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Blood Oct 1977The polymerization of thrombin and ancrod fibrin monomers was studied with a standardized technique that evaluated turbidity changes and protein incorporation into the... (Comparative Study)
Comparative Study
The polymerization of thrombin and ancrod fibrin monomers was studied with a standardized technique that evaluated turbidity changes and protein incorporation into the clot. Ancrod fibrin monomers were found to polymerize more slowly and form less turbid clots (at identical protein concentrations). Changes in ionic strength and pH influences ancrod fibrin monomer polymerization to a greater extent than thrombin fibrin monomer polymerization. Benzyltriethylammonium chloride was shown to be a potent inhibitor of fibrin monomer polymerization, with a greater inhibitory effect on ancrod fibrin monomers than on thrombin fibrin monomers. The differences between ancrod and thrombin fibrin may play a role in the infrequent thrombotic complications reported with ancrod therapy.
Topics: Ancrod; Chemical Phenomena; Chemistry; Electrophoresis, Polyacrylamide Gel; Fibrin; Humans; Hydrogen-Ion Concentration; Osmolar Concentration; Polymers; Thrombin
PubMed: 20184
DOI: No ID Found -
The Biochemical Journal Sep 1993The 1.54 kb cDNA for ancrod, a thrombin-like enzyme, was cloned from a lambda ZAP cDNA library derived from the venom glands of Calloselasma (Agkistrodon) rhodostoma....
The 1.54 kb cDNA for ancrod, a thrombin-like enzyme, was cloned from a lambda ZAP cDNA library derived from the venom glands of Calloselasma (Agkistrodon) rhodostoma. The cDNA sequence reveals that ancrod is synthesized as a pre-zymogen of 258 amino acids, including a putative secretory peptide of 18 amino acids and a proposed zymogen peptide of 6 amino-acid residues. The amino-acid sequence of the predicted active form of the enzyme exhibits a high degree of sequence similarity to those of mammalian serine proteases (trypsin and pancreatic kallikrein) and other thrombin-like enzymes (batroxobin and flavoxobin). Key amino-acid residues (His43, Asp88, Ser182 and Asp176) that are thought to be involved in the substrate cleavage and in the substrate-binding reaction are conserved. Ancrod contains 13 cysteine residues. Based on alignment with the amino-acid sequences of trypsin and batroxobin, six disulphide bridges can be predicted to be present in the ancrod protein. The existence of a free cysteine, which changes the common sequence surrounding the Ser182 active site from Gly-Asp-Ser-Gly-Gly-Pro to Cys-Asp-Ser-Gly-Gly-Pro, is unusual for a serine protease.
Topics: Amino Acid Sequence; Ancrod; Base Sequence; Cloning, Molecular; Crotalid Venoms; DNA; Molecular Sequence Data; Polymerase Chain Reaction; Restriction Mapping; Sequence Analysis; Sequence Analysis, DNA
PubMed: 8373353
DOI: 10.1042/bj2940387 -
British Medical Journal (Clinical... Jul 1983
Topics: Ancrod; Aspirin; Cerebrovascular Disorders; Dextrans; Dipyridamole; Drug Administration Schedule; Heparin; Humans; Myocardial Infarction; Recurrence; Sulfinpyrazone; Thrombosis
PubMed: 6191822
DOI: 10.1136/bmj.287.6386.196 -
The Journal of Biological Chemistry Jan 2007The ARF tumor suppressor signals through p53 and other poorly defined anti-proliferative pathways to block carcinogenesis. In a search for new regulators of ARF...
The ARF tumor suppressor signals through p53 and other poorly defined anti-proliferative pathways to block carcinogenesis. In a search for new regulators of ARF signaling, we discovered a novel nuclear protein that we named NIAM (nuclear interactor of ARF and MDM2) for its ability to bind both ARF and the p53 antagonist MDM2. NIAM protein is normally expressed at low to undetectable levels in cells because of, at least in part, MDM2-mediated ubiquitination and proteasomal degradation. When reintroduced into cells, NIAM activated p53, caused a G1 phase cell cycle arrest, and collaborated with ARF in an additive fashion to suppress proliferation. Notably, NIAM retains growth inhibitory activity in cells lacking ARF and/or p53, and knockdown experiments revealed that it is not essential for ARF-mediated growth inhibition. Thus, NIAM and ARF act in separate anti-proliferative pathways that intersect mechanistically and suppress growth more effectively when jointly activated. Intriguingly, silencing of NIAM accelerated chromosomal instability, and microarray analyses showed reduced NIAM mRNA expression in numerous primary human tumors. This study identifies a novel protein with tumor suppressor-like behaviors and functional links to ARF-MDM2-p53 signaling.
