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)

Thrombin-treated tumor cells induce a metastatic phenotype in experimental pulmonary murine metastasis. Thrombin binds to a unique protease-activated receptor (PAR-1) that requires N-terminal proteolytic cleavage for activation by its tethered end. A 14-mer thrombin receptor activation peptide (TRAP) of the tethered end induces the same cellular changes as thrombin. Four murine tumor cells (Lewis lung, CT26 colon CA, B16F10 melanoma, and CCL163 fibroblasts) contain PAR-1, as detected by reverse transcriptase-polymerase chain reaction (RT-PCR). B16F10 cells did not contain the two other thrombin receptors, PAR-3 and glycoprotein Ib. TRAP-treated B16F10 tumor cells enhance pulmonary metastasis 41- to 48-fold (n = 17). Thrombin-treated B16F10 cells transfected with full-length murine PAR-1 sense cDNA (S6, S7, S14, and S22) enhanced their adhesion to fibronectin 1.5- to 2.4-fold (n = 5, P <.04), whereas thrombin-treated wild-type cells do not. S6 (adhesion index, 1.5-fold) and S14 (index, 2.4-fold) when examined by RT-PCR and Northern analysis showed minimal expression of PAR-1 for S6 over wild-type and considerable expression for S14. Immunohistochemistry showed greater expression of PAR-1 for S14 compared with wild-type or empty-plasmid transfected cells. In vivo experiments with the thrombin-treated S14 transfectant showed a fivefold to sixfold increase in metastases compared with empty-plasmid transfected thrombin-treated naive cells or S6 cells (n = 20, P =.0001 to .02). Antisense had no effect on thrombin-stimulated tumor mass. Thus, PAR-1 ligation and expression enhances and regulates tumor metastasis.
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PMID:Protease-activated receptor 1 (PAR-1) is required and rate-limiting for thrombin-enhanced experimental pulmonary metastasis. 980 63

Our study concerns the biological effects of abciximab (c7E3 Fab, ReoPro), a powerful new antiplatelet drug that blocks glycoprotein (GP) IIb-IIIa complexes. Samples were examined from 6 patients with coronary artery disease who received a bolus of abciximab followed by a 10- microg/min infusion for at least 18 hours before percutaneous transluminal coronary angioplasty. Inhibition of ADP-induced PA was maximal for 4 patients but partial (79% and 53%) for 2 others during the infusion. Flow cytometry performed with monoclonal antibodies (PAC-1, AP-6, and F26) specific for the "activated" GP IIb-IIIa complex revealed large decreases in the expression of activation markers on platelets during therapy, but these decreases were less marked when inhibition of ADP-induced PA was incomplete. Residual aggregation was seen for all patients during the infusion when TRAP 14-mer peptide or thrombin was the stimulus. Unblocked GP IIb-IIIa complexes were detected on thrombin-stimulated platelets from the patients by immunoelectron microscopy performed using the monoclonal antibody AP-2. Unblocked GP IIb-IIIa complexes were also detected by flow cytometry when platelets preincubated for 1 hour in vitro with abciximab under saturating conditions were (1) incubated with TRAP 14-mer or (2) permeabilized with Triton X-100. In confirming interpatient variation in the platelet response to a standard dose of abciximab, our results also show that an uninhibited internal pool of GP IIb-IIIa complexes may mediate a residual response to strong agonists.
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PMID:Expression of markers of platelet activation and the interpatient variation in response to abciximab. 997

The thrombin aptamer is a 15-mer oligodeoxyribonucleotide that folds into a unimolecular quadruplex consisting of a stack of two guanine quartets connected by two external loops and one central loop and possesses a high affinity for thrombin. We have undertaken a systematic examination, in KCl, of the thermodynamic stability of thrombin aptamer analogues containing sequence modifications in one or more of the loops, as well as in the number of quartets. UV melting studies have been carried out to obtain the relevant thermodynamic parameters for these aptamers. van't Hoff analysis of these data, with a two-state model for unimolecular denaturation, gave excellent fits to the experimental observations. Thermodynamic analysis indicates that the central loop sequence in the parent aptamer is optimal for stability. Modifications in this or other loops can effect either DeltaH degrees, DeltaS degrees, or both. Addition of a single G at the 5'-end decreases stability while addition of a G at the 3'-end increases stability. Differential scanning calorimetry experiments on the thrombin aptamer reveal that a heat capacity change, not detected by UV measurements, accompanies the unfolding of the aptamer.
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PMID:Effect of loop sequence and size on DNA aptamer stability. 1068 28

