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)

Purified platelet Factor XIII was radioiodinated and then partially degraded by thrombin or trypsin, and a fibrin-binding fragment was identified by autoradiography and immunoblotting following separation by SDS/polyacrylamide-gel electrophoresis. Limited proteolysis of 125I-Factor XIII by thrombin or trypsin produced an 125I-51 kDa fragment and an unlabelled 19 kDa fragment. The 51 kDa fragment was purified by h.p.l.c. on a TSK-125 gel-filtration column. Partial amino acid sequence analysis of the 51 kDa fragment indicated that it was similar in sequence to the Gly38-Lys513 segment in placental Factor XIII a-chain. More than 70% of the 51 kDa fragment bound to fibrin, whereas the 19 kDa fragment did not bind. The active site was localized to the 51 kDa fragment since this fragment expressed transglutaminase activity, cross-linked fibrin and fibrinogen and incorporated iodo[14C]acetamide into the active-site cysteine residue. Isolation of a fibrin-binding fragment expressing transglutaminase activity demonstrates that each a-chain of the dimeric Factor XIIIa could function independently to cross-link fibrin. The fibrin-binding site could play an important role in localizing Factor XIIIa to the fibrin clot.
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PMID:Isolation of a fibrin-binding fragment from blood coagulation factor XIII capable of cross-linking fibrin(ogen). 306 50

Fibrinogen (approximately 10(-5) M) labilizes heterologous interactions within the thrombin-modified factor XIII zymogen (i.e., XIII' = a2'b2) so that, in the time frame (ca. 10 min) of normal clotting in plasma (37 degrees C, mu = 0.15, pH 7.5), 1.5 mM Ca2+ is sufficient to cause the release of the noncatalytic b subunits and also the unmasking of 1 equiv of iodo[1-14C]acetamide-titratable group per catalytic a subunit. Under similar conditions, but in the absence of fibrinogen, approximately 10 mM Ca2+ would be needed to achieve the same effect. Thus, by promoting the conversion of XIII' to XIIIa (i.e., a2'b2 leads to a2* + b2), fibrinogen functions as a physiologically important Ca2+-modulator protein. Total plasmin digests of fibrinogen display the regulatory phenomenon nearly as well as the parent protein. In an attempt to identify the structural domain on the fibrinogen which is responsible for this novel function of the molecule, it was found that two overlapping fragments derived from the midsections of the alpha chains, either by CNBr cleavage (residues 243-476) or by plasmin digestion (residues 242-424), are active.
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PMID:Alpha-chain domain of fibrinogen controls generation of fibrinoligase (coagulation factor XIIIa). Calcium ion regulatory aspects. 611 70

Partially purified (approx. 5000-fold), low molecular weight human antihemophilic factor, free of detectable Von Willebrand factor (ristocetin cofactor activity or Von Willebrand antigen), was prepared from fresh citrated plasma by limited reduction with 1 mM dithiothreitol and chromatography on Sepharose CL-4B, Sephadex G-100, and polyelectrolyte E-5. The ratio of antihemophilic factor activity to Von Willebrand factor activity or antigen was greater than 27 000 : 1. The antihemophilic factor activity could be neutralized with homologous antibody and could be further increased with thrombin. The Mr (approx. 116 000) was determined by calibrated gel permeation chromatography, electrophoresis in 5% polyacrylamide gels with sodium dodecyl sulfate and by electrophoresis in large-pore acrylamide gels without it. Since the low Mr antihemophilic factor could be prepared by treating fresh rather than fresh-frozen plasma with dithiothreitol, it was concluded that partial reduction of the antihemophilic factor with this reagent helped to maintain the antihemophilic factor in a low Mr form. When iodo[l-14C]acetamide was used to alkylate the reduced plasma proteins prior to purification, the molecular weight of the purified antihemophilic factor remained low despite numerous purification steps. By this means, one of four radioactive proteins (Mr 116 000) in the final preparation was bound specifically to homologous antihemophilic factor antibody and attributed to 14C-labeled antihemophilic factor. While the data suggest that antihemophilic factor in fresh plasma contains one or more dithiothreitol-sensitive intramolecular disulfide bonds, the possibility of disulfide linkages with other proteins(s) cannot be excluded.
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PMID:Partial purification of biologically active, low molecular weight, human antihemophilic factor free of Von Willebrand factor. I. Partial characterization and evidence for disulfide bond(s) susceptible to limited reduction. 678 56

