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)

The GTPase activity of a G protein alpha subunit functions as a timer to control the lifetime of the activated conformation of the protein. Expression of the GTPase-deficient Gi2 alpha subunit oncogene, gip2 (alpha i2Q205L), in Chinese hamster ovary cells inhibited the stimulation of adenylylcyclase and altered the calcium regulation of the Gi2-phospholipase A2 (PLA2) effector complex. The phenotypic consequence of the activated alpha i2 mutant on hormonal stimulation of PLA2 varied depending on the cytoplasmic calcium transient elicited by different Gi2-linked receptors. The stimulation of PLA2 by thrombin, which mobilized calcium only from internal stores, was markedly attenuated in gip2-expressing cells. In contrast, the attenuation of the PLA2 response to ATP, a purinergic agonist which mobilizes calcium from both extracellular space and internal stores, was significantly less than that observed for thrombin. Ionomycin, a calcium ionophore, stimulated PLA2 activity in clones which expressed gip2 to a level similar to that observed in wild-type Chinese hamster ovary cells. Thus, the dominant GTPase-deficient gip2 polypeptide will constitutively inhibit adenylylcyclase but differentially modulate enzymes regulated by calcium and coupled to Gi2.
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PMID:GTPase-deficient G alpha i2 oncogene gip2 inhibits adenylylcyclase and attenuates receptor-stimulated phospholipase A2 activity. 190 71

A recombinant variant of hirudin, the blood-clotting inhibitor of the leech Hirudo medicinalis, has been characterized employing spectroscopic and hydrodynamic techniques. Conditions have been defined for efficient reconstitution of the native, disulfide-bonded inhibitor from completely unfolded, reduced polypeptide chains. The spectral properties of the native inhibitor are consistent with previous results on the solution structure of hirudin. Extremely low circular dichroism in the far ultraviolet ([theta]Mr,220 nm = -8 +/- 1 x 10(2) deg.cm2.dmol-1) indicates a very low content of regular secondary structure. Although both tyrosine residues of the recombinant inhibitor titrate around pH 10.6, typical for solvent-exposed tyrosines, fluorescence emission and near-ultraviolet circular dichroism suggest that at least one of the tyrosines is partially shielded from solvent quench, and immobilized in an asymmetric environment. Reversible thermal unfolding of hirudin around 65 degrees C is indicated by the disappearance of its dichroic absorption in the near ultraviolet and by a fourfold increase in ellipticity at 225 nm. The transition can be approximated by a two-state model with a transition enthalpy of delta Hvan't Hoff = 159 kJ/mol and a transition entropy of 464 J.mol-1.K-1. Reduced hirudin at room temperature is largely unfolded and inactive as an inhibitor of thrombin assayed with a low-molecular-mass substrate. Refolding and reoxidation are observed at alkaline pH in the presence of a mixture of glutathione and glutathione disulfide. Spectroscopy, thrombin inhibition, and reversed-phase HPLC indicate reconstitution yields close to 100% and that the reconstituted inhibitor is identical to the native starting material.
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PMID:Characterization, stability and refolding of recombinant hirudin. 193 81

Natural compounds have been the first historical source of antithrombotic compounds (heparin, vitamin K antagonists, streptokinase, urokinase); molecules extracted from plants or animals still provide some of the most original and promising approaches for the discovery of new drugs in this class. In this review, we will briefly describe three examples of current research trends that could lead to the development of new antithrombotic drugs of natural origin. Flavonoids have been shown to be inhibitors of cyclic nucleotide phosphodiesterase; this enzymatic activity is one of the main mechanisms of inhibition of aggregation of blood platelets by flavonoids. Some of these compounds could represent templates for the development of new inhibitors of platelet activation. Garlic (Allium sativum) has been shown to inhibit platelet aggregation in vivo and in vitro; a number of active principles has now been identified and their mechanisms of action are currently being explored. An ancient remedy, the medicinal leech (Hirudo medicinalis), has been found to contain several potent anticoagulant proteins. Among them, hirudin, a polypeptide of 65 amino acids, has been identified as one of the most potent inhibitors of thrombin. The production of sufficient amounts of hirudin through molecular biology techniques has now allowed the performance of clinical trials. These three examples show that careful consideration of biochemical, ethnopharmacological, or toxicological properties of natural products can still constitute a valuable basis for the development of new drugs.
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PMID:Old and new natural products as the source of modern antithrombotic drugs. 195 60

