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)

Ecotin, a serine protease inhibitor found in the periplasm of Escherichia coli, has been characterized as an extremely potent anticoagulant and reversible tight-binding inhibitor of human factor Xa (FXa). The ecotin gene was cloned by PCR, highly expressed in E. coli, and purified from the E. coli periplasm. The binding of ecotin to FXa was stoichiometric with an equilibrium dissociation constant Ki of 54 pM. The association rate constant was 1.35 x 10(6) M-1 s-1, and the dissociation rate constant, measured in the presence of human leukocyte elastase (HLE) to prevent reassociation of ecotin with FXa, was 6.5 x 10(-5) s-1. Ecotin prolonged clotting time ca. 10-fold at 0.3 microM and at 2 microM in activated partial thromboplastin time and prothrombin time assays, respectively. Ecotin did not effectively inhibit the human plasma proteases thrombin, tissue factor.factor VIIa, factor XIa, activated protein C, plasmin, or tissue plasminogen activator (t-PA); however, it did potently inhibit factor XIIa, plasma kallikrein, HLE, and bovine trypsin and chymotrypsin. Coincubation of ecotin and FXa at 10 microM each resulted in a (ecotin)2.(FXa)2 complex as determined by gel filtration. Dimerization of ecotin alone was measured by fluorescence titration which yielded a Kd of ca. 390 nM. FXa cleaved ecotin slowly at pH 4.0 between M84 and M85. Replacement of the P1 Met84 residue with Arg and Lys led to FXa inhibitors with Ki values of 11 and 21 pM, respectively. The P1 Arg and Lys mutants also significantly inhibited thrombin, factor XIa, activated protein C, plasmin, factor XIIa, kallikrein, and bovine trypsin and chymotrypsin but did not inhibit tissue factor.factor VIIa, t-PA, or HLE.
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PMID:Ecotin is a potent anticoagulant and reversible tight-binding inhibitor of factor Xa. 814 99

A series of new peptidyl (alpha-aminoalkyl)phosphonate diphenyl esters containing the 4-amidinophenyl group were synthesized and tested as irreversible inhibitors for thrombin and other trypsin-like enzymes. These phosphonates irreversibly inhibited several coagulation enzymes and trypsin. Boc-D-Phe-Pro-(4-AmPhGly)P(OPh)2 is the best human thrombin inhibitor in the series with a k(obs)/[I] value of 11,000 M-1 s-1, and it inhibits thrombin more than 5-fold more effectively than the other enzymes tested. Z-(4-AmPhGly)P(OPh)2 is the best inhibitor for plasma kallikrein with a k(obs)/[I] value of 18,000 M-1 s-1. Generally, the (4-AmPhGly)P(OPh)2 derivatives are better inhibitors of thrombin and trypsin than the corresponding (4-AmPhe)P(OPh)2 derivatives which contain an extra CH2 separating the amidinophenyl group from the peptide backbone. The amidino phosphonates did not inhibit acetylcholinesterase and were chemically stable in neutral buffers. In addition, the inhibited trypsin derivative did not regain any enzyme activity after removal of excess inhibitor and incubation in a pH 7.5 buffer for 1 day. Boc-D-Phe-Pro-(4-AmPhGly)P(OPh)2 and D-Phe-Pro-(4-AmPhe)P(OPh)2 prolonged the prothrombin time ca. 2-fold and prolonged the activated partial thromboplastin time ca. 3-4-fold in human plasma at concentrations of 63 and 125 microM, respectively. The novel amidine-containing peptidyl phosphonates reported here are thus effective anticoagulants in vitro, and they may have utility for use in vivo.
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PMID:Novel amidine-containing peptidyl phosphonates as irreversible inhibitors for blood coagulation and related serine proteases. 829 9