Hyaluronan was partially depolymerized on a large-scale quantity using bacterial hyaluronidase (E.C. 4.2.2.1) for preparation of chemically fully O-sulfated oligosaccharides. The hyaluro-oligosaccharide (HAoligo) mixture obtained by partial digestion was repeatedly applied to low pressure gel permeation chromatographic separation to purify the size-unified oligosaccharide ranged from 4- to 20-mer. The purity and size of each HAoligo was confirmed by using proton nuclear magnetic resonance ((1)H NMR) spectroscopy, capillary electrophoresis (CE) on normal polarity mode, and a newly established separation method by normal phase chromatography with Amide-80 column. The purified HAoligos ranged 4- to 20-mer were applied to chemically fully O-sulfation. Characterization of chemically fully O-sulfated HAoligos was performed by both chemical compositional analyses after hydrolysis and (1)H NMR spectroscopy. While the anti-factor IIa activity of 4- to 20-mer O-sulfated HAoligos was less than 3.1 units/mg, the inhibitory action for hyaluronidase (bovine testicular hyaluronidase (E.C.3.2.1.35)) of the oligosaccharides ranged 16- to 20-mer were corresponding to 79% of that shown by fully O-sulfated hyaluronan (MW 100 kDa) through both competitive and noncompetitive effects.
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PMID:Preparation and inhibitory activity on hyaluronidase of fully O-sulfated hyaluro-oligosaccharides. 1118 62

Proliferation and migration of vascular smooth muscle cells (VSMCs) are involved in the processes of atherosclerosis and restenosis. The protein product of the growth arrest-specific gene 6 (Gas-6) has recently been identified as a ligand for the Axl/Rse/Mer tyrosine kinase receptor family, which may be involved in proliferation and migration of VSMCs. Here we show that Gas-6 gene expression is increased in proliferating VSMCs in tissue culture (2.5-fold increase by Northern blot) and following neointimal proliferation in a rabbit balloon-injury model (3-fold increase by Western blot). Neither platelet-derived growth factor (PDGF) nor thrombin stimulate the expression of Gas-6 in cultured VSMCs despite the ability of the PDGF, but not thrombin, to stimulate proliferation in growth-arrested cells. These data suggest a role for the Gas-6 regulatory system in VSMC proliferation, which may be a target for therapeutic interventions in the atherosclerotic process and restenosis after angioplasty.
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PMID:Growth arrest-specific gene 6 expression in proliferating rabbit vascular smooth muscle cells in vitro and in vivo. 1127 3

Angiogenesis is required for tumor growth and metastasis. It has recently been suggested that thrombin is a potent promoter of angiogenesis. We therefore examined the possibility that thrombin could be inducing the expression of vascular endothelial growth factor (VEGF), which promotes endothelial growth. Primary human FS4 fibroblasts as well as tumor cell lines: prostate DU145 and megakaryocyte CHRF were incubated with thrombin (0.25-1 unit/ml) for 1-8 hrs and then examined for mRNA by Northern Analysis. Enhanced mRNA (approximately 3-4 fold over base line) was noted at 2-4 hrs, with 0.5 u/ml thrombin. The effect was specific for thrombin activity on its PAR-1 receptor, since equal units of hirudin completely inhibited the response and the thrombin effect could be mimicked with the 14 mer thrombin receptor activation peptide (TRAP). Upregulation of mRNA was associated with enhanced VEGF protein synthesis and secretion as assayed by immunoblot. Enhanced expression of VEGF mRNA was not secondary to enhanced transcription (nuclear run on experiments), but due to an >3 fold stabilization of mRNA (Actinomycin D chase experiment). Enhanced VEGF mRNA stabilization is promoted by the PI3Kinase and serine/threonine kinase pathways, since thrombin-induced mRNA expression is inhibited by Wortmanin and H7. No effect was noted with the MAPKinase inhibitor, PD98059. Thus, thrombin-induced tumorigenesis and metastasis is associated with enhanced VEGF protein synthesis and secretion via the stabilization of VEGF mRNA promoted by the PI3Kinase and serine/threonine kinase pathways. This could help explain how thrombin promotes angiogenesis.
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PMID:Thrombin induces increased expression and secretion of VEGF from human FS4 fibroblasts, DU145 prostate cells and CHRF megakaryocytes. 1168 29

