroforesis , Femenino , Fibrinógeno/aislamiento & purificación , Fibrinógeno/metabolismo , Humanos

Asunto(s)

Trastornos de la Coagulación Sanguínea/congénito , Fibrinógeno/aislamiento & purificación , Ancrod/metabolismo , Batroxobina/metabolismo , Pruebas de Coagulación Sanguínea , Electroforesis de las Proteínas Sanguíneas , Niño , Endopeptidasas/metabolismo , Femenino , Productos de Degradación de Fibrina-Fibrinógeno/análisis , Fibrinógeno/análisis , Fibrinopéptido A/metabolismo , Humanos , Inmunoelectroforesis , Relación Estructura-Actividad , Trombina/metabolismo

RESUMEN

The action of ancrod on fibrinogen and prothrombin metabolism was studied in six healthy individuals by the use of 131I-fibrinogen and 125I-prothrombin and by measurement of blood levels of fibrinopeptide A. Two untreated healthy controls were studied at the same time. Rapid defibrinogenation occurred during the initial 3 hr ancrod infusion, and fibrinogen levels were maintained near zero throughout the study. Large quantities of non-thrombin-clottable TCA-precipitable 131I material could be demonstrated in the circulation, reaching a maximum 3 to 6 hr after ancrod infusion and clearing with a half-life of 6 hr. Gel filtration of 6 hr plasmas demonstrated the presence of complexes larger than fibrinogen, as well as degradation products of fibrinogen-fibrin. Prothrombin concentration and metabolism were unchanged by ancrod treatment. Fibrinopeptide A levels in the ancrod group were greather than 4,000 ng/ml during the initial defibrinogenation, declined to greater than 80 ng/ml, and then increased to high levels after 3 days. These studies provide explanations of previous observations concerning the specificity of ancrod and demonstrate that rapid clotting of fibrinogen and dissolution of fibrin can occur in vivo without recruitment of the classic coagulation mechanism.

Asunto(s)

Ancrod/farmacología , Fibrinógeno/metabolismo , Protrombina/metabolismo , Adulto , Ancrod/metabolismo , Coagulación Sanguínea/efectos de los fármacos , Cromatografía en Gel , Productos de Degradación de Fibrina-Fibrinógeno/análisis , Fibrinopéptido A/metabolismo , Semivida , Humanos , Radioisótopos de Yodo , Masculino

RESUMEN

The snake venom enzymes Ancrod and Batroxobin marajoensis are able to activate human plasma factor XIII as shown by the formation of the gamma-dimers. The concentration of gamma-dimers increases with the concentration of the activating enzymes. Factor XIII activated by Ancrod or Batroxobin marajoensis is, however, unable to catalyse the incorporation of the amine dansyl-cadaverine into casein. The partially activated factor XIII is therefore not demonstrable by means of the artificial test system. This factor XIII loses little activity and remains activable by thrombin.

Asunto(s)

Ancrod/metabolismo , Endopeptidasas/metabolismo , Factor XIII/metabolismo , Venenos de Serpiente/metabolismo , Coagulación Sanguínea , Cadaverina/metabolismo , Caseínas/metabolismo , Compuestos de Dansilo/metabolismo , Activación Enzimática , Fibrina/metabolismo , Humanos , Hidrólisis , Trombina/metabolismo

RESUMEN

The gelation time, opacity, light scattering, and elastic moduli of human fibrin gels clotted in the presence of thrombin, Ancrod, and Reptilase have been compared. At low ionic strength lateral association to thick fibers is observed in all cases. At all ionic strengths thrombin fibrin forms thicker fibers than does Ancrod fibrin. We have demonstrated that an increase in the extent of lateral association is linked to an increase in its velocity and to a decrease in the gelation time. One may consider the removal of fibrinopeptide B to act as a switch: after it is removed fibrin assembles rapidly to thick fibers and gelation is fast; but when this peptide is still attached, there is a slow assembly of thin fibers, and gelation, especially of dilute fibrin, is delayed. We believe that this delay is critical for the complete digestion by plasmin of fibrin formed during in vivo defibrination with Ancrod and of fibrin produced by very small amounts of thrombin (which would still contain fibrinopeptide B), and that slow release of fibrinopeptide B is part of a control mechanism for the regulation of fibrin formation and the prevention of intravascular coagulation.

Asunto(s)

Ancrod/metabolismo , Batroxobina/metabolismo , Endopeptidasas/metabolismo , Fibrina , Fibrinógeno , Fibrinopéptido B , Péptido Hidrolasas/metabolismo , Trombina/metabolismo , Calcio/farmacología , Elasticidad , Fibrinógeno/metabolismo , Fibrinopéptido B/metabolismo , Humanos , Cinética , Luz , Nefelometría y Turbidimetría , Concentración Osmolar , Conformación Proteica , Dispersión de Radiación

RESUMEN

Plasma from patients with both acute and chronic liver disease has been examined for evidence of acquired dysfibrinogenaemia, using electrophoretic methods and coagulation tests. An examination of isolated fibrins upon SDS polyacryamide gel electrophoresis failed to demonstrate any molecular or structural defect associated with the polypeptide chains of the patients' fibrinogen or fibrinogen derivatives produced by thrombin or plasmin. However, purified fibrin monomers isolated from plasma using both Reptilase and thrombin exhibited delayed polymerization rates and the occurrence of acquired dysfibrinogenaemia in liver disease is therefore confirmed.

Asunto(s)

Trastornos de la Coagulación Sanguínea/etiología , Fibrina/metabolismo , Fibrinógeno/metabolismo , Hepatopatías/complicaciones , Enfermedad Aguda , Ancrod/metabolismo , Batroxobina/metabolismo , Pruebas de Coagulación Sanguínea , Enfermedad Crónica , Humanos , Trombina/metabolismo

Asunto(s)

Ancrod , Endopeptidasas , Aminoácidos/análisis , Ancrod/aislamiento & purificación , Ancrod/metabolismo , Animales , Coagulación Sanguínea , Carbohidratos/análisis , Cromatografía de Afinidad , Endopeptidasas/aislamiento & purificación , Esterasas/aislamiento & purificación , Esterasas/metabolismo , Métodos , Venenos de Serpiente