Cyclotheonamide A (CA), a cyclic peptide isolated from the marine sponge of the genus Theonella was shown to be a slow-binding inhibitor of several trypsin-like serine proteinases. Values of 4.6 x 10(4), 4.8 x 10(4), 9.3 x 10(3), 2.1 x 10(3) and 2.7 x 10(2) M-1 s-1 were determined for the second-order rate constants for formation of CA complexes with thrombin, trypsin, plasmin, 2-chain t-PA and factor Xa, respectively. The equilibrium constant (Ki) was measured for dissociation of CA from the CA complex with human thrombin (Ki = 1.0 nM), bovine trypsin (Ki = 0.2 nM), human plasmin (Ki = 12 nM), human factor Xa (Ki = 50 nM) and human 2-chain tissue plasminogen activator (t-PA) (Ki = 40 nM). CA produces dose dependent increases in clotting time assays. The clotting time in the thrombin time, activated partial thromboplastin time and prothrombin time assays, were doubled by 1.5, 0.9 and 48 microM CA, respectively. A model for the binding of CA to the active site of thrombin is proposed.
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PMID:Inhibition of thrombin and other trypsin-like serine proteinases by cyclotheonamide A. 832 86

Increases in intrinsic fluorescence (delta I), reflecting changes in tryptophan environments, occur upon bond cleavages necessary for prothrombin (II) activation to thrombin (IIa) by prothrombinase. Cleavage at Arg274-Thr275 (numbering based on bovine prothrombin sequence, with chymotrypsinogen numbering in braces) between the amino-terminal fragment 1.2 and protease (Pre2) domains of prothrombin yields delta I = 5%, and cleavage within the Pre2 domain at Arg323-Ile324 to form IIa yields delta I = 35%, while cleavage at both yields delta I = 25%. Since the change in fluorescence upon activation of prothrombin can be largely attributed to a change within the Pre2 domain, the susceptibilities of each of the 9 Trp residues of IIa and its immediate precursor Pre2 to oxidation by N-bromosuccinimide (NBS) were compared. Pre2 and IIa were titrated with increasing amounts of NBS (0.5-5 equiv of NBS/TRP), aliquots were removed and fully digested with trypsin, and tryptophan-containing peptides were separated and quantitated by RP-HPLC with fluorescence detection. Tryptic digests yielded 9 tryptophan-containing peptides, which were identified by amino acid composition. Tryptophan residues in IIa and Pre2 displayed a 10-fold range of sensitivity to modification. Tryptophans 337 and 360 (W29, W51) were modified less readily in IIa than in Pre2, while residues 373, 542, and 550 (W60D, W207, W215) were modified more readily, and other residues were equally susceptible. Residues 360 and 373 (W29, W60D) flank the active site histidine. From the crystal structure, residues 373 and 550 (W60D, W215) are implicated in substrate binding.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Structural changes in the protease domain of prothrombin upon activation as assessed by N-bromosuccinimide modification of tryptophan residues in prethrombin-2 and thrombin. 845 46

The wealth of structural information now available for thrombin, its precursors, its substrates, and its inhibitors allows a rationalization of its many roles. alpha-thrombin is a rather rigid molecule, binding to its target molecules with little conformational change. Comparison of alpha-thrombin with related trypsin-like serine proteinases reveals an unusually deep and narrow active site cleft, formed by loop insertions characteristic of thrombin. This canyon structure is one of the prime causes for the narrow specificity of thrombin. The observed modularity of thrombin allows a diversity in this specificity; its "mix-and-match" nature is exemplified by its interactions with macromolecules (Fig. 20). The apposition of the active site to a hydrophobic pocket (the apolar binding site) on one side and a basic patch (the fibrinogen recognition exosite) on the other allows for a fine tuning of enzymatic activity, as seen for fibrinogen. Thrombin receptor appears to use the same sites, but in a different way. Protein C seems only able to interact with thrombin if the recognition exosite is occupied by thrombomodulin. These two sites are also optimally used by hirudin, allowing the very tight binding observed; thrombin inhibition is effected by blocking access to the active site. On the other hand, antithrombin III makes little use of the recognition exosite; instead, its interactions are tightened with the help of heparin, which binds to a second basic site (the heparin binding site). Thrombin's modularity is a result of the conjunction of amino acid residues of like properties, such as charge or hydrophobicity. The charge distribution plays a role, not only in the binding of oppositely charged moieties of interacting molecules, but also in selection and preorientation of them. Nonproteolytic cellular properties are attributed to 1) the rigid insertion loop at Tyr60A, and 2) a partially inaccessible RGD sequence. The former can interact with cells in the native form; the latter would appear to be presented only in an (at least partially) unfolded state. The membrane binding properties of prothrombin can be understood from the ordered arrangement of calcium ions on binding to the Gla domain. Kringle F2 binds to thrombin at the heparin binding site through charge complementarity; a conformational change appears to occur on binding. The observed rigidity of the thrombin molecule in its complexes makes thrombin ideal for structure based drug design. Thrombin can be inhibited either at the active site or at the fibrinogen recognition exosite, or both.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:A player of many parts: the spotlight falls on thrombin's structure. 846 68

