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)

We determined the role of specific thrombin "exosites" in the mechanism of inhibition by the plasma serine proteinase inhibitors heparin cofactor II (HC) and antithrombin (AT) in the absence and presence of a glycosaminoglycan by comparing the inhibition of alpha-thrombin to epsilon- and gamma T-thrombin (produced by partial proteolysis of alpha-thrombin by elastase and trypsin, respectively). All of the thrombin derivatives were inhibited in a similar manner by AT, either in the absence or presence of heparin, which confirmed the integrity of both heparin binding abilities and serpin reactivities of epsilon- and gamma T-thrombin compared to alpha-thrombin. Antithrombin activities of HC in the absence of a glycosaminoglycan with alpha-, epsilon, and gamma T-thrombin were similar with rate constants of 3.5, 2.4, and 1.2 x 10(4) M-1 min-1, respectively. Interestingly, in the presence of glycosaminoglycans the maximal inhibition rate constants by HC with heparin and dermatan sulfate, respectively, were as follows: 30.0 x 10(7) and 60.5 x 10(7) for alpha-thrombin, 14.6 x 10(7) and 24.3 x 10(7) for epsilon-thrombin, and 0.017 x 10(7) and 0.034 x 10(7) M-1 min-1 for gamma T-thrombin. A hirudin carboxyl-terminal peptide, which binds to anion-binding exosite-I of alpha-thrombin, dramatically reduced alpha-thrombin inhibition by HC in the presence of heparin but not in its absence. We analyzed our results in relation to the recently determined x-ray structure of D-Phe-Pro-Arg-chloromethyl ketone-alpha-thrombin (Bode, W., Mayr, I., Baumann, U., Huber, R., Stone, S. R., and Hofsteenge, J. (1989) EMBO J. 8, 3467-3475). Our results suggest that the beta-loop region of anion-binding exosite-I in alpha-thrombin, which is not present in gamma T-thrombin, is essential for the rapid inhibition reaction by HC in the presence of a glycosaminoglycan. Therefore, alpha-thrombin and its derivatives would be recognized and inhibited differently by HC and AT in the presence of a glycosaminoglycan.
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PMID:Role of thrombin exosites in inhibition by heparin cofactor II. 174 Apr 13

Antithrombin (AT) Vicenza has been previously identified as a functionally abnormal antithrombin associated with familial thrombosis (Finazzi et al, 1985). It binds normally to heparin, but loses its affinity following interaction with thrombin: it is a poor inhibitor of thrombin. AT Vicenza was isolated from plasma by heparin-Sepharose and thrombin-Sepharose chromatography, fragmented with cyanogen bromide (CNBr) and its tryptic peptides were analysed by fast atom bombardment mass spectrometry mapping. An abnormal peptide mass 1112 was identified. Edman degradation confirmed a substitution of Ala to Pro in the sequence Ala 383-Arg 393. Polymerase chain reaction amplification of exon 6 of the gene followed by genomic sequencing, localized the mutation to codon 384, GCA to CCA. The same mutation has recently been reported in AT Charleville (Mohlo-Sabatier et al, 1989). Sodium dodecyl-sulphate polyacrylamide gel electrophoresis of AT Vicenza (/Charleville) under non-reducing conditions revealed an apparent increase in mol. wt following interaction with thrombin: under reducing conditions the mol. wt was less than that of normal AT. This indicated cleavage and unfolding of the molecule. The site of cleavage was determined by incubation of AT Vicenza (/Charleville) with thrombin-Sepharose, reduction and S-carboxymethylation and reverse phase FPLC. A peptide was identified with the NH2-terminal sequence beginning Ser-Leu-Asn, demonstrating the cleavage had occurred at the reactive site of the variant. It is concluded that the Ala 384 to Pro substitution transforms AT Vicenza (/Charleville) from an inhibitor into a substrate.
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PMID:Antithrombin Vicenza, Ala 384 to Pro (GCA to CCA) mutation, transforming the inhibitor into a substrate. 199 1