Heparin accelerates the rate of reaction of antithrombin with thrombin, an effect which is abolished by mild reduction of the antithrombin with dithiothreitol. Reduced antithrombin incorporates 1.7 mol of [14C]acetamide/mol of protein, with cysteine as the only amino acid modified. Tryptic digestion of the reduced and alkylated antithrombin results in the formation of only two labeled peptides. In the absence of heparin, the second order rate constant for the reaction of thrombin with both reduced and native antithrombin is 5.9 to 9.6 x 10(5) M-1 min-1. In the presence of heparin, the rate constant for the reaction between reduced antithrombin and thrombin is 8.3 to 12.2 x 10(5) M-1 min-1, while the rate of reaction between native antithrombin and thrombin is too fast to follow under the conditions used. Reduced antithrombin elutes from a heparin-Sepharose column at 0.5 M NaCl, contrast to 10 M NaCl required for elution of the native protein. The intrinsic tryptophan fluorescence enhancement caused by heparin binding to native antithrombin is not observed with reduced antithrombin. These data indicate that cleavage of one of the three antithrombin disulfide bonds results in reduced affinity for heparin and the loss of heparin-accelerated antithrombin activity and imply that heparin and thrombin bind at different sites on the antithrombin molecule.
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PMID:A disulfide bond in antithrombin is required for heparin-accelerated thrombin inactivation. 736 49

Replacement of the amidinopiperidine P1 group of 3-benzylsulfonylamino-6-methyl-2-pyridinone acetamide thrombin inhibitor L-373,890 (2) with a mildly basic 5-linked 2-amino-6-methylpyridine results in an equipotent compound L-374,087 (5, Ki = 0.5 nM). Compound 5 is highly selective for thrombin over trypsin, is efficacious in the rat ferric chloride model of arterial thrombosis and is orally bioavailable in dogs and cynomolgus monkeys. The structural basis for the critical importance of both methyl groups in 5 was confirmed by X-ray crystallography.
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PMID:L-374,087, an efficacious, orally bioavailable, pyridinone acetamide thrombin inhibitor. 987 47

Thrombin and factor Xa (fXa) are the only serine proteases for which small, potent, selective, noncovalent inhibitors have been developed, which are ultimately intended as drug development candidates (in this case as anticoagulants). Noncovalent inhibitors may be more selective and chemically and metabolically less reactive than covalent inhibitors. In addition, noncovalent inhibitors are more likely to have fast-binding kinetics which is particularly important in the development of thrombin inhibitors. TAME derived noncovalent thrombin inhibitors argatroban, napsagatran, and UK 156,406 have entered clinical trials as anticoagulants, the latter as an orally active agent. Serine trap deletion from substrate-like peptides led to the development of inogatran and melagatran, both of which have entered clinical trials as intravenous agents. The use of 3-aminopyridinone and pyrazinone acetamide peptidomimetic templates has resulted in the development of L-375,378 which has been chosen for clinical development as an orally active anticoagulant. Recently, compounds which do not have the conventional hydrogen bonding capabilities of peptides have begun to appear in the thrombin literature. Publications on noncovalent fXa inhibitors cover this type of peptidomimetic almost exclusively.
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PMID:Small, noncovalent serine protease inhibitors. 1018 77

