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Kidney International Aug 2004Tissue factor initiated glomerular fibrin deposition is an important mediator of injury in crescentic glomerulonephritis. Recent data have suggested noncoagulant roles...
BACKGROUND
Tissue factor initiated glomerular fibrin deposition is an important mediator of injury in crescentic glomerulonephritis. Recent data have suggested noncoagulant roles for tissue factor in inflammation.
METHODS
To test the hypothesis that in addition to its effects in initiating coagulation, tissue factor has proinflammatory effects in glomerulonephritis, rabbits given crescentic anti-glomerular basement membrane (GBM) antibody-induced glomerulonephritis were defibrinogenated with ancrod. One group of defibrinogenated rabbits was also given anti-tissue factor antibodies. Comparisons were made between these groups, as well as a third group that was neither defibrinogenated with ancrod nor given anti-tissue factor antibodies.
RESULTS
Defibrinogenation alone abolished glomerular fibrin deposition, reduced crescent formation, and limited renal impairment (ancrod-treated, serum creatinine 274 +/- 37 micromol/L; untreated 415 +/- 51 micromol/L; P < 0.01). Tissue factor inhibition in defibrinogenated rabbits resulted in further protection of renal function (creatinine 140 +/- 19 micromol/L, P < 0.01) and reduced proteinuria (0.4 +/- 0.2g/day, untreated 2.6 +/- 0.4 g/day, P <0.01), which was significantly increased by defibrinogenation alone (ancrod-treated, 5.6 +/- 1.2 g/day). Anti-tissue factor antibodies (but not defibrinogenation alone) attenuated glomerular T-cell and macrophage recruitment, and major histocompatibility complex (MHC) class II expression.
CONCLUSION
These results demonstrate important proinflammatory effects of tissue factor in crescentic glomerulonephritis that are fibrin independent and provide in vivo evidence for tissue factor's proinflammatory effects on MHC class II expression and leukocyte accumulation.
Topics: Ancrod; Animals; Anti-Glomerular Basement Membrane Disease; Antibodies; Anticoagulants; Fibrin; Fibrinogen; Histocompatibility Antigens Class II; Kidney Glomerulus; Male; Rabbits; Thromboplastin
PubMed: 15253718
DOI: 10.1111/j.1523-1755.2004.00785.x -
Journal of Cellular and Molecular... Sep 2009This study has used immunohistochemical examination of tissue obtained from Alzheimer's disease (AD) brains and rat hippocampus injected with Abeta(1-42) peptide to...
This study has used immunohistochemical examination of tissue obtained from Alzheimer's disease (AD) brains and rat hippocampus injected with Abeta(1-42) peptide to determine effects of induced inflammatory reactivity on integrity of blood-brain barrier (BBB) and viability of neurons. Tissue from AD, but not non-demented, brains exhibited a diffuse pattern of staining for fibrinogen and immunoglobulin (IgG) indicative of BBB leakiness with considerable fibrinogen immunoreactivity (ir) appearing in association with Abeta deposits. Immunostaining for the endothelial cell specific glycoprotein, von Willebrand factor, showed morphological evidence for altered blood vessels in AD tissue. AD brains also demonstrated extensive areas of fibrinogen ir in association with microglial reactivity. In vivo, intra-hippocampal injection of Abeta(1-42) caused time-dependent (1-7 days after injection) increases in double staining of fibrinogen with areas of microgliosis. Two independent pharmacological strategies were employed to examine how Abeta(1-42) stimulation (7 days injection) may be linked to neurodegeneration. The defibrinogenating compound, ancrod, reduced inflammatory reactivity, levels of parenchymal fibrinogen and IgG, and was neuroprotective. These results prompted use of Abeta(1-42) plus fibrinogen as a novel in vivo inflammatory stimulus and this combination significantly enhanced inflammatory reactivity, vascular perturbations and neuronal damage compared to Abeta(1-42) alone. A second approach, using anti-Mac-1 (antibody for antigen CD11b) to block activation of microglia, was highly effective in attenuating effects of Abeta(1-42) plus fibrinogen amplification of inflammatory and vascular responses and conferred significant neuroprotection. The overall findings from study of AD tissue and in vivo in Abeta(1-42) and Abeta(1-42) plus fibrinogen stimulated rat hippocampus suggest microglial responses to promote increased extravasation of blood protein as a critical component in amplifying inflammatory reactivity and causing neuronal damage in inflamed AD brain.