We describe a 20-kDa phosphorylated polypeptide, which is secreted constitutively at the apical surface of the kidney-derived Madin-Darby canine kidney cell line. Using polyclonal antibodies raised against this protein, we show that it is generated from a 60-kDa O-glycosylated, sulfated, and phosphorylated precursor protein by an intracellular proteolytic maturation step, which is pH-sensitive. Amino acid sequence analysis of the 20-kDa secreted polypeptide demonstrated that it displays 70% identity with the carboxyl-terminal amino acids of human osteopontin. The amino-terminal amino acid of the 20-kDa polypeptide corresponds to amino acid 213 of human osteopontin. Thrombin has been shown to cleave rat osteopontin in vivo and in vitro at amino acid 153, yielding two fragments of 28 and 26 kDa. A similar cleavage product can be detected by thrombin treatment of the 60-kDa precursor, suggesting that the precursor is identical or closely related to osteopontin. In the rat nephron, the protein has been localized along the luminal surfaces of the proximal and distal tubule and the collecting duct cells. These results show that in the kidney-derived cell line Madin-Darby canine kidney osteopontin or a closely related protein is proteolytically processed to a 20-kDa polypeptide, raising the possibility that diverse functions of osteopontin in various tissues might be attributed to specific processing to distinct polypeptides.
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PMID:Biosynthesis and secretion of an osteopontin-related 20-kDa polypeptide in the Madin-Darby canine kidney cell line. 199 15

The thrombin-specific inhibitor, hirudin variant rHV2-Lys 47 (rHirudin), is a 65-amino acid polypeptide produced by recombinant DNA technology in yeast. Previous studies have shown that the acidic C-terminal segment of hirudin is susceptible to enzymic degradation. To address the question of C-terminal-truncated forms of the protein in terms of by-products or metabolites, well-defined reference compounds are needed. We prepared nine derivatives by carboxypeptidase Y digestion of rHirudin followed by a two-step chromatographic purification. Liquid secondary ion mass spectrometric measurements performed on peptides collected after reversed-phase high-performance liquid chromatography showed three pure forms (1-64, 1-63 and 1-56) and three mixtures of two forms each (1-62 + 1-61, 1-58 + 1-57 and 1-55 + 1-54), which were readily distinguished from one another by their mass spectra. Further purification of these co-eluted samples was achieved by ion-exchange chromatography and their structures were confirmed by liquid secondary ion mass spectrometry. Preliminary studies conducted on intact rHirudin indicated that this is an excellent analytical tool for mass measurements of hirudin-related proteins. Indeed, it allowed rapid (within 10-15 min), precise (0.50 a.m.u. relative to expected value), reproducible (mean MH+ = 6907.64 +/- 0.42 a.m.u.), sensitive (up to 500 ng, i.e. 72 pmol) and specific measurement of the quasi-molecular ion (MH+) of the protein, and was thus readily applicable to the analysis of several derivatives.
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PMID:Liquid secondary ion mass spectrometry applied to structural confirmation of enzymically prepared C-terminal-truncated derivatives of recombinant hirudin. 202 8

Hirudin is a 65-amino acid polypeptide with three disulfide linkages. It is stable under extreme pH (1.47-12.9), high temperature (95 degrees C), and in the presence of denaturants (6 M guanidinium chloride or 8 M urea). The thrombin inhibitory activity of hirudin remains unaffected even after cleavage of an internal peptide bond (Lys36-Asn37). One condition which effectively and irreversibly inactivates hirudin is the combination of elevated temperature and alkaline pH. Structural analysis reveals that inactivation is a consequence of base-catalyzed beta-elimination of the disulfide bonds. The reaction leads to the conversion of hirudin to a mixture of highly heterogeneous polymers (from monomer to heptamer) which are intra- and intermolecularly cross-linked by cystine (20%), lanthionine (50%), and lysinoalanine (30%).
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PMID:Stability of hirudin, a thrombin-specific inhibitor. The structure of alkaline-inactivated hirudin. 204 Jun 4

We have obtained evidence that the ligand-recognition region of the integrin beta-subunit, platelet glycoprotein IIIa (GPIIIa), is discontinuous. Receptor function can be localized to residues near the N-terminus and to the central region of the polypeptide chain. The epitope recognized by our monoclonal antibody, CS-1, which substantially inhibits fibrin(ogen) binding to ADP- and thrombin-stimulated platelets [Ramsamooj, Doellgast & Hantgan (1990) Thromb. Res. 58, 577-592], is contained within residues 349-422 of GPIIIa. This sequence is adjacent to a proteinase-resistant domain of GPIIIa which is linked by disulphide bond(s) to an N-terminal segment near to the putative Arg-Gly-Asp recognition site [D'Souza, Ginsberg, Burke, Lam & Plow (1988) Science 242, 91-93]. Limited trypsin digestion of purified platelet GPIIIa yielded a mixture of two-chain molecules comprised of an N-terminal fragment disulphide-bonded to one of four fragments, which began at residues 299, 303, 353 or 423. Tryptic cleavage of the 300-422 segment correlated with loss of immunoreactivity with anti-GPIIIa monoclonal antibody, CS-1. Chymotrypsin cleavage of GPIIIa resulted in an N-terminal 19 kDa fragment joined by at least one intrachain cystine residue to a 46 kDa polypeptide beginning at residue 349. Partial reduction with dithiothreitol released the larger chymotryptic fragment with its epitope for CS-1 intact. These results have enabled us to localize the epitope recognized by our inhibitory monoclonal antibody, CS-1, to residues 349-422 of GPIIIa. Our data are consistent with a structure in which both the N-terminal and central regions of GPIIIa, which may be in close proximity in the functional GPIIb-IIIa complex, participate in ligand binding.
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PMID:Evidence that the central region of glycoprotein IIIa participates in integrin receptor function. 206 10

