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:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Antithrombin activity has been identified in intact washed human platelets. An apparent activity was demonstrated at platelet concentrations above 0.31 X 10(9)/ml, when platelet suspensions were incubated with 2.0 NIH units/ml of thrombin. Neither red cells nor white cells revealed antithrombin activity. No significant loss of the platelet antithrombin activity was observed after ten successive washings or after treatment of platelets with antibodies to antithrombin III or alpha2-macroglobulin. Almost the same amount of antithrombin activity as normal platelets was demonstrated in the platelets from an afibrinogenemic patient. Pre-treatment of platelets with trypsin, papain, and neuroaminidase reduced the activity significantly, whereas lipase was without effect. The platelet antithrombin reacted with thrombin in less than 3 seconds, and this rapid reaction of platelet antithrombin was different from that of plasma antithrombin III or fibrinogen. The thrombin-like clotting activity of ancrod was inhibited by fibrinogen but not platelets. Also, unlike plasma antithrombin III or fibrinogen, brief exposure to heat (56 degrees C or 60 degrees C) reduced considerable amounts of platelet antithrombin activity. These results suggest that platelets possess a specific antithrombin with different characteristics from other known antithrombins.
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PMID:Antithrombin activity of intact human platelets. 5 97

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

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 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 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 is a major proteinase inhibitor of the blood coagulation system. Its inherited deficiency or abnormality is often associated with thromboembolism. Antithrombin "Northwick Park," a functionally inactive variant antithrombin, has recently been shown by us (Lane, D.A., Flynn, A., Ireland, H., Erdjument, H., Samson, D., Howarth, D., and Thompson, E. (1987) Br. J. Haematol. 65,451-456) to be present in plasma, in part, as a high Mr (approximately 120,000) component which has a characteristic electrophoretic mobility in agarose gels in the absence of denaturing agents. In this communication, we present evidence that this Mr approximately 120,000 variant component is comprised of an antithrombin-albumin covalent disulfide-linked complex. This proposal is supported by results of: (a) fast atom bombardment mass spectrometry of the isolated reduced, S-carboxymethylated, trypsin-digested Mr approximately 120,000 complex; (b) sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this complex and its reduced and S-carboxymethylated constituents; (c) immunoblotting of these polyacrylamide gels with antisera specific for antithrombin and albumin; (d) NH2-terminal sequence analysis of one of the isolated, S-carboxymethylated proteins that comprise the Mr approximately 120,000 complex; and (e) fast atom bombardment mass spectrometry of its tryptic peptides.
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PMID:Formation of a covalent disulfide-linked antithrombin-albumin complex by an antithrombin variant, antithrombin "Northwick Park". 365 16

Antithrombin (AT) is the principal inhibitor of thrombin in human plasma, and a member of the serine proteinase (serpin) family of proteins. Previously, we have described a point mutation in the human AT gene that converted amino acid 392 from glycine to aspartic acid which was associated with thrombotic disease in a Swedish family [(1992) Blood 79, 1428-1434]. This observation prompted us to investigate the consequences of other substitutions at this position, termed P2 with respect to the reactive centre. Site-directed mutagenesis was employed to generate seven mutants (Pro, Met, Gln, Val, Lys, Glu, and Asp), whose properties were compared with wild-type recombinant AT, following in vitro transcription and cell-free expression in a rabbit reticulocyte lysate system. With only one exception, the variant forms were less active than the wild-type in forming complexes with either alpha-thrombin, factor Xa, or trypsin. Hydrophobic (Val) or negatively charged (Asp or Glu) substitutions were particularly disruptive, in that these variants exhibited less than 10% wild-type antithrombin or antitrypsin activity. In contrast, the formation of complexes with the various proteases of the Pro variant was essentially unimpaired. We conclude that the P2 residue of AT plays a role in optimal presentation of the reactive centre to its cognate protease, and propose that the observed requirement of Gly or Pro at this position is suggestive of a bend in the polypeptide backbone that aids in this presentation.
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PMID:Site-directed mutagenesis of the P2 residue of human antithrombin. 831 64

Human plasma alpha1-antitrypsin inhibits human pancreatic trypsin, chymotrypsin and elastase, which are massively released into the blood stream during acute pancreatitis. To examine whether the plasma proteins of individuals with genetic deficiency of alpha1-antitrypsin are protected against the deleterious action of these enzymes by other inhibitors, we have tested their inhibition by alpha2-antiplasmin and antithrombin. We have determined the inhibition rate constants kass and calculated d(t), the in vivo inhibition time. Surprisingly, trypsin is inhibited faster by alpha2-antiplasmin [kass=2.5 x 10(6) M(-1)S(-1), d(t)=2.3 s] and antithrombin [kass=1.7 x 10(5) M(-1)s(-1), d(t)=5.8 s] than by alpha1-antitrypsin [d(t)=17 s or 116 s in alpha1-antitrypsin-sufficient or alpha1-antitrypsin-deficient individuals, respectively]. Low molecular weight heparin accelerates the inhibition of trypsin by antithrombin by a factor of 16 [d(t)=0.36 s]. Antithrombin and alpha2-antiplasmin are not physiological inhibitors of chymotrypsin and elastase. These enzymes are, however, physiologically inhibited by alpha1-antitrypsin and alpha1-antichymotrypsin even in alpha1-antitrypsin-deficient individuals. We conclude that (i) low molecular weight heparin may be helpful in the management of acute pancreatitis, and (ii) genetically determined alpha1-antitrypsin deficiency probably does not lead to a significantly increased risk of plasma protein degradation during this disease.
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PMID:Inhibition of human pancreatic proteinases by human plasma alpha2-antiplasmin and antithrombin. 1519 3


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