Topics: Aged; Aged, 80 and over; Alzheimer Disease; Amyloid beta-Peptides; Ancrod; Animals; Astrocytes; Blood Vessels; Blood-Brain Barrier; Brain; Dementia; Fibrinogen; Fluorescence; Humans; Inflammation; Macrophage-1 Antigen; Male; Microglia; Middle Aged; Peptide Fragments; Permeability; Rats; Rats, Sprague-Dawley; von Willebrand Factor
PubMed: 18657226
DOI: 10.1111/j.1582-4934.2008.00434.x -
The Journal of Biological Chemistry Aug 1975The two stages in the activation of human plasminogen by urokinase have been examined kinetically in order to evaluate the significance of each stage in the activation...
The two stages in the activation of human plasminogen by urokinase have been examined kinetically in order to evaluate the significance of each stage in the activation process. The cleavage of the preactivation peptide from the NH2 terminus of native plasminogen (NH2-terminal glutamic acid) is clearly catalyzed by urokinase and is the rate-limiting first step in activation (Stage 1); this reaction is 20-fold slower than the conversion of the intermediate plasminogen (NH2-terminal lysine) to plasmin (Stage 2). Both lysine and its analogoue, epsilon-aminocaproic acid, exert two effects on the activation of native plasminogen. At low concentrations of these agents, activation is greatly accelerated. Analysis of activation in the presence and absence of these agents by sodium dodecyl sulfate gel electrophoresis indicates that the activation pathway is the same in both cases with the formation of a transient intermediate plasminogen; only the kinetics of proteolysis are altered. This enhancement in the rate of activation results solely from acceleration of the Stage 1 reaction; Stage 2 is essentially unaffected at low concentrations. Stage 1 is maximally enhanced (75-fold) at either 0.0025 M epsilon-aminocaproic acid or 0.025 M lysine and occurs 4 times more rapidly than Stage 2, which becomes the rate-limiting step at these concentrations. Plasmin also cleaves the preactivation peptide from native plasminogen and this reaction rate is enhanced by the same concentrations of lysine and epsilon-aminocaproic acid. These data suggest that lysine and epsilon-aminocaproic acid, which are known to bind to plasminogen and significantly alter its conformation, may thereby enhance preactivation peptide cleavage and consequently, plasminogen activation. At high concentrations, both Stages 1 and 2 are similarly inhibited by these agents, which suggests that this effect may be exerted by the direct inhibition of urokinase. The relative rates of preactivation peptide cleavage by the enzymes urokinase, plasmin, thrombin, and ancrod were also determined. Urokinase is 10 times more effective than plasmin in catalyzing this reaction and 1.8 X 10(4) times more effective than thrombin, while ancrod does not exert an effect. No plasmin is formed by either thrombin or ancrod.
Topics: Amino Acid Sequence; Amino Acids; Aminocaproates; Binding Sites; Dansyl Compounds; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Humans; Kinetics; Lysine; Peptides; Plasminogen; Protein Binding; Urokinase-Type Plasminogen Activator
PubMed: 1150667
DOI: No ID Found -
European Journal of Biochemistry Apr 1996The thrombin-like serine protease ancrod from the Malayan pit viper Agkistrodon rhodostoma was expressed in mouse epithelial cells (C127). Oligosaccharide constituents...
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.
Topics: Agkistrodon; Ancrod; Animals; Carbohydrate Sequence; Carbohydrates; Cell Line; Cloning, Molecular; Glycosylation; Mice; Molecular Sequence Data; Polysaccharides; Recombinant Proteins; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
PubMed: 8620863
DOI: 10.1111/j.1432-1033.1996.0113n.x -
Biochimica Et Biophysica Acta Nov 1995The mechanism of morphologic change of human cultured umbilical vein endothelial cells (HUVECs) caused by fibrin was investigated. Ancrod, a thrombin-like enzyme, did...
The morphologic change of endothelial cells by ancrod-generated fibrin is triggered by alpha v beta 3 integrin binding and the subsequent activation of a G-protein coupled phospholipase C.
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.
