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)

Hirulog-1 [D-Phe-Pro-Arg-Pro-[Gly]4-desulphohirudin-(53-64) (HV1)] was designed to bind by its first four and last 12 residues to the alpha-thrombin catalytic site and anion-binding exosite for fibrin(ogen) recognition respectively, with a [Gly]4 bridge and an Arg-Pro bond at the scissional position. Human alpha-, gamma- and zeta-thrombins, as well as bovine trypsin, readily hydrolyse Spectrozyme-TH (D-hexahydrotyrosyl-Ala-Arg p-nitroanilide) at pH 7.4 and approx. 23 degrees C. Both alpha- and zeta-thrombins, which have high fibrinogen-clotting activities (greater than 3000 kunits/g), were inhibited with this substrate by hirulog-1 [Ki = 2.56 +/- 0.35 nM (n = 3) and 1.84 +/- 0.15 nM (n = 3) respectively] and slowly cleaved the inhibitor [k = 0.326 +/- 0.082 min-1 (n = 12) and 0.362 +/- 0.056 min-1 (n = 18) respectively], whereas gamma-thrombin, which has essentially no clotting activity (approx. 4 kunits/g), and trypsin were not inhibited with greater than 1000-fold molar excess of hirulog-1. Similar inhibition parameters were also obtained for hirulog-1 incubated with alpha-thrombin or zeta-thrombin at approx. 23 degrees C and by measuring thrombin activity with fibrinogen in the clotting assay at 37 degrees C. Cleavage of the Arg-3-Pro-4 bond in hirulog-1 by either alpha- or zeta-thrombin was shown by identical cleavage products of either thrombin on h.p.l.c. and by sequence analysis of the alpha-thrombin products. These data demonstrate that hirulog-1 is a specific inhibitor of thrombin forms with high fibrinogen-procoagulant activities and that its Arg-3-Pro-4 bond is slowly cleaved by these thrombin forms.
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PMID:Thrombin-specific inhibition by and slow cleavage of hirulog-1. 144 27

Thrombomodulin is an endothelial glycoprotein that serves as a cofactor for protein C activation. To examine the ligand specificity of human thrombomodulin, we performed equilibrium binding assays with human thrombin, thrombin S205A (wherein the active site serine is replaced by alanine), meizothrombin S205A, and human factor Xa. In competition binding assays with CV-1(18A) cells expressing cell surface recombinant human thrombomodulin, recombinant wild type thrombin and thrombin S205A inhibited 125I-diisopropyl fluorophosphate-thrombin binding with similar affinity (Kd = 6.4 +/- 0.5 and 5.3 +/- 0.3 nM, respectively). However, no binding inhibition was detected for meizothrombin S205A or human factor Xa (Kd greater than 500 nM). In direct binding assays, 125I-labeled plasma thrombin and thrombin S205A bound to thrombomodulin with Kd values of 4.0 +/- 1.9 and 6.9 +/- 1.2 nM, respectively. 125I-Labeled meizothrombin S205A and human factor Xa did not bind to thrombomodulin (Kd greater than 500 nM). We also compared the ability of thrombin and factor Xa to activate human recombinant protein C. The activation of recombinant protein C by thrombin was greatly enhanced in the presence of thrombomodulin, whereas no significant activation by factor Xa was detected with or without thrombomodulin. Similar results were obtained with thrombin and factor Xa when human umbilical vein endothelial cells were used as the source of thrombomodulin. These results suggest that human meizothrombin and factor Xa are unlikely to be important thrombomodulin-dependent protein C activators and that thrombin is the physiological ligand for human endothelial cell thrombomodulin.
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PMID:Ligand specificity of human thrombomodulin. Equilibrium binding of human thrombin, meizothrombin, and factor Xa to recombinant thrombomodulin. 131 33

According to present models, thrombin activates platelets by cleaving its receptors after Arg41, creating a new N terminus which acts as a tethered ligand. In support of this model, a peptide (SFLLRNPNDKYEPF or TRP42/55) corresponding to residues 42-55 has been shown to activate the receptor. In the present studies, the structural basis for thrombin receptor activation was examined using fragments of this peptide, as well as variants of the peptide with selected amino acid substitutions. The results show that the features of SFLLRNPNDKYEPF required to mimic the effects of thrombin reside within the first 6 residues, SFLLRN. A hexapeptide comprised of these residues was approximately 5 times more potent than the parent peptide in assays of platelet aggregation and, in addition, caused tyrosine phosphorylation, inhibition of cAMP formation, and an increase in cytosolic Ca2+. Omission of either the Ser residue or the Arg and Asn residues greatly diminished peptide activity, as did the substitution of Ala for Phe or Arg. Substitution of Ala for Ser or the initial Leu, on the other hand, had little adverse effect. The inactive peptides SALLRN and NPNDKYEPF had no effect on platelet activation initiated by SFLLRN, but FLLRN inhibited platelet aggregation in response to both SFLLRN and thrombin. These results suggest that within SFLLRN the Phe and Arg residues are particularly important and that Phe must be preceded by another amino acid, the identity of which is not tightly constrained. This observation and comparisons with the homologous domains of proteins whose tertiary structure is known were used to predict the conformation of the SFLLR sequence. The model which emerged suggests that the SFLLR domain may be part of an extended beta structure in the intact receptor and that cleavage by thrombin causes it to contract and assume a modified helical configuration. In this predicted conformation the side chains of Phe and Arg point in the same direction, potentially into a pocket formed by the remainder of the receptor.
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PMID:Structure-function relationships in the activation of platelet thrombin receptors by receptor-derived peptides. 131 29

