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British Journal of Cancer Aug 1988Coumarins inhibit metastasis in a number of animal models, but the mechanism of this effect remains unclear. We have investigated the relationship between the...
Coumarins inhibit metastasis in a number of animal models, but the mechanism of this effect remains unclear. We have investigated the relationship between the coagulation system and metastasis using a new model system, involving i.v. injection of Mtln3 rat mammary carcinoma cells into Fischer 344 rats, and subsequent estimation of pulmonary seeding. Injection of factors II, VII, IX and X elevated the median number of surface pulmonary seedlings per animal to 182, and injection of factors II, IX and X to 181, compared with a median for control animals of 12 (P less than 0.001). Injection of factor VII alone, or of bovine serum albumin did not significantly affect pulmonary seeding. In a second experiment, arvin defibrination reduced the mean plasma fibrinogen concentration to 76.8 mg dl-1 from a control value of 228 mg dl-1. This degree of defibrination had no significant effects on pulmonary seeding, nor on the enhancing effects of factor complex injection (median numbers of seedlings per animal; control 15, arvin 21, arvin plus factors II, VII, IX and X 170, factors II, VII, IX and X only, 157). Factor complex injections did not detectably shorten thrombotest clotting times. In vitro testing suggested that Mtln3 cells contain little or no conventional factor X activating cancer procoagulant. The complex of coagulation factors II, IX and X appears to contain a component which greatly enhances metastasis in this model. This may explain the previously reported antimetastatic effect of coumarin anticoagulants, which suppress factors II, VII, IX and X. The enhancing effect of the factor complex does not appear to be altered by significant reductions in fibrin forming capacity, and defibrination itself has no effect on metastasis. These findings suggest the possibility that the effect of this factor complex on metastasis may be mediated via mechanisms other than the formation of a fibrin clot.
Topics: Ancrod; Animals; Anticoagulants; Blood Coagulation Factors; Female; Fibrin; Fibrinogen; Lung Neoplasms; Mammary Neoplasms, Experimental; Neoplasm Metastasis; Rats; Rats, Inbred F344; Tumor Cells, Cultured
PubMed: 3166906
DOI: 10.1038/bjc.1988.184 -
Blood Aug 1983A new case of congenital dysfibrinogenemia, in which the patient has severe thrombotic disease, is reported. The abnormal fibrinogen molecules are characterized by...
A new case of congenital dysfibrinogenemia, in which the patient has severe thrombotic disease, is reported. The abnormal fibrinogen molecules are characterized by normal fibrinopeptide release with thrombin and defective polymerization in the formation of fibrin. Clotting times with ancrod and reptilase are significantly prolonged. All other coagulation tests (except those for fibrinogen function) are normal, and the patient has no other underlying disease. The apparent paradox of defective fibrinogen, which clots abnormally and is yet associated with thrombotic disease, can be explained by further analysis of the patient's fibrinogen. The two important functional properties of this fibrinogen are: (1) it forms fibrin gels that are extremely rigid, and (2) the fibrin is highly resistant to lysis by plasmin. Thus, although the abnormal fibrinogen forms defective clots, the fibrin that is formed cannot be removed by the fibrinolytic system. These results provide a molecular explanation for the thrombotic disease in this patient. This abnormal fibrinogen appears to have unique characteristics and has been designated as fibrinogen Chapel Hill Ill.
Topics: Adolescent; Blood Coagulation Disorders; Coumarins; Dextrans; Fibrinogen; Humans; Leg; Male; Pulmonary Embolism; Thrombophlebitis
PubMed: 6191801
DOI: No ID Found -
Blood Mar 1987Plasma levels of a fast-acting plasminogen activator inhibitor (PAI), which neutralizes both tissue plasminogen activator (t-PA) and urokinase, are markedly increased in...
Plasma levels of a fast-acting plasminogen activator inhibitor (PAI), which neutralizes both tissue plasminogen activator (t-PA) and urokinase, are markedly increased in endotoxin-treated rabbits. The ability of this inhibitor to prevent the fibrinolysis that occurs after a thrombogenic stimulus was investigated in a rabbit model. Normal and endotoxin-treated male New Zealand rabbits were infused with ancrod, an enzyme that causes noncrosslinked fibrin formation in vivo. Ancrod stimulated t-PA activity by 90% in normal rabbits and caused hypofibrinogenemia but did not increase PAI levels or induce fibrin deposition in target organs. Rabbits injected with endotoxin (10 micrograms/kg) showed an increase in PAI from less than 1 to 32 U/mL 4 hours later. When ancrod was infused at this time, 90% of the rabbits developed renal fibrin thrombi. Fibrin deposition was recorded in 40% of the rabbits that received a lower dose of endotoxin (1.0 microgram/kg) and had a PAI level of 14 U/ml at the time of ancrod infusion 4 hours later. Fibrin deposition did not occur in the endotoxin-treated rabbits that received normal saline. These data suggest that high levels of PAI inhibit fibrinolysis in vivo, thereby promoting fibrin clot deposition following a thrombogenic stimulus.
