Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.5 (thrombin)
33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Detailed coagulation studies were done prospectively on 43 patients with biliary atresia who had undergone Kasai operation (hepatic portoenterostomy). Patients were divided into three groups based on levels of factor V, factor II, and Echis II and/or response to vitamin K: no coagulopathy (46.5% of patients); coagulopathy of liver disease (30.2% of patients); and coagulopathy of vitamin K deficiency (23.3% of patients). Patients with the coagulopathy of liver disease had significantly lower levels of factors XII, V, and antithrombin III as well as longer thrombin times than patients with no coagulopathy or vitamin K deficiency. Factor V levels were decreased only in patients with more advanced liver disease; normal levels of factor V were not usually helpful in differentiating liver disease and vitamin K deficiency. The prothrombin time, factor VII-X levels, and factor II levels were significantly different for all three groups; the most abnormal values occurred in the vitamin K-deficient group. Comparison of the Echis II level to factor II coagulant activity was helpful in deciding whether a coagulopathy was due to liver disease, vitamin K deficiency, or both. Factor VIII levels were elevated in all groups. Factor VIII coagulant activity was significantly higher by the two-stage (TGT) method than by the one-stage (PTT) method. Hypersplenism causing neutropenia and thrombocytopenia was commonly seen after the age of 5 years. Vitamin E deficiency was more common than vitamin K deficiency; however, all vitamin K-deficient patients were vitamin E deficient. Coagulation status correlated well with hepatobiliary scan data, but not serum bilirubin levels. Recommendations for treatment of patients with vitamin K deficiency and/or liver disease are discussed.
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PMID:The multiple coagulopathies of biliary atresia. 669 16

We have used two-dimensional 1H NMR spectroscopy to study the conformation of the thrombin-binding aptamer d(GGTTGGTGTGGTTGG) in solution. This is one of a series of thrombin-binding DNA aptamers with a consensus 15-base sequence that was recently isolated and shown to inhibit thrombin-catalyzed fibrin clot formation in vitro [Bock, L. C., Griffin, L. C., Latham, J. A., Vermaas, E. H. & Toole, J. J. (1992) Nature (London) 355, 564-566]. The oligonucleotide forms a unimolecular DNA quadruplex consisting of two G-quartets connected by two TT loops and one TGT loop. A potential T.T bp is formed between the two TT loops across the diagonal of the top G-quartet. Thus, all of the invariant bases in the consensus sequence are base-paired. This aptamer structure was determined by NMR and illustrates that this molecule forms a specific folded structure. Knowledge of this structure may be used in the further development of oligonucleotide-based thrombin inhibitors.
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PMID:Thrombin-binding DNA aptamer forms a unimolecular quadruplex structure in solution. 847 24

The architecture of G-G-G-G tetrad-aligned DNA quadruplexes in monovalent cation solution is dependent on the directionality of the four strands, which in turn are defined by loop connectivities and the guanine syn/anti distribution along individual strands and within individual G-G-G-G tetrads. The smallest unimolecular G-quadruplex belongs to the d(G2NnG2NnG2NnG2) family, which has the potential to form two stacked G-tetrads linked by Nn loop connectivities. Previous studies have focused on the thrombin-binding DNA aptamer d(G2T2G2TGTG2T2G2), where Nn was T2 for the first and third connecting loops and TGT for the middle connecting loop. This DNA aptamer in K(+) cation solution forms a unimolecular G-quadruplex stabilized by two stacked G(syn)-G(anti)-G(syn)-G(anti) tetrads, adjacent strands which are antiparallel to each other and edge-wise connecting T2, TGT and T2 loops. We now report on the NMR-based solution structure of the d(G2T4G2CAG2GT4G2T) sequence, which differs from the thrombin-binding DNA aptamer sequence in having longer first (T4) and third (GT4) loops and a shorter (CA) middle loop. This d(G2T4G2CAG2GT4G2T) sequence in Na(+) cation solution forms a unimolecular G-quadruplex stabilized by two stacked G(syn)-G(syn)-G(anti)-G(anti) tetrads, adjacent strands which have one parallel and one antiparallel neighbors and distinct non-edge-wise loop connectivities. Specifically, the longer first (T4) and third (GT4) loops are of the diagonal type while the shorter middle loop is of the double chain reversal type. In addition, the pair of stacked G-G-G-G tetrads are flanked on one side by a G-(T-T) triad and on the other side by a T-T-T triple. The distinct differences in strand directionalities, loop connectivities and syn/anti distribution within G-G-G-G tetrads between the thrombin-binding DNA aptamer d(G2T2G2TGTG2T2G2) quadruplex reported previously, and the d(G2T4G2CAG2GT4G2T) quadruplex reported here, reinforces the polymorphic nature of higher-order DNA architectures. Further, these two small unimolecular G-quadruplexes, which are distinct from each other and from parallel-stranded G-quadruplexes, provide novel targets for ligand recognition. Our results demonstrate that the double chain reversal loop connectivity identified previously by our laboratory within the Tetrahymena telomere d(T2G4)4 quadruplex, is a robust folding topology, since it has now also been observed within the d(G2T4G2CAG2GT4G2T) quadruplex. The identification of a G-(T-T) triad and a T-T-T triple, expands on the available recognition alignments for base triads and triples.
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PMID:A double chain reversal loop and two diagonal loops define the architecture of a unimolecular DNA quadruplex containing a pair of stacked G(syn)-G(syn)-G(anti)-G(anti) tetrads flanked by a G-(T-T) Triad and a T-T-T triple. 1141 45