Warfarin overdose leads to hypoprothrombinemia and bleeding diathesis. We report here the case of a 47 year old woman who ingested an overdose of a non-steroidal anti-inflammatory drug, sold in Mexico under the name of Wobenzym (R), and containing, according to the manufacturer: pancreatin, bromelin, papain, lipase, amylase, trypsin, alpha chymotrypsin and rutin. She developed skin, urinary and gastrointestinal bleeding and was found to be apparently under the effect of a coumadin overdose, i.e. prolonged prothrombin time, prolonged activated thromboplastin time, and low functional and antigenic levels of prothrombin. A platelet count, and the thrombin, reptilase and bleeding times were normal. All laboratory and clinical abnormalities reverted to normal by using fresh frozen plasma and parenteral vitamin K. In addition, we were able to show that the commercial preparation could prolong the prothrombin time in rabbits and, by high-performance liquid chromatography, a pike consonant with purified coumadin was found in the drug. It is concluded that this drug is probably contaminated by coumadin, and that physicians must be aware of its potential side effects.
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PMID:[Probable coumarin poisoning upon ingestion of an anti-inflammatory agent]. 896 87

A simple and efficient activation-affinity purification system was developed to obtain thrombin from recombinant CHO cells expressing human prothrombin. In this method, a controllable process for the activation of recombinant prothrombin is directly coupled with a purification strategy for the recombinant thrombin generated. At a constant flow rate and with a contact time limited to few seconds, recombinant prothrombin was filtered through immobilized trypsin. In a closed flow system, the recombinant thrombin generated was filtered through newly designed thrombin-specific affinity gels. Hirudin, the most specific thrombin inhibitor, and hirudin-based peptides were covalently immobilized to Sepharose, thus creating thrombin-specific affinity gels that immediately absorb the thrombin generated from the activation mixture. Prothrombin and incompletely activated molecules did not bind to the affinity gel and were recirculated for a further activation cycle. Due to the specificity of the affinity gels for thrombin and the elimination of thrombin from the activation mixture, proteolytic degradation and autocatalytic inactivation of the recombinant thrombin was prevented. Recombinant thrombin was isolated from the hirudin-based affinity gels by chaotrope salt elution, resulting in high yields of highly pure, active thrombin. Affinity purification of thrombin was not deleteriously affected by contamination of the starting material with other proteins. Activation and affinity purification were equally effective for recombinant and human plasma-derived prothrombin as well as for human and recombinant thrombin.
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PMID:Immobilized hirudin and hirudin-based peptides used for the purification of recombinant human thrombin prepared from recombinant human prothrombin. 881 54