Topics: Adenocarcinoma; Ancrod; Animals; Breast Neoplasms; Cell Division; Cell Line, Tumor; Cell Nucleus; Chromosomes; DNA-Binding Proteins; Fibroblasts; Humans; Intracellular Signaling Peptides and Proteins; Mice; Molecular Sequence Data; Nuclear Proteins; Osteosarcoma; Proto-Oncogene Proteins c-mdm2; RNA, Messenger; Tumor Suppressor Protein p14ARF; Tumor Suppressor Protein p53; Ubiquitin
PubMed: 17110379
DOI: 10.1074/jbc.M609612200 -
The Journal of Experimental Medicine Dec 1993Although "biocompatible" polymeric elastomers are generally nontoxic, nonimmunogenic, and chemically inert, implants made of these materials may trigger acute and...
Although "biocompatible" polymeric elastomers are generally nontoxic, nonimmunogenic, and chemically inert, implants made of these materials may trigger acute and chronic inflammatory responses. Early interactions between implants and inflammatory cells are probably mediated by a layer of host proteins on the material surface. To evaluate the importance of this protein layer, we studied acute inflammatory responses of mice to samples of polyester terephthalate film (PET) that were implanted intraperitoneally for short periods. Material preincubated with albumin is "passivated," accumulating very few adherent neutrophils or macrophages, whereas uncoated or plasma-coated PET attracts large numbers of phagocytes. Neither IgG adsorption nor surface complement activation is necessary for this acute inflammation; phagocyte accumulation on uncoated implants is normal in hypogammaglobulinemic mice and in severely hypocomplementemic mice. Rather, spontaneous adsorption of fibrinogen appears to be critical: (a) PET coated with serum or hypofibrinogenemic plasma attracts as few phagocytes as does albumin-coated material; (b) in contrast, PET preincubated with serum or hypofibrinogenemic plasma containing physiologic amounts of fibrinogen elicits "normal" phagocyte recruitment; (c) most importantly, hypofibrinogenemic mice do not mount an inflammatory response to implanted PET unless the material is coated with fibrinogen or the animals are injected with fibrinogen before implantation. Thus, spontaneous adsorption of fibrinogen appears to initiate the acute inflammatory response to an implanted polymer, suggesting an interesting nexus between two major iatrogenic effects of biomaterials: clotting and inflammation.
Topics: Ancrod; Animals; Biocompatible Materials; Cell Adhesion; Chemotaxis, Leukocyte; Fibrin; Fibrinogen; Humans; In Vitro Techniques; Inflammation; Mice; Mice, Inbred BALB C; Phagocytes; Polyethylene Terephthalates
PubMed: 8245787
DOI: 10.1084/jem.178.6.2147 -
Toxicon : Official Journal of the... Jan 2011Gyroxin is a serine protease enzyme component of the South American rattlesnake (Crotalus durissus terrificus) venom. This toxin displays several activities, including...
Gyroxin is a serine protease enzyme component of the South American rattlesnake (Crotalus durissus terrificus) venom. This toxin displays several activities, including the induction of blood coagulation (fibrinogenolytic activity), vasodilation and neurotoxicity, resulting in an effect called barrel rotation. The mechanisms involved in this neurotoxic activity are not well known. Because gyroxin is a member of a potentially therapeutic family of enzymes, including thrombin, ancrod, batroxobin, trypsin and kallicrein, the identification of the mechanism of gyroxin's action is extremely important. In this study, gyroxin was isolated from crude venom by affinity and molecular exclusion chromatography. Analysis of the isolated gyroxin via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a single protein band with a molecular weight of approximately 28 kDa, confirming the identity of the molecule. Furthermore, intravenous administration of purified gyroxin (0.25 μg/g of body weight) to mice resulted in symptoms compatible with barrel rotation syndrome, confirming the neurotoxic activity of the toxin. Mice treated with gyroxin showed an increase in the concentration of albumin-Evans blue in brain extracts, indicating an increase in the blood-brain barrier (BBB) permeability. This gyroxin-induced increase in BBB permeability was time-dependent, reaching a peak within 15 min after exposure, similar to the time span in which the neurotoxic syndrome (barrel rotation) occurs. This work provides the first evidence of gyroxin's capacity to temporarily alter the permeability of the BBB.