A new water-soluble color reagent, 4-N,N-dimethylaminoazobenzene-4'-isothiocyano-2'-sulfonic acid (S-DABITC), was used to identify lysine residues of antithrombin III which participate in the binding of heparin. Antithrombin, modified with S-DABITC in the presence and absence of low molecular weight heparin (Mr 5000) was reduced, carboxymethylated, and digested with trypsin. The digest was analyzed by high-performance liquid chromatography and monitored at 465 nm. In the absence of heparin, four major colored peptides (T1, T2, T3, and T4) were identified. When antithrombin was preincubated with heparin (2-fold by weight), followed by S-DABITC modification, the recovery of peptide T4 remained unchanged, but the recoveries of T1, T2, and T3 were reduced by 93, 86, and 98%, respectively. In addition, a new colored peptide, TA, appeared. Amino acid sequencing of peptides T1, T2, T3, and TA localized S-DABITC modification sites as Lys-136, Lys-125, Lys-107, and Lys-236, respectively. Thus, binding of heparin to human antithrombin diminished S-DABITC modification at Lys-107, Lys-125, and Lys-136, but at the same time enhanced S-DABITC modification at Lys-236. This phenomenon was further characterized by varying the molar ratio of heparin/antithrombin (from 0.04 to 20). The shielding of Lys-125 and Lys-136 was inversely proportional to the activation of Lys-236. At a heparin/antithrombin molar ratio of 1, the extent of shielding of Lys-125 and Lys-136 and the unmasking of Lys-236 were 25-33%. This shielding-unmasking effect correlated with enhanced antithrombin inhibition of thrombin. We conclude that Lys-107, Lys-125, and Lys-136 are situated within the heparin-binding site of human antithrombin and that binding of heparin to antithrombin causes a conformational change of antithrombin that leads to the exposure of Lys-236 for S-DABITC modification.
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PMID:Binding of heparin to human antithrombin III activates selective chemical modification at lysine 236. Lys-107, Lys-125, and Lys-136 are situated within the heparin-binding site of antithrombin III. 249 30

Antithrombin is a major plasma protein inhibitor of proteinases generated during blood coagulation; it plays an important role in the regulation of thrombin in blood. The anticoagulant heparin greatly accelerates the rate of inactivation of proteinases by antithrombin, predominantly through its well defined, highly specific binding reaction with the inhibitor, but also through a less strictly defined interaction with some of the proteinases (such as thrombin). There is evidence for an analogous acceleratory mechanism in vivo, that functions by the binding of antithrombin to a subpopulation of heparan sulphate proteoglycans intercalated in the surface of endothelial cells. The location and structure of the gene for antithrombin are known. Both its overall organization and the structure of the subdomains of the expressed protein can be considered in terms of their relationships to a serine proteinase inhibitor superfamily, which is believed to have evolved from a common ancestor. The region of the antithrombin gene 5' to the coding region has been characterized. Unlike other members of the serpin family, there is no TATA-like promoter sequence. Two enhancer sequences have been identified that are homologous to enhancer regions of other genes. There are two polymorphisms: an intragenic polymorphism arising from a translationally silent A to G transition in codon 305, and a length polymorphism arising from the presence of 32 bp or 108 bp non-homologous sequences 345 bp upstream from the translation initiation codon. Inherited deficiency of antithrombin is associated with familial thromboembolism. The molecular genetic basis of some subtypes of deficiency is increasingly yielding to investigation. It is interesting to note that a number of mutations have been identified in CpG dinucleotides, supporting the suggestion that this dinucleotide sequence may represent a mutation hotspot in the human genome.
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PMID:Antithrombin: structure, genomic organization, function and inherited deficiency. 268 61