Topics: Ancrod; Animals; Endotoxins; Fibrin; Fibrinolysis; Glycoproteins; Male; Plasminogen Inactivators; Rabbits; Thrombosis; Tissue Plasminogen Activator; Urokinase-Type Plasminogen Activator
PubMed: 3101764
DOI: No ID Found -
The Biochemical Journal Jun 1987Magnetically induced birefringence was used to monitor fibrin polymerization after the release of the small negatively charged A fibrinopeptides from human fibrinogen by...
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.
Topics: Ancrod; Batroxobin; Birefringence; Fibrin; Fibrinogen; Fibrinopeptide A; Humans; Kinetics; Macromolecular Substances; Magnetics; Models, Biological
PubMed: 3446182
DOI: 10.1042/bj2440633 -
Blood Mar 1987Whereas crude bovine thrombin activated single-chain urokinase-type plasminogen activator (scu-PA), otherwise called pro-urokinase (pro-UK), purified human thrombin...
Inactivation of single-chain urokinase (pro-urokinase) by thrombin and thrombin-like enzymes: relevance of the findings to the interpretation of fibrin-binding experiments.
Whereas crude bovine thrombin activated single-chain urokinase-type plasminogen activator (scu-PA), otherwise called pro-urokinase (pro-UK), purified human thrombin converted pro-UK (scu-PA) to a two-chain form that had no amidolytic activity. The two chains (Mr approximately 33,000 and 22,000) were disulfide linked and resistant to subsequent activation by plasmin. By contrast, thrombin did not inactivate tissue plasminogen activator or two-chain urokinase. The enzyme from snake venom Agkistrodon contortrix, relatively specific for fibrinopeptide B, had an effect similar to thrombin, whereas the enzyme from Agkistrodon rhodostoma (ancrod), specific for fibrinopeptide A, did not. When pro-UK (scu-PA) was present during thrombin clotting of fibrinogen, degradation of 125I-pro-UK (scu-PA) in the clot supernatant was seen, whereas virtually full recovery (95%) of radioactivity was found. A loss of latent amidolytic activity in the clot supernatant was also found, the extent of which could be correlated with the degree of degradation of the radiolabeled probe. It was concluded that thrombin inactivation of pro-UK (scu-PA) accounts for the loss of amidolytic activity in the clot supernatant, which has been attributed to fibrin binding. Further confirmation was obtained from experiments in which ancrod was used as the clotting agent. Full recovery of both radioactivity and latent amidolytic activity of pro-UK (scu-PA) in the supernatant was obtained under these conditions. These findings indicate that thrombin may introduce an artifact in the results of certain experiments designed to study the fibrin affinity or fibrinolytic effect of pro-UK (scu-PA).
Topics: Ancrod; Blood Coagulation; Crotalid Venoms; Enzyme Activation; False Positive Reactions; Fibrin; Fibrinogen; Fibrinolysin; Plasminogen Activators; Plasminogen Inactivators; Protein Denaturation; Thrombin; Tissue Plasminogen Activator; Urokinase-Type Plasminogen Activator
PubMed: 2949785
DOI: No ID Found -
Blood Dec 1993A 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...
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).
Topics: Adolescent; Amino Acid Sequence; Ancrod; Female; Fibrinogens, Abnormal; Fibrinopeptide A; Glutamates; Glutamic Acid; Glycine; Humans; Kinetics; Male; Molecular Sequence Data; Point Mutation; Thrombin
PubMed: 7903170
DOI: No ID Found -
Proceedings of the Royal Society of... Apr 1975
Topics: Ancrod; Anticoagulants; Fibrinolysis; Heparin; Humans; Leg; Phlebography; Streptokinase; Thrombophlebitis
PubMed: 1197330
DOI: No ID Found -
British Journal of Experimental... Aug 1974Recipients which have already rejected allografts of skin react rapidly and violently against a subsequently allotransplanted kidney. This is the renal allotransplant...