Factor VIII heavy chain (FVIII HC) polypeptides have been studied in both normal plasma and FVIII concentrates on exposure to three coagulation proteases. FVIII samples were incubated with labelled affinity-purified anti-FVIII Fab' fragments, immunocomplexes formed were visualized by autoradiography after sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and apparent relative molecular masses (Mr) of each band assigned. FVIII HC polypeptides were detected in all types of samples, including plasma, without further purification. Normal plasma contained a range of polypeptides with the largest dominant band at a net apparent Mr of 250-300 kD, and the smallest at 80-90 kD: the bands visualized correspond to the 90-210 kD HC species seen on conventional analysis of purified FVIII. No bands were produced from samples of haemophilic plasma. Treatment of plasma or FVIII concentrate with low concentrations (1 IU/ml) of thrombin removed the 250-300 kD and other intermediate bands, intensified then removed the 80-90 kD polypeptide and produced a band at 40-50 kD. Thrombin-associated rise and fall in FVIII clotting activity by one-stage assay correlated with intensity of the 80-90 kD polypeptide. A polypeptide of Mr 40-50 kD was also produced after incubation with activated factor X: activated factor VII plus thromboplastin had no effect on HC structure. FVIII polypeptides were visualized in prothrombin complex concentrates, with a more degraded profile seen in a deliberately 'activated' product.
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PMID:Proteolysis of factor VIII heavy chain polypeptides in plasma and concentrates. 206 61

An anticoagulant proteinase, lebetase, was isolated from the venom of Vipera lebetina by gel filtration and anion-exchange chromatography. The proteinase consists of a single polypeptide chain with a molecular weight of 23,700. Amino acid analysis indicates that lebetase contains 214 residues. It consists of several isoforms in the pH range 4.6-5.4, all having fibrinolytic activity. Lebetase hydrolyzes casein, fibrinogen and fibrin, and oxidized insulin B-chain in the positions Ala14-Leu15 and Tyr16-Leu17. Its fibrinolytic activity is inhibited by EDTA and lost upon heating at 70 degrees C for 10 min. The enzyme readily hydrolyzes the A alpha-chain and more slowly the B beta-chain of fibrinogen. In fibrin, the same chains are attacked. Thus, the enzyme is an A alpha, B beta-fibrinogenase. The fibrinolytic activity of lebetase is direct, without any plasminogen activation. E280 1% is 13.0 for lebetase. The enzyme showed low hemorrhagic activity.
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PMID:Purification and characterization of lebetase, a fibrinolytic enzyme from Vipera lebetina (snake) venom. 206 76

The effects of the vasoactive perivascular neuropeptides calcitonin gene-related peptide (CGRP), neurokinin A (NKA), neuropeptide Y (NPY), and vasoactive intestinal polypeptide (VIP) on proliferation of cultured human umbilical vein endothelial cells (HUVECs) were investigated. CGRP was shown to increase both cell number and DNA synthesis, whereas NKA, NPY, and VIP were ineffective. 125I-labeled CGRP was shown to bind to HUVECs and this binding was displaced by addition of unlabeled CGRP, suggesting the existence of specific CGRP receptors. The effect of CGRP on formation of adenosine 3',5'-cyclic monophosphate (cAMP) and inositol phosphates (InsP), two intracellular messengers known to be involved in regulation of cell proliferation, was investigated. CGRP stimulated cAMP formation but was without effect on the formation of InsP. Proliferation, as well as cAMP formation, was also stimulated by cholera toxin. Basic fibroblast growth factor stimulated growth without affecting cAMP or InsP formation, whereas thrombin, which increased InsP formation, did not stimulate proliferation. We thus suggest that CGRP may act as a local factor stimulating proliferation of endothelial cells; that the mechanism of action is associated with cAMP formation; and that this effect of CGRP may be important for formation of new vessels during physiological and pathophysiological events such as ischemia, inflammation, and wound healing.
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PMID:Calcitonin gene-related peptide stimulates proliferation of human endothelial cells. 215 44


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