We found two heterozygous dysfibrinogenemias, designated fibrinogen Kosai and fibrinogen Ogasa. Kosai was associated with arteriosclerosis obliterans but Ogasa showed no bleeding or thrombotic tendencies. The plasma fibrinogen concentrations from the two propositi (Ogasa and Kosai) were much lower when determined by the thrombin-time method (0.94 and 1.06 g L(-1), respectively) than when determined by the immunological method (2.87 and 2.72 g L(-1), respectively). We performed DNA sequencing and functional analyses to clarify the relationship between the structural and functional abnormalities. Genetic analysis of PCR-amplified DNA from the propositi identified the heterozygous substitution Bbeta15Gly-->Cys (GGT-->TGT). Western blotting analysis of purified fibrinogen revealed the existence of albumin-fibrinogen complexes. Functional analyses indicated that compared with the normal control, the propositi's fibrinogen released only half the normal amount of fibrinopeptide B and showed markedly impaired polymerization. In addition, the observation of thinner fibers in fibrin clots (by scanning electron microscopy) indicated markedly defective lateral aggregation in the variant fibrinogens. The impaired functions may be due to the substitution of Cys for Bbetao15Gly plus the existence of some additional disulfide-bonded forms.
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PMID:Fibrinogens Kosai and Ogasa: Bbeta15Gly-->Cys (GGT-->TGT) substitution associated with impairment of fibrinopeptide B release and lateral aggregation. 1287 1

The structure of a complex between thrombin and a GGTTGGTGTGGTTGG DNA 15-mer has been analyzed crystallographically. The solution NMR structure of the 15-mer has two stacked G-quartets similar to that found in the previous X-ray structure determination of the 15-mer-thrombin complex [Padmanabhan, Padmanabhan, Ferrara, Sadler & Tulinsky (1993). J. Biol. Chem. 268, 17651-17654]; the strand polarity, however, is reversed from that of the crystallographic structure. The structure of the complex here has been redetermined with better diffraction data confirming the previous crystallographic structure but also indicating that the NMR solution structure fits equally well. Both 15-mer complex structures refined to an R value of about 0.16 presenting a disconcerting ambiguity. Since the two 15-mer structures associate with thrombin in different ways (through the TGT loop in the X-ray and TT loop in the NMR model), other independent lines of physical or chemical evidence are required to resolve the ambiguity.
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PMID:An ambiguous structure of a DNA 15-mer thrombin complex. 1529

Hydrogen exchange rates of the imino protons of the thrombin-binding 15 mer DNA aptamer d(G(1)G(2)T(3)T(4)G(5)G(6)T(7)G(8)T(9)G(10)G(11)T(12)T(13)G(14)G(15)) in the presence of Sr(2+) were measured. In the temperature range 15-35 degrees C, the exchange rates of the eight iminos in the quadruplex core were not uniform, with the G(2), G(11) and G(15) iminos exchanging faster, the G(1), G(5), G(10) and G(14) iminos exchanging slower, and the G(6) imino exchanging at a medium rate. In the quadruplex G(1), G(5), G(10) and G(14) adopted syn glycosidic conformation, while G(2), G(6), G(11) and G(15) adopted anti-conformation. It was found that the four slowly exchanging iminos, which were all the syn-iminos, happened to be located in the TT loops that were not easy to open to the solvent. The anti-iminos exchanged faster, but the G(6) imino exchanged slower than other anti-iminos, because its hydrogen bond with the G(10)O6 was stabilized by the TGT loop. The fact that the G(6) imino exchanged at a faster rate than those syn-iminos in the TT loops suggested that the TGT loop was less stable than the TT loops. Unfolding mechanism for the quadruplex was thus proposed: The quadruplex first uncoupled the three base pairs: G(1)-G(15), G(2)-G(14) and G(5)-G(11), which were not protected by any loops. Then it opened the TGT loop. Finally, it opened the TT loops and the sequence became an unstructured random coil that exchanged with the quadruplex conformation. The conformational exchange between the quadruplex and random coil had been detected.
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PMID:NMR study of the folding-unfolding mechanism for the thrombin-binding DNA aptamer d(GGTTGGTGTGGTTGG). 1561 22