Topics: Ancrod; Calcium; Cell Adhesion; Endothelium, Vascular; Fibrin; GTP-Binding Proteins; Humans; Inositol Phosphates; L-Lactate Dehydrogenase; Morphogenesis; Pertussis Toxin; Phosphatidylinositols; Receptors, Vitronectin; Signal Transduction; Type C Phospholipases; Virulence Factors, Bordetella
PubMed: 7488643
DOI: 10.1016/0167-4889(95)00099-e -
Journal of Biochemistry May 1996A thrombin-like enzyme, calobin, has been purified to homogeneity from the venom of Agkistrodon caliginosus by a procedure involving Bio-Gel P-100, Mono S, and Pro-RPC....
A thrombin-like enzyme, calobin, has been purified to homogeneity from the venom of Agkistrodon caliginosus by a procedure involving Bio-Gel P-100, Mono S, and Pro-RPC. The enzyme was identified as a monomer with a molecular weight of 34,000 on SDS-PAGE, and its isoelectric point was 6.2. Calobin acted on fibrinogen to form fibrin with a specific activity of 226 NIH equivalent units, and also exhibited arginine esterase activity. The enzyme predominantly cleaved the alpha-chain of fibrinogen with little degradation of the beta-chain. It contained abundant asparagine/aspartic acid residues, but very few tyrosine or methionine residues. The proteolytic activity of the enzyme with TAME as a substrate was higher than that of thrombin. However, it showed neither lysine esterase nor caseinolytic activity. The enzyme activity was strongly inhibited by PMSF, and moderately by benzamidine and soybean trypsin inhibitor, indicating it is a serine protease. On the other hand, the enzyme activity was not inhibited by hirudin or aprotinin. cDNA (1.6 kb) for calobin has been cloned from an A. caliginosus cDNA library. The cDNA sequence indicates that calobin is synthesized as a pre-zymogen of 262 amino acids, including a putative secretory signal peptide of 18 amino acids and a proposed zymogen peptide of 6 amino acid residues. The cDNA sequence encodes a 238-amino acid residue molecule exhibiting strong amino acid sequence homology to those of ancrod, batroxobin, and flavoxobin isolated from other snake venoms. Calobin contains 12 cysteine residues. As judged on alignment of the amino acid sequences of other thrombin-like enzymes (batroxobin, ancrod, and flavoxobin), calobin constitute the formation of six disulfide bridges. Amino acid residues, His43, Asp88, and Ser182, which are thought to be the catalytic active site are highly conserved. As calobin is a glycoprotein, its possible glycosylation site, Asn-X-Thr, is located at amino acid residues 81-83.
Topics: Agkistrodon; Amino Acid Sequence; Animals; Base Sequence; Cloning, Molecular; Crotalid Venoms; Isoelectric Point; Molecular Sequence Data; Molecular Weight; Platelet Aggregation; Rats; Sequence Homology, Amino Acid; Serine Endopeptidases; Substrate Specificity; Thrombin
PubMed: 8797081
DOI: 10.1093/oxfordjournals.jbchem.a021319 -
FEBS Letters Feb 1992The thrombin-like serine protease and antithrombotic agent, Ancrod, was rapidly purified from the crude venom of Akistrodon rhodostoma by agmatine-Sepharose affinity...
The thrombin-like serine protease and antithrombotic agent, Ancrod, was rapidly purified from the crude venom of Akistrodon rhodostoma by agmatine-Sepharose affinity chromatography followed by MonoQ anion exchange chromatography. N-Terminal sequencing and analysis of overlapping proteolytic fragments of purified Ancrod by automated Edman degradation in combination with tandem mass spectroscopy allowed the determination of the 234 amino acid sequence of the protease. Glycosylation sites at all five canonical N-linked glycosylation sites were inferred from the appearance of blank sequencer cycles in the amino acid sequence and were confirmed by mass spectroscopic analysis of the N-glycanase-treated peptides. Monoclonal antibodies raised against the denatured protein and HF-deglycosylated protein recognized Ancrod on Western blots. Sequence comparison to other thrombin-like serine proteases and reptilian fibrinogenases revealed a number of similarities, most notably the catalytic triad and many conserved cysteine positions.