Recipients which have already rejected allografts of skin react rapidly and violently against a subsequently allotransplanted kidney. This is the renal allotransplant second-set reaction, the primary manifestation of which is a sudden severe afferent vasoconstriction. As a result of this vasomotor disturbance, red blood cells and platelets sometimes aggregate in the glomerular capillaries and the renal tubule cells suffer necrosis. The precipitating cause of the vasomotor disturbance has not been determined but platelet aggregation can be ruled out since in ancrod (Arvin)-treated recipients this does not occur although a second-set reaction develops. No evidence of glomerular basement membrane damage, due either to anti-glomerular basement membrane antibodies or antigen antibody complexes, was detected by the current ultrastructural studies. Since in these experiments the recipients were previously sensitized by skin allografts, a cytotoxic factor which is directed against specific renal components should not be expected to mediate the renal allotransplant second-set reaction. There is, at some structural level, common antigenicity between skin, heart and kidney and, since all three tissues succumb to changes in their vascular systems, it suggests some common antigenic factor in blood vessels. The features of the renal second-set reaction have been compared with other established renal immune disease states but none can compare in rapidity and severity of action with the second-set reaction.
Topics: Antigen-Antibody Complex; Antigen-Antibody Reactions; Anuria; Basement Membrane; Cell Movement; Erythrocytes; Graft Rejection; HLA Antigens; Humans; Immunologic Memory; Kidney; Kidney Glomerulus; Kidney Transplantation; Microscopy, Electron; Necrosis; Platelet Aggregation; Transplantation Immunology; Transplantation, Homologous; Vasomotor System
PubMed: 4611463
DOI: No ID Found -
Clinical and Experimental Immunology Jul 1979Recent studies in experimental crescentic glomerulonephritis, using the technique of glomerular culture, have shown that the macrophage is a major cell type present...
Recent studies in experimental crescentic glomerulonephritis, using the technique of glomerular culture, have shown that the macrophage is a major cell type present within the glomeruli and developing crescents. It has been suggested that their accumulation is a consequence of glomerular fibrin deposition. The effect of defibrination with ancrod on the cellular events occurring in experimental crescentic glomerulonephritis in the rabbit was therefore assessed in this disease using the techniques of culture of isolated glomeruli, electronmicroscopy or renal tissue, and light microscopy. Defibrinated animals developed only minimal renal impairment, virtually no fibrin deposition in Bowman's Space and only a mild degree of crescent formation, in contrast to the severe renal failure, fibrin deposition and crescent formation that occurred in the untreated animals. The culture of isolated glomeruli and electronmicroscopy of intact renal tissue demonstrated large numbers of macrophages within and emerging from glomeruli of both defibrinated and untreated animals. However, only in untreated animals were macrophages seen to migrate into Bowman's Space, phagocytose fibrin, transform into epithelioid cells and accumulate to form crescents. These studies suggest that fibrin deposition in Bowman's Space is the major stimulus to the macrophage migration from capillary loops and accumulation in Bowman's Space. However, fibrin deposition does not appear to be the stimulus to macrophage accumulation within capillary loops as this event was not affected by defibrination.
Topics: Animals; Culture Techniques; Female; Fibrin; Glomerulonephritis; Kidney Glomerulus; Macrophages; Microscopy, Electron; Rabbits
PubMed: 487657
DOI: No ID Found -
European Journal of Biochemistry May 1992The carbohydrate side chains of the thrombin-like serine protease ancrod from the venom of the Malayan pit viper Agkistrodon rhodostoma were liberated from tryptic...
Carbohydrate structure of a thrombin-like serine protease from Agkistrodon rhodostoma. Structure elucidation of oligosaccharides by methylation analysis, liquid secondary-ion mass spectrometry and proton magnetic resonance.
The carbohydrate side chains of the thrombin-like serine protease ancrod from the venom of the Malayan pit viper Agkistrodon rhodostoma were liberated from tryptic glycopeptides by treatment with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F and fractionated by high-performance liquid chromatography. Glycans obtained were characterized by digestion with exoglycosidases, methylation analysis and, in part, by liquid secondary-ion mass spectrometry and 1H-NMR spectroscopy. The results reveal that this snake venom glycoprotein contains partially truncated di-, tri- and tetraantennary complex type N-glycans carrying Fuc(alpha 1-6) residues at the innermost N-acetylglucosamine and solely (alpha 2-3)-linked sialic acid substituents. As a characteristic feature, ancrod oligosaccharides comprise mainly sialylated Gal beta 3GlcNAc beta lactosamine antennae. Furthermore, a small proportion of the sugar chains were found to carry a NeuAc alpha 3GalNAc beta 4GlcNAc beta antenna exclusively linked to C-2 of Man(alpha 1-3) residues of the pentasaccharide core. Thus, many of the glycans found represent novel glycoprotein-N-glycan structures.
Topics: Carbohydrate Sequence; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Electrophoresis, Polyacrylamide Gel; Magnetic Resonance Spectroscopy; Mass Spectrometry; Methylation; Molecular Sequence Data; Oligosaccharides; Protons; Serine Endopeptidases; Thrombin; Viper Venoms
PubMed: 1315684
DOI: 10.1111/j.1432-1033.1992.tb16863.x