Lebetins from Macrovipera lebetina snake venom constitute a new class of inhibitors of platelet aggregation. There are two groups of peptides: lebetin 1 (L1; 11- to 13-mer) and lebetin 2 (L2; 37- to 38-mer). The short lebetins are identical to the N-terminal segments of the longer ones. They inhibit platelet aggregation induced by various agonists (e.g. thrombin, PAF-acether or collagen). The shortest lebetin (11-mer) shows potent inhibition of rabbit (IC(50) = 7 nM) and human (IC(50) = 5 nM) platelets. They prevent collagen-induced thrombocytopenia in rats. N- and C-terminal-truncated synthetic L1gamma (sL1gamma; 11-mer) is less active in inhibiting platelet aggregation than the native peptide. Results from Ala scan studies of the sL1gamma peptide indicated that replacement of the residues (P3, G7, P8, P9 or N10) resulted in a remarkable drop in the activity, whereas replacement of residues K2, P4 or K6 by Ala resulted in enhancement of the antiplatelet activity by at least 10-fold. To examine the activity of multimeric L1gamma, several multimeric peptides were synthesized using the multiple-antigen peptide system assembled on a branched lysine core and their antiplatelet activity was evaluated in vitro. The largest multimeric peptides showed a 1,000-fold increase in antiplatelet activity.
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PMID:Lebetin peptides: potent platelet aggregation inhibitors. 1191 Jan 86

Here we report the in vitro selection of novel small peptide motifs that bind to human alpha-thrombin. We have applied mRNA display to select for thrombin binding peptides from an unbiased library of 1.2 x 10(11) different 35-mer peptides, each containing a random sequence of 15 amino acids. Two clones showed binding affinities ranging from 166 to 520 nM. A conserved motif of four amino acids, DPGR, was identified. Clot formation of human plasma is inhibited by the selected clones, and they downregulate the thrombin-mediated activation of protein C. The identified peptide motifs do not share primary sequence similarities to any of the known natural thrombin binding motifs. As new inhibitors for human thrombin open interesting possibilities in thrombosis research, our newly identified peptides may provide further insights into this field of investigation and may be possible candidates for the development of new anti-thrombotic agents.
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PMID:A novel class of small functional peptides that bind and inhibit human alpha-thrombin isolated by mRNA display. 1257

We screened the DNA aptamer inhibiting thrombin with novel method using algorithm mimicking evolution. For screening, we first randomly designed and synthesized ten 15-mer oligonucleotides supposed to form G-quartet structure and then measured the inhibitory activity for thrombin. The aptamers showing the high activities were selected and we shuffled and mutated those sequences in silico to generate 10 new sequences of aptamers for next generation. After repeating 5 cycles, we successfully obtained the same aptamers reported previously showing high inhibitory activity. In addition, we added 8-mer oligonucleotides to the both 5' and 3' ends of the selected 15-mer aptamer and then repeated evolution in silico. After 2 cycles, we were able to obtain the aptamer showing better inhibitory activity than the 15-mer aptamer.
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PMID:Novel strategy for DNA aptamers inhibiting enzymatic activity using algorithm mimicking evolution. 1451 Apr 52

The formation of complexes between various thrombin preparations and a 30-mer aptamer DNA was comparatively studied, and a correlation between the complex formation and the fibrinogen-hydrolyzing activity of thrombin was found. The aptamer DNA was shown to inhibit the fibrin formation from fibrinogen.
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PMID:[Aptameric DNA--a new type of thrombin inhibitor]. 1460 4


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