Topics: Amino Acid Sequence; Amino Acids; Ancrod; Blotting, Western; Molecular Sequence Data; Sequence Alignment; Viper Venoms
PubMed: 1544412
DOI: 10.1016/0014-5793(92)80559-y -
Clinical and Experimental Immunology Feb 1975The protective effects of anticoagulants in nephrotoxic nephritis in rabbits have been studied, using various doses of heparin and defibrination with ancrod. Massive... (Comparative Study)
Comparative Study
The protective effects of anticoagulants in nephrotoxic nephritis in rabbits have been studied, using various doses of heparin and defibrination with ancrod. Massive doses of heparin (2000 units/kg/day) were required before significant reduction in glomerular fibrin deposition, extracepillary cell proliferation and urea retention occurred. Doses of 300 and 1000 units/kg/day were insufficient to modify fibrin deposition and cell proliferation. Defibrination with ancrod provided protection, judged by histological and functional criteria, comparable to 2000 units of heparin/kg/day; but fibrin could still be demonstrated in the glomeruli of animals treated with 2000 units of heparin/kg/day, contrasting with the virtual absence of fibrin in animals given ancrod.
Topics: Ancrod; Animals; Basement Membrane; Blood Coagulation Tests; Complement C3; Dose-Response Relationship, Drug; Endopeptidases; Fibrin; Heparin; Immunoglobulin G; Kidney Glomerulus; Male; Nephritis; Rabbits
PubMed: 1212801
DOI: No ID Found -
Thorax Jul 1970Ancrod (Arvin) on slow intravenous administration converts fibrinogen into fibrin at a rate which does not cause intravascular thrombosis. The fibrin is eliminated from...
Ancrod (Arvin) on slow intravenous administration converts fibrinogen into fibrin at a rate which does not cause intravascular thrombosis. The fibrin is eliminated from the circulation and the blood is thus rendered incoagulable. To test the efficacy of ancrod in the prevention of thrombosis after prosthetic replacement of the heart valves, the tricuspid valve was replaced with a polypropylene mitral valve in 17 calves, using cardiopulmonary bypass. Five calves were eliminated from the study. Eleven calves were treated with ancrod, and one untreated calf was used as a control. Four out of five prosthetic valves in calves treated with ancrod for up to 72 hours were free of thrombus formation, and one out of three at one week. In the long-term study, fibrinogen titres rose after varying intervals, in spite of continual treatment with ancrod. This `escape' of fibrinogen titres from the control of ancrod may be due to species resistance or to the development of immunity. Further study is in progress to elucidate this point and to gain further knowledge for the clinical application of ancrod.
Topics: Animals; Anticoagulants; Cattle; Fibrinogen; Heart Valve Prosthesis; Male; Mitral Valve; Peptide Hydrolases; Thrombosis; Venoms
PubMed: 5485009
DOI: 10.1136/thx.25.4.472 -
British Journal of Cancer Nov 1973After the intravenous injection of Walker 256 tumour cells into rats the platelet count decreased rapidly and remained low during the following period of observation....
After the intravenous injection of Walker 256 tumour cells into rats the platelet count decreased rapidly and remained low during the following period of observation. The platelet decrease was closely related to the number of cells injected. Intra-arterial tumour cell injections required a considerably higher tumour cell count to produce a comparable thrombocytopenia. Non-viable tumour cells and tumour cell fragments induced a similar decrease of circulating platelets. Neither viable tumour cells nor tumour cell fragments aggregated rat platelets in vitro. The presence of fibrin monomers in tumour cell injected animals suggested intravascular fibrin deposition; the plasma fibrinogen level, however, did not decrease significantly. Isotope studies using (51)Cr labelled platelets revealed a rapid disappearance of the platelets from the circulation and their trapping in the lung-the primary site of tumour cell lodgement. Dipyridamole and ancrod pretreatment did not influence the decrease of platelets and their accumulation in the lung after tumour cell injection. In contrast, heparin completely prevented the thrombocytopenia and the platelet trapping in the lung. From the present experiments it is concluded that embolic tumour cells lead to early endothelial damage, resulting in local thrombin formation with subsequent irreversible platelet aggregation.
Topics: Animals; Blood Cell Count; Blood Platelets; Carcinoma 256, Walker; Chromium; Chromium Isotopes; Dipyridamole; Embolism; Endothelium; Fibrin; Fibrinogen; Heparin; Injections, Intra-Arterial; Injections, Intravenous; Kidney; Liver; Lung; Neoplasm Metastasis; Platelet Adhesiveness; Rats; Spleen; Thrombin; Thrombocytopenia; Time Factors
PubMed: 4758369
DOI: 10.1038/bjc.1973.168