The dual-labeled oligonucleotide derivative, FAT-0, carrying 6- carboxyfluorescein (FAM) and 6-carboxytetramethylrhodamine (TAMRA) labels at the 5' and 3' termini of the thrombin-binding aptamer (TBA) sequence 5'-GGT TGG TGT GGT TGG-3', and its derivatives, FAT-n (n=3, 5, and 7) with a spacer at the 5'-end of a TBA sequence of T(m)A (m=2, 4, and 6) have been designed and synthesized. These fluorescent probes were developed for monitoring K(+) concentrations in living organisms. Circular dichroism, UV-visible absorption, and fluorescence studies revealed that all FAT-n probes could form intramolecular tetraplex structures after binding K(+). Fluorescence resonance energy transfer and quenching results are discussed taking into account dye-dye contact interactions. The relationship between the fluorescence behavior of the probes and the spacer length in FAT-n was studied in detail and is discussed.
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PMID:G quadruplex-based FRET probes with the thrombin-binding aptamer (TBA) sequence designed for the efficient fluorometric detection of the potassium ion. 1700 71

The solution structure of a new modified thrombin binding aptamer (TBA) containing a 5'-5' inversion of polarity site, namely d(3'GGT5'-5'TGGTGTGGTTGG3'), is reported. NMR and CD spectroscopy, as well as molecular dynamic and mechanic calculations, have been used to characterize the 3D structure. The modified oligonucleotide is characterized by a chair-like structure consisting of two G-tetrads connected by three edge-wise TT, TGT and TT loops. d(3'GGT5'-5'TGGTGTGGTTGG3') is characterized by an unusual folding, being three strands parallel to each other and only one strand oriented in opposite manner. This led to an anti-anti-anti-syn and syn-syn-syn-anti arrangement of the Gs in the two tetrads. The thermal stability of the modified oligonucleotide is 4 degrees C higher than the corresponding unmodified TBA. d(3'GGT5'-5'TGGTGTGGTTGG3') continues to display an anticoagulant activity, even if decreased with respect to the TBA.
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PMID:A new modified thrombin binding aptamer containing a 5'-5' inversion of polarity site. 1714 16

In this work, we report the solution structure, thermodynamic studies, and the pharmacological properties of a new modified thrombin binding aptamer (TBA) containing a G-LNA residue, namely d(5'-GGTTGGTGTGGTTGg-3'), where upper case and lower case letters represent DNA and LNA residues, respectively. NMR and CD spectroscopy, as well as molecular dynamics and mechanic calculations, has been used to characterize the three-dimensional structure. The modified oligonucleotide is characterized by a chair-like structure consisting of two G-tetrads connected by three edge-wise TT, TGT, and TT loops. d(5'-GGTTGGTGTGGTTGg-3') is characterized by the same folding of TBA, being two strands parallel to each other and two strands oriented in opposite manner. This led to a syn-anti-syn-anti and anti-syn-anti-syn arrangements of the Gs in the two tetrads. d(5'-GGTTGGTGTGGTTGg-3') possesses an anticoagulant activity, even if decreased with respect to the TBA.
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PMID:A novel thrombin binding aptamer containing a G-LNA residue. 1759 Mar 40

The G-quadruplex DNA structure has been suggested to be a potential target for anticancer therapies. Therefore, there is increasing interest in the development of drugs that could modulate the stability of G-quadruplex structures. In the current work, the interaction between the thrombin-binding aptamer (TBA, 5'-GGT TGG TGT GGT TGG-3'), which can form an intramolecular G-quadruplex structure, and the porphyrin 5,10,15,20-tetrakis-(N-methyl-4-pyridyl)-21,23H-porphyrin tetratosylate (TmPyP4) was studied. The application of a high-performance liquid chromatography-photodiode array (HPLC-PDA) detector-based method to study this kind of interaction was tested. Molecular absorption data recorded along the chromatographic runs were analyzed by means of multivariate data analysis methods. Moreover, biospecific interaction analysis (BIA) by surface plasmon resonance (SPR) and melting and mole ratio experiments monitored by UV-visible molecular absorption and circular dichroism spectroscopies, were applied to confirm and expand the chromatographic studies. The results showed the formation of an interaction complex with a stoichiometry 1:1 (TmPyP4/TBA) and logarithm of the equilibrium constant equal to 5.7+/-0.2. Melting and circular dichroism data reflected that the initial G-quadruplex structure of TBA is stabilized in the interaction complex, being slightly distorted by the presence of the ligand.
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PMID:Study of the interaction between the G-quadruplex-forming thrombin-binding aptamer and the porphyrin 5,10,15,20-tetrakis-(N-methyl-4-pyridyl)-21,23H-porphyrin tetratosylate. 1849 81


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