Antithrombin is a plasma protein inhibitor that can be grouped within a serine proteinase inhibitor superfamily. Antithrombin Pescara is a functional variant of antithrombin found in a family with a high incidence of thrombosis. Preliminary functional analysis has suggested that the abnormality resides in the reactive site rather than in the heparin binding domain of the molecule. Accordingly, we have isolated the variant from plasma using heparin-Sepharose chromatography, followed by chromatography upon thrombin-Sepharose to remove the normal antithrombin that is present (the propositus is heterozygous for the variant). The variant protein was reduced, S-carboxy-methylated, and fragmented with CNBr. A pool ("CNBr pool 4") containing the reactive site region was isolated by reverse-phase high performance liquid chromatography and sequentially treated with trypsin and V8 protease. Fast atom bombardment-mass spectrometric analysis of this subdigest identified a novel peptide of mass 1708. Four steps of Edman degradation together with further analysis by fast atom bombardment-mass spectroscopy identified the NH2-terminal sequence of this peptide as Ala-Ala-Ala-Ser. The mass of the novel peptide and its changing mass in response to Edman degradation are only compatible with its identity as Ala382-Arg399, with the reactive site Arg393 replaced by Pro. Using specific oligonucleotide hybridization, we demonstrated that the molecular defect of antithrombin Pescara is caused by a CGT to CCT mutation in codon 393. These findings may be of broad interest, as other members of the serine protease inhibitor superfamily contain arginine at their reactive sites and may be expected to undergo a similar mutation.
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PMID:A novel amino acid substitution in the reactive site of a congenital variant antithrombin. Antithrombin pescara, ARG393 to pro, caused by a CGT to CCT mutation. 272 64

Antithrombin Chicago is a functionally inactive antithrombin variant whose inheritance is associated with thrombotic disease. The variant antithrombin was isolated from plasma of the propositus by chromatography on heparin-Sepharose, followed by passage through thrombin-Sepharose to remove the normal antithrombin component that is present. A pool of fragments ("CNBr pool 4") containing the reactive site region was prepared from the reduced and S-carboxymethylated variant by cleavage with cyanogen bromide followed by reverse-phase HPLC. Sequential treatment of CNBr pool 4 with trypsin and V8 protease produced peptides whose molecular masses were then determined by fast atom bombardment mass spectrometry. The variant protein digests were characterised by a reduction of a peptide of mass 1086, corresponding to the normal antithrombin sequence Ala382-Arg393. However, they contained a peptide of mass 1748, which arises when Arg393 is replaced by His in the sequence Ala382-Arg399. It is concluded that the functional and clinical abnormalities of antithrombin Chicago are all probably caused by a single amino acid substitution, Arg393 to His.
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PMID:Antithrombin Chicago, amino acid substitution of arginine 393 to histidine. 278 9

Antithrombin is a protease inhibitor that neutralizes the activity of the serine proteases of the coagulation cascade, such as factors IXa, Xa, XIa, XIIa, and thrombin by forming a 1:1 stoichiometric complex between enzyme and inhibitor via a reactive site (arginine)-active center (serine interaction). Heparin binds to lysyl residues on antithrombin and accelerates the rate of complex formation. Studies of the binding parameters and kinetic characteristics of the heparin-antithrombin-hemostatic enzyme interactions have revealed that binding of heparin to antithrombin is responsible for a approximately 1000-fold acceleration of the thrombin-antithrombin or factor IXa-antithrombin and factor Xa-antithrombin interactions (allosteric effect). The reactions between free thrombin or free factor IXa and heparin provide an additional 4- to 15-fold enhancement in the rate of these processes (approximation effect) and account for 1-2% of the total rate of enhancement. It has been shown that commercial heparin is composed of anticoagulantly active and anticoagulantly inactive species. The anticoagulantly active mucopolysaccharide contains a unique antithrombin-binding site. Anticoagulantly inactive heparin does not possess this structure and does not bind to the protease inhibitor. Anticoagulantly active heparin also contains a critical region required for the acceleration of the various enzyme-inhibitor interactions. The two different domains of the heparin molecule interact with separate areas of antithrombin and induce distinct conformational transitions within the protease inhibitor. Anticoagulantly active heparinlike molecules (most likely a heparan sulfate with an appropriate sequence for anticoagulant activity) are found on the luminal surface of the endothelium. This heparinlike substance appears to alter the conformation of antithrombin in a manner virtually identical to that of commercial heparin. Both anticoagulantly active heparin and inactive heparin are able to suppress smooth muscle cell proliferation in vitro and in vivo and can reverse the effects of mitogenic factors such as platelet-derived growth factor. Furthermore, it has been shown that bovine aortic endothelial cells produce heparinlike molecules with growth inhibitory potency.
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PMID:Role of heparin and heparinlike molecules in thrombosis and atherosclerosis. 315 97