Factor VII is a vitamin K-dependent zymogen of a serine protease that participates in the initial phase of blood coagulation. A factor VII molecular variant (factor VII Central) was identified in a 24-year-old male with severe factor VII deficiency and whose plasma factor VII antigen was 38% of normal, but expressed <1% factor VII procoagulant activity. DNA sequence analysis of the patient's factor VII gene revealed a thymidine to cytidine transition at nucleotide 10907 in exon VIII that results in a novel amino acid substitution of Phe328 to Ser. The patient was homozygous for this mutation, whereas each parent of the patient was heterozygous for this mutation. To investigate the molecular properties of this variant, a recombinant F328S factor VII mutant was prepared and analyzed in relation to wild-type factor VII. F328S factor VII exhibited <1% factor VII procoagulant activity and a 2-fold decreased affinity for tissue factor and failed to activate factor X or IX in the presence of tissue factor following activation by factor Xa. In addition, F328S factor VIIa exhibited no detectable amidolytic activity in the presence of tissue factor. The rate of F328S factor VII activation by factor Xa was markedly decreased relative to the rate of wild-type factor VII activation as revealed by densitometry scanning of SDS gels. Temporal analysis of this reaction by SDS-polyacrylamide gel electrophoresis also revealed the formation of two novel F328S factor VII degradation products (40 and 9 kDa) resulting from factor Xa proteolysis of the Arg315-Lys316 peptide bond in intact F328S factor VII. Computer modeling and molecular dynamics simulations of the serine protease domain of factor VIIa suggested that the inability of F328S factor VIIa to cleave substrates may result from the apparent formation of a hydrogen bond between Tyr377 and Asp338, a residue at the bottom of the substrate-binding pocket important for the interaction of substrate arginine side chains with the enzyme. These findings suggest that Phe328, which is conserved in prothrombin, factor IX, factor X, factor VII, and trypsin, is important for factor VIIa catalysis.
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PMID:Factor VII central. A novel mutation in the catalytic domain that reduces tissue factor binding, impairs activation by factor Xa, and abolishes amidolytic and coagulant activity. 894 45

The Erythrina variegata Kunitz family trypsin inhibitors, ETIa and ETIb, prolonged the activated partial thromboplastin time (APTT) and also the prothrombin time (PT) of human plasma, but the Kunitz family chymotrypsin inhibitor, ECI, and Bowman-Birk family inhibitor, EBI, from E. variegata hardly prolonged these times. Trypsin inhibitors ETIa and ETIb inhibited the amidolytic activity of factor Xa, and ETIb but not ETIA inhibited plasma kallikrein. Neither ETIa nor ETIb exhibited any inhibitory activity toward beta-factor XIIa and thrombin. Furthermore, trypsin inhibitors ETIa and ETIb inhibited plasmin, a serine proteinase in the fibrinolytic system, whereas ECI and EBI did not. These results indicate that Erythrina Kunitz proteinase inhibitors possess different potency toward serine proteinases in the blood coagulation and fibrinolytic systems, in spite of their high similarity in amino acid sequence.
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PMID:Inhibitory potency of Erythrina variegata proteinase inhibitors toward serine proteinases in the blood coagulation and fibrinolytic systems. 898 61

Beta2-glycoprotein I (beta2GPI) is a plasma glycoprotein with unknown physiological function(s). In in vitro experiments it has been demonstrated that beta2GPI has both anticoagulant properties, such as the inhibition of factor X and prothrombin activation and procoagulant properties, such as the inhibition of the anticoagulant activity of activated protein C. Besides this, beta2GPI bound to cardiolipin is recognized by antiphospholipid antibodies (aPL). In this study we demonstrate that beta2GPI is very sensitive for cleavage between Lys317 and Thr318 by plasmin, resulting in two immunologically different cleaved forms. In vitro experiments show that these plasmin cleaved forms of beta2GPI bind to negatively charged phospholipids with much lower affinity compared to intact beta2GPI. Similar to plasmin, trypsin and elastase can also induce this proteolytical cleavage in beta2GPI, whereas thrombin and factor Xa do not cleave beta2GPI. The in vivo occurrence of the proteolytical cleavage was demonstrated by the finding that in plasmas of patients with disseminated intravascular coagulation (DIC) and in plasmas of patients treated with streptokinase, significant amounts of cleaved beta2GPI (up to 12 microg/ml) are present. During the development of DIC, the increase in levels of cleaved beta2GPI is accompanied by a 70% decrease in the levels of intact beta2GPI, whereas in streptokinase treated patients levels of intact beta2GPI stay within the normal range. This study demonstrates for the first time that during in vivo activation of fibrinolysis beta2GPI is cleaved. which results in the formation of a form of beta2GPI with much lower affinity for negatively charged phospholipids. Plasmin is most likely responsible for this modification.
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PMID:Beta2-glycoprotein I is proteolytically cleaved in vivo upon activation of fibrinolysis. 1034 17


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