Chondroitin sulfates proteoglycans were isolated from human placenta. For the identification of enzymatic digestion products of isolated proteoglycan, strong anion, exchange-high performance liquid chromatography (SAX-HPLC) was performed. By the action of chondroitin ABC and chondroitin B lyase, three unsaturated disaccharides 2-acetamide-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-D-galactose (deltaDi-OS), 2-acetamide-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose (deltaDi-6S) and 2-acetamide-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose (deltaDi-4S) were produced from the human placenta proteoglycan. The anticoagulant activity of chondroitin sulfate proteoglycan was evaluated by activated partial thromboplastin time (aPTT) assay and thrombin time (TT) assay. The clotting times of aPTT and TT were increased from 72 to 144 sec and 19 to 27 sec, respectively. The immuno-modulating activity of chondroitin sulfate proteoglycan was examined by cell proliferation assay and these results suggest that it may play a role in suppression of the function of immune-related cells.
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PMID:Isolation and characterization of proteoglycan derived from human placenta and its biological activities. 1083 48

Use of a chlorophenoxyacetamide P1 group with a pyridinone acetamide P2/P3 gave an exceptionally potent thrombin inhibitor (K(i)=40 pM). Truncation of the molecule at the N-terminus gave unique, low nanomolar, non-covalent thrombin inhibitors which do not have a group to fill thrombin's 'distal binding pocket'. A co-crystal structure indicates the importance of an intramolecular hydrogen bond between the P1 side chain and P1/P2 amide link in this series.
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PMID:Small, low nanomolar, noncovalent thrombin inhibitors lacking a group to fill the 'distal binding pocket'. 1248 15

Recent efforts in the field of thrombin inhibitor research have focused on the identification of compounds with good oral bioavailability and pharmacokinetics. In this manuscript we describe a metabolism-based approach to the optimization of the 3-(2-phenethylamino)-6-methylpyrazinone acetamide template (e.g., 1) which resulted in the modification of each of the three principal components (i.e., P1, P2, P3) comprising this series. As a result of these studies, several potent thrombin inhibitors (e.g., 20, 24, 25) were identified which exhibit high levels of oral bioavailability and long plasma half-lives.
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PMID:Metabolism-directed optimization of 3-aminopyrazinone acetamide thrombin inhibitors. Development of an orally bioavailable series containing P1 and P3 pyridines. 1257 Mar 69

Compound I, (2-[3-[(2,2-difluoro-2(2-pyridyl)ethyl)amino]-6-methyl-2-oxohydropyrazinyl]-N-[(3-fluoro(2-pyridyl))methyl]acetamide, is a potent competitive inhibitor of thrombin that reacts stoichiometrically with the protease. Compounds of this class possess therapeutic potential as anticoagulation agents. During the metabolic characterization of compound I, evidence was obtained for extensive metabolic activation of the pyrazinone ring system. Following administration of (14)C-labeled I to rats, significant levels of irreversibly bound radioactivity to proteins were detected in rat plasma and liver. LC/MS/MS analysis of metabolites formed in rat and human liver microsomes fortified with glutathione (GSH) revealed the presence of two structurally distinct GSH adducts. It is proposed that the first of these GSH conjugates derives from a two electron oxidation of the 6-methyl-2-oxo-3-aminopyrazinone moiety to afford an electrophilic imine-methide intermediate, while the second is formed by addition of GSH to an epoxide formed by P-450-mediated oxidation of the double bond at the 5-6 position of the pyrazinone ring. The addition of GSH to the proposed epoxide facilitates opening of the pyrazinone ring and a rearrangement to afford a stable, rearranged imidazole-containing metabolite. Elucidation of the metabolic activation pathways of I provides structural guidance for the design of thrombin inhibitors with decreased potential for the generation of chemically reactive intermediates.
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PMID:Metabolic activation of a pyrazinone-containing thrombin inhibitor. Evidence for novel biotransformation involving pyrazinone ring oxidation, rearrangement, and covalent binding to proteins. 1258 91


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