Antithrombin Northwick Park and antithrombin Glasgow are functionally variant antithrombins with impaired abilities to interact with thrombin. Thrombosis is associated with their inheritance. Both of the purified, reduced, and S-carboxymethylated variant antithrombins were treated with cyanogen bromide and the major pools of each containing the amino acid sequence Gly339-Met423 were isolated. Following treatment of these pools with trypsin, fast atom bombardment mass spectrometry identified tryptic peptides (found also in normal antithrombin treated in the same way) that corresponded to amino acid sequences Gly339-Lys370 and Val400-Met423. The tryptic peptides, corresponding to amino acid sequences Ala371-Arg393 and Ser394-Arg399 were present in both variant preparations in greatly reduced amounts compared to a normal antithrombin preparation. However, two novel tryptic peptides of molecular mass (M + H)+ 2976 and 2952 were identified in the digests of antithrombin Northwick Park and Glasgow, respectively. Further analyses of these novel tryptic peptides were carried out by V8 protease treatment and sequential Edman degradation coupled with mass spectrometric analysis of the shortened peptides. This established that these peptides comprised the amino acid sequence Ala371-Arg399, but with single amino acid substitutions at the reactive site, Arg393 replaced by Cys (in antithrombin Northwick Park) and by His (in antithrombin Glasgow).
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PMID:Single amino acid substitutions in the reactive site of antithrombin leading to thrombosis. Congenital substitution of arginine 393 to cysteine in antithrombin Northwick Park and to histidine in antithrombin Glasgow. 316 33

Antithrombin Milano is an unusual antithrombin variant, exhibiting an abnormal, fast moving component on crossed immunoelectrophoresis (in the absence of heparin). Antithrombin isolated from the propositus could be resolved into two peaks on anion-exchange chromatography; antithrombin Milano peak 1 of Mr approximately 60,000 which could inhibit thrombin, and antithrombin Milano peak 2 of Mr approximately 120,000 which was inactive. The latter component also reacted with antisera to both antithrombin and albumin on immunoblotting. Under reducing conditions, the approximately 120,000 Mr component migrated on SDS-PAGE as two distinct bands with Mr approximately 60,000, one of which reacted with antiserum to antithrombin and the other (of slower mobility) of which reacted with antiserum to albumin only. These and other results established the approximately 120,000 Mr component to be an inactive, disulphide-linked variant antithrombin and albumin complex. The variant antithrombin was isolated, following reduction and S-carboxymethylation, by reverse-phase HPLC and then it was fragmented with CNBr. A major CNBr pool containing the sequence Gly339-Met423 was treated with trypsin, followed by V8 protease. The resulting peptides were analysed by fast atom bombardment mass spectrometry (Fab-MS) mapping. A peptide of molecular mass 1086, corresponding to the normal sequence Ala382-Arg393, was almost absent from the mass spectrum, but an additional peptide of mass number 1772 was present. These results are almost identical to those found in another variant antithrombin, Northwick Park (Erdjument et al., J Biol Chem, 262: 13381, 1987; Erdjument et al., J. Biol Chem, 263: 5589-5593, 1988), indicating the same single amino acid substitution of Arg393 to Cys.
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PMID:Antithrombin Milano, single amino acid substitution at the reactive site, Arg393 to Cys. 323 50

Antithrombin Glasgow is a hereditary abnormal antithrombin that has lost thrombin inhibitory activity. It was isolated from the plasma of a 41-year-old male with a history of thrombotic events. Antithrombin Glasgow was purified from plasma using heparin-Sepharose chromatography at pH 7.4 eluting with increasing concentrations of NaCl. The normal protein eluted with 0.9 mol/l NaCl and Glasgow with 1.05 mol/l NaCl. Electrophoresis in agarose at pH 8.6 showed the variant to migrate more anodally than normal. The C-terminal small fragment resulting from catalytic cleavage with elastase between P3 and P4 of the reactive loop was isolated and sequenced. This showed the replacement of the arginine at residue 3 by a histidine. This is residue 393 in the intact molecule. The findings suggest that heparin, on binding, interacts indirectly with the reactive centre region of antithrombin.
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PMID:Antithrombin Glasgow, 393 Arg to His: a P1 reactive site variant with increased heparin affinity but no thrombin inhibitory activity. 336 Jan 40


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