Topics: Animals; Behavior, Animal; Blood-Brain Barrier; Cattle; Chemical Fractionation; Chromatography, Affinity; Crotalid Venoms; Electrophoresis, Agar Gel; Evans Blue; Male; Mice; Neurotoxins; Serum Albumin
PubMed: 20637222
DOI: 10.1016/j.toxicon.2010.06.027 -
Blood May 1975This study examines the role of neutrophils (PMN) in the pathogenesis of endotoxin-induced microclot formation. It is intended to clarify whether granulocytes are...
This study examines the role of neutrophils (PMN) in the pathogenesis of endotoxin-induced microclot formation. It is intended to clarify whether granulocytes are involved in endotoxin-induced activation of intravascular coagulation (generation of soluble fibrin) and/or in endotoxin-induced precipitation of soluble fibrin. Precipitation of soluble fibrin was achieved by injection of endotoxin into ancrod-infused rabbits with circulating soluble fibrin (first model). Activation of intravascular coagulation was elicited by two intravenous injections of endotoxin into rabbits (second model). Seventy-two and ninety-six hours after injection of nitrogen mustard, leukopenic rabbits had PMN counts between 0 and 50 cells per mul. Neutropenia did not prevent the occurrence of glomerular microclots after infusion of ancrod and injection of endotoxin (first model). Neutropenia influenced neither the decrease in mean fibrinogen concentrations nor the drop in mean platelet counts after ancrod and endotoxin administration. In contrast to the first model, neutropenia prevented the occurrence of glomerular microclots and of circulating soluble fibrin after two injections of endotoxin (second model). It did not, however, protect rabbits from the decrease in mean platelet counts after endotoxin administration. These data indicate that granulocytes are involved in endotoxin-induced activation of intravascular coagulation and the production of soluble fibrin but are not essential to endotoxin-induced precipitation of soluble fibrin.
Topics: Animals; Blood Cell Count; Blood Platelets; Disseminated Intravascular Coagulation; Endotoxins; Female; Fibrin; Fibrinogen; Granulocytes; Hematocrit; Kidney Glomerulus; Leukocyte Count; Leukocytes; Male; Neutropenia; Neutrophils; Nitrogen Mustard Compounds; Rabbits; Salmonella enteritidis; Solubility; Tryptophan; Venoms
PubMed: 1091310
DOI: No ID Found -
European Journal of Biochemistry Feb 2000Kinetics for the hydrolysis of the chromogenic active-site titrant N alpha-(N,N-dimethylcarbamoyl)-alpha-azaornithine p-nitrophenyl ester (Dmc-azaOrn-ONp) catalysed by... (Comparative Study)
Comparative Study
Kinetics for the hydrolysis of the chromogenic active-site titrant N alpha-(N,N-dimethylcarbamoyl)-alpha-azaornithine p-nitrophenyl ester (Dmc-azaOrn-ONp) catalysed by bovine beta-trypsin, bovine alpha-thrombin, bovine Factor Xa, human alpha-thrombin, human Factor Xa, human Lys77-plasmin, human urinary kallikrein, Mr 33 000 and Mr 54 000 species of human urokinase, porcine pancreatic beta-kallikrein-A and -B and Ancrod (the coagulating serine proteinase from the Malayan pit viper Agkistrodon rhodostoma venom) have been obtained between pH 6.0 and 8.0, at 21.0 degrees C, and analysed in parallel with those for the enzymatic cleavage of N alpha-(N,N-dimethylcarbamoyl)-alpha-azalysine p-nitrophenyl ester (Dmc-azaLys-ONp). The enzyme kinetics are consistent with the minimum three-step catalytic mechanism of serine proteinases, the rate-limiting step being represented by the deacylation process. Bovine beta-trypsin kinetics are modulated by the acid-base equilibrium of the His57 catalytic residue (pKa approximately 6.9). Dmc-azaOrn-ONp and Dmc-azaLys-ONp bind stoichiometrically to the serine proteinase active site, and allow the reliable determination of the active enzyme concentration between 1.0 x 10-6 M and 3.0 x 10-4 M. The affinity and the reactivity for Dmc-azaOrn-ONp (expressed by Ks and k+2/Ks, respectively) of the serine proteinases considered are much lower than those for Dmc-azaLys-ONp. The very different affinity and reactivity properties for Dmc-azaOrn-ONp and Dmc-azaLys-ONp have been related to the different size of the ornithine/lysine side chains, and to the ensuing different positioning of the active-site titrants upon binding to the enzyme catalytic centre (i.e. to P1-S1 recognition). These data represent the first detailed comparative investigation on the catalytic properties of serine proteinases towards an ornithine derivative (i. e. Dmc-azaOrn-ONp).