To elucidate the role of the COOH-terminal region of antithrombin III, we studied the effects of synthetic peptides corresponding to its sequence on the amidolytic and proteolytic activities of thrombin and Factor Xa in the presence or absence of the inhibitor, antithrombin III. The peptides ANRPFLVFI and IIFMGRVANP corresponding to residues Ala404 to Ile412 and Ile420 to Pro429, respectively, blocked the inhibition by antithrombin III. The effect of IIFMGRVANP was reduced in the presence of heparin. Both peptides at a concentration of 1 mM blocked complex formation between antithrombin III and thrombin or Factor Xa. The two peptides, particularly IIFMGRVANP, directly enhanced the amidolytic activity of thrombin and Factor Xa on the synthetic substrate Boc-Ala-Gly-Arg-MCA (where Boc is t-butoxycarbonyl and MCA is 4-methylcoumarin), which corresponds to residues P3-P1 of the reactive site of antithrombin III, and also on other substrates due to increased Vmax. IIFMGRVANP also shortened the thrombin-induced fibrinogen clotting time, whereas ANRPFLVFI inhibited the thrombin-catalyzed activation of protein C both in the presence and absence of thrombomodulin. The direct effect of ANRPFLVFI and IIFMGRVANP on thrombin was confirmed by enhancement of the incorporation of dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide into thrombin. These findings suggest that the COOH-terminal region of antithrombin III interacts with thrombin and Factor Xa to increase the reactivity of the enzyme, which may enhance acyl-bond formation between the inhibitor and the enzyme.
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PMID:The role of the COOH-terminal region of antithrombin III. Evidence that the COOH-terminal region of the inhibitor enhances the reactivity of thrombin and factor Xa with the inhibitor. 133 Oct 47

Cleavage of the human platelet thrombin receptor by thrombin exposes a new N-terminal which acts as a putative tethered ligand. A synthetic peptide--"SFLL" (SFLLRNPNDKYEPF), corresponding to the new N-terminal region, activates and induces platelet aggregation and serotonin secretion. We have found that the pentapeptide--SFLLR is the minimal peptide length which retains full activity in inducing [14C]serotonin secretion. Structure-activity relationship studies were performed on this pentameric peptide. Systematic replacement of all amino acids with L-Ala indicated the importance of F-2, L-3 and R-5 for activity. Further studies demonstrated that the positive charge at the N-terminus, but not at the C-terminus of the pentapeptide, is crucial for activity.
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PMID:Structure-activity studies of the thrombin receptor activating peptide. 133 4

Buried water molecules in the structurally homologous family of eukaryotic serine proteases were examined to determine whether buried waters and their protein environments are conserved in these proteins. We found 16 equivalent water sites conserved in trypsin/ogen, chymotrypsin/ogen, elastase, kallikrein, thrombin, rat tonin and rat mast cell protease, and 5 additional water sites in enzymes which share the primary specificity of trypsin. Based on an alignment of 30 serine protease sequences, it appears that the protein environments of these 21 conserved buried waters are highly conserved. The protein environments of buried waters are comprised primarily of atoms from highly conserved residues or main chain atoms from nonconserved residues. In one instance, the protein environment of a water is conserved even in the presence of an unlikely Pro/Ala substitution. We also note 3 instances in which a histidine side chain substitutes for water, suggesting that the structural role of water at these sites is satisfied by the presence of an alternative hydrogen bonding partner. Buried waters appear to be integral structural components of these proteins and should be incorporated into protein structures predicted on the basis of sequence homology to this family, including the catalytic domains of coagulation proteases.
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PMID:Buried water in homologous serine proteases. 133 31

Structural studies on a hereditary abnormal prothrombin, prothrombin Salakta, have been performed to identify the difference responsible for its reduced fibrinogen clotting activity and its reduced esterase activity. Amino acid composition and sequence analyses of a peptide isolated from a lysylendopeptidase digest of the abnormal thrombin indicated that Glu-466 had been replaced by Ala. This amino acid substitution can result from a single nucleotide change in the codon for Glu-466 (GAG----GCG). The model building and the molecular dynamics simulation of thrombin Salakta suggest that the Glu-466----Ala substitution would change the proper conformation around the substrate binding site containing Trp-468, which is a unique surface loop on the thrombin molecule. This is the experimental and theoretical evidence supporting the role of the surface loop containing Trp-468 for the proper conformation of the substrate binding site.
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PMID:Prothrombin Salakta: substitution of glutamic acid-466 by alanine reduces the fibrinogen clotting activity and the esterase activity. 135 85