Topics: Animals; Aza Compounds; Binding Sites; Catalytic Domain; Histidine; Humans; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Lysine; Models, Molecular; Ornithine; Serine Endopeptidases; Serine Proteinase Inhibitors; Titrimetry
PubMed: 10672036
DOI: 10.1046/j.1432-1327.2000.01120.x -
Neuron Dec 2017Blood-brain barrier (BBB) disruption alters the composition of the brain microenvironment by allowing blood proteins into the CNS. However, whether blood-derived...
Blood-brain barrier (BBB) disruption alters the composition of the brain microenvironment by allowing blood proteins into the CNS. However, whether blood-derived molecules serve as extrinsic inhibitors of remyelination is unknown. Here we show that the coagulation factor fibrinogen activates the bone morphogenetic protein (BMP) signaling pathway in oligodendrocyte progenitor cells (OPCs) and suppresses remyelination. Fibrinogen induces phosphorylation of Smad 1/5/8 and inhibits OPC differentiation into myelinating oligodendrocytes (OLs) while promoting an astrocytic fate in vitro. Fibrinogen effects are rescued by BMP type I receptor inhibition using dorsomorphin homolog 1 (DMH1) or CRISPR/Cas9 activin A receptor type I (ACVR1) knockout in OPCs. Fibrinogen and the BMP target Id2 are increased in demyelinated multiple sclerosis (MS) lesions. Therapeutic depletion of fibrinogen decreases BMP signaling and enhances remyelination in vivo. Targeting fibrinogen may be an upstream therapeutic strategy to promote the regenerative potential of CNS progenitors in diseases with remyelination failure.
Topics: Activin Receptors, Type I; Animals; Blood Vessels; Bone Morphogenetic Proteins; Fibrinogen; Lysophosphatidylcholines; Mice; Mice, Inbred C57BL; Mice, Knockout; Microarray Analysis; Myelin Sheath; Oligodendrocyte Precursor Cells; Plasmids; Remyelination; Signal Transduction
PubMed: 29103804
DOI: 10.1016/j.neuron.2017.10.008 -
The Journal of Experimental Medicine Aug 2007Cerebrovascular dysfunction contributes to the pathology and progression of Alzheimer's disease (AD), but the mechanisms are not completely understood. Using transgenic...
Cerebrovascular dysfunction contributes to the pathology and progression of Alzheimer's disease (AD), but the mechanisms are not completely understood. Using transgenic mouse models of AD (TgCRND8, PDAPP, and Tg2576), we evaluated blood-brain barrier damage and the role of fibrin and fibrinolysis in the progression of amyloid-beta pathology. These mouse models showed age-dependent fibrin deposition coincident with areas of blood-brain barrier permeability as demonstrated by Evans blue extravasation. Three lines of evidence suggest that fibrin contributes to the pathology. First, AD mice with only one functional plasminogen gene, and therefore with reduced fibrinolysis, have increased neurovascular damage relative to AD mice. Conversely, AD mice with only one functional fibrinogen gene have decreased blood-brain barrier damage. Second, treatment of AD mice with the plasmin inhibitor tranexamic acid aggravated pathology, whereas removal of fibrinogen from the circulation of AD mice with ancrod treatment attenuated measures of neuroinflammation and vascular pathology. Third, pretreatment with ancrod reduced the increased pathology from plasmin inhibition. These results suggest that fibrin is a mediator of inflammation and may impede the reparative process for neurovascular damage in AD. Fibrin and the mechanisms involved in its accumulation and clearance may present novel therapeutic targets in slowing the progression of AD.
Topics: Alzheimer Disease; Animals; Blood-Brain Barrier; Disease Models, Animal; Disease Progression; Fibrin; Fibrinogen; Inflammation; Mice; Mice, Transgenic; Models, Biological; Permeability; Plasminogen; Tranexamic Acid
PubMed: 17664291
DOI: 10.1084/jem.20070304