Matrix metalloproteinase 9 (MMP-9) has been purified as an inactive zymogen of M(r) 92,000 (proMMP-9) from the culture medium of HT 1080 human fibrosarcoma cells. The NH2-terminal sequence of proMMP-9 is Ala-Pro-Arg-Gln-Arg-Gln-Ser-Thr-Leu-Val-Leu-Phe-Pro, which is identical to that of the 92-kDa type IV collagenase/gelatinase. The zymogen can be activated by 4-aminophenylmercuric acetate, yielding an intermediate form of M(r) 83,000 and an active species of M(r) 67,000, the second of which has a new NH2 terminus of Met-Arg-Thr-Pro-Arg-(Cys)-Gly-Val-Pro-Asp-Leu-Gly-Arg-Phe-Gln-Thr- Phe-Glu. Immunoblot analyses demonstrate that this activation process is achieved by sequential processing of both NH2- and COOH-terminal peptides. TIMP-1 complexed with proMMP-9 inhibits the conversion of the intermediate form to the active species of M(r) 67,000. The proenzyme is fully activated by cathepsin G, trypsin, alpha-chymotrypsin, and MMP-3 (stromelysin 1) but not by plasmin, leukocyte elastase, plasma kallikrein, thrombin, or MMP-1 (tissue collagenase). During the activation by MMP-3, proMMP-9 is converted to an active species of M(r) 64,000 that lacks both NH2- and COOH-terminal peptides. In addition, HOCl partially activates the zymogen by reacting with an intermediate species of M(r) 83,000. The enzyme degrades type I gelatin rapidly and also cleaves native collagens including alpha 2 chain of type I collagen, collagen types III, IV, and V at undenaturing temperatures. These results indicate that MMP-9 has different activation mechanisms and substrate specificity from those of MMP-2 (72-kDa gelatinase/type IV collagenase).
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PMID:Matrix metalloproteinase 9 (92-kDa gelatinase/type IV collagenase) from HT 1080 human fibrosarcoma cells. Purification and activation of the precursor and enzymic properties. 140 Apr 81

Site-specific substitutions of the first five amino acids of the thrombin inhibitor hirudin have been made and the effects of these substitutions on the kinetics of formation of the thrombin-hirudin complex evaluated. The effects of different substitutions of Val1 indicate that nonpolar interactions play a major role in the binding of this residue. In the second position (Val2), polar amino acids were better accommodated than in the first. The mutant with arginine in the second position bound particularly well to thrombin; its dissociation constant was 9-fold lower than that of wild-type recombinant hirudin. Comparison of the effects of single and double mutations involving Val1 and Val2 indicates that there was no cooperativity in the binding of these two residues. Elimination of the hydrophobic interactions made by the aromatic ring of Tyr3 of hirudin resulted in a large loss of binding energy (12.7 kJ mol-1). Replacement of Thr4 of hirudin by serine and alanine suggested that both the gamma-methyl and the hydroxyl group of the threonine were important in the stabilization of the thrombin-hirudin complex. Replacement of Asp5 of hirudin by alanine and glutamate caused about the same loss in binding energy (5 kJ mol-1). The effects of site-specific substitutions are discussed in terms of the crystal structure of the thrombin-hirudin complex. Molecular modeling provided plausible explanations for many of the observed effects. For instance, such studies suggested that the improved binding of the mutant with arginine in the second position could be due to an interaction of the arginine with the primary specificity pocket.
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PMID:Interaction of the N-terminal region of hirudin with the active-site cleft of thrombin. 158 11

In an abnormal fibrinogen (fibrinogen Naples) associated with congenital thrombophilia we have identified a single base substitution (G----A) in the B beta chain gene that results in an amino acid substitution of alanine by threonine at position 68 in the B beta chain of fibrinogen. The propositus and two siblings were found to be homozygous for the mutation, whereas the parents and another sibling were found to be heterozygous. Individuals homozygous for the defect had a severe history of both arterial and venous thrombosis; heterozygous individuals had no clinical symptoms. The three homozygotes had a prolonged thrombin clotting time in plasma, whereas the heterozygotes had a normal thrombin clotting time. Fibrinopeptide A and B (FpA and FpB) release from purified fibrinogen by human alpha-thrombin was delayed in both the homozygous propositus and a heterozygous family member. Release of FpA from the normal and abnormal amino-terminal disulfide knot (NDSK) corresponded to that found with the intact fibrinogens, indicating a decreased interaction of thrombin with the NDSK part of fibrinogen Naples. Binding studies showed that fibrin from homozygous abnormal fibrinogen bound less than 10% of active site inhibited alpha-thrombin as compared with normal fibrin, while fibrin formed from heterozygous abnormal fibrinogen bound approximately 50% of alpha-thrombin. These results suggest that the mutation of B beta Ala 68----Thr affects the binding of alpha-thrombin to fibrin, and that defective binding results in a decreased release of FpA and FpB in both homozygous and heterozygous abnormal fibrinogens.
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PMID:Molecular basis of fibrinogen Naples associated with defective thrombin binding and thrombophilia. Homozygous substitution of B beta 68 Ala----Thr. 163 10


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