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)

The X-ray crystallographic structure of Ac-(D)Phe-Pro-boroArg-OH [DuP714, Ki = 0.04 nM; Kettner, C., Mersinger, L., & Knabb, R. (1990) J. Biol. Chem. 265, 18289] complexed with human alpha-thrombin shows the boron atom covalently bonded to the side-chain oxygen of the active site serine, Ser195. The boron adopts a nearly tetrahedral geometry, and the boronic acid forms a set of interactions with the protein that mimic the tetrahedral transition state of serine proteases. Contributions of the arginine side chain to inhibitor affinity were examined by synthesis of the ornithine, lysine, homolysine, and amidine analogs of DuP714. The basic groups interact with backbone carbonyl groups, water molecules, and an aspartic acid side chain (Asp189) located in the thrombin S1 specificity pocket. The variation in inhibition constant by 3 orders of magnitude appears to reflect differences in the energetics of interactions made with thrombin and differences in ligand flexibility in solution.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Kinetic and crystallographic studies of thrombin with Ac-(D)Phe-Pro-boroArg-OH and its lysine, amidine, homolysine, and ornithine analogs. 789 72

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

A major component of thrombus formation is the activation of blood platelets and binding of soluble fibrinogen. Glycoprotein IIb-IIIa (GPIIb-IIIa) is the dominant receptor on platelets mediating this activity. This study describes how GPIIb-IIIa is differentially displayed on resting and activated human platelets and how this display is alternatively masked and enhanced by arginine-glycine-aspartic acid (RGD) containing or mimicking compounds. Results indicate that thrombin strongly increases GPIIb-IIIa display on platelet membrane surfaces while other platelet activators enhance GPIIb-IIIa display to lesser degrees. This upregulated display was masked by compounds RGDS, GPenGRGDSPCA and L-367,073. Conversely, membrane GPIIb-IIIa display was markedly enhanced on otherwise resting platelets by incubation with these same compounds. This study indicates that small ligands that bind to RGD recognition sites can modulate GPIIb-IIIa membrane surface display differentially, depending on the nature of their cellular targets.
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PMID:Ligand interactions with the RGD recognition site alter binding of GPIIb-IIIa reactive monoclonal antibody to human platelets. 837 15

Mammalian aspartyl-tRNA synthetase occurs in the multienzyme complex of aminoacyl-tRNA synthetases, while bacterial and yeast aspartyl-tRNA synthetases exist as free soluble enzymes. Cloning and sequencing of mammalian aspartyl-tRNA synthetase revealed a newly evolved N-terminal 32-amino-acid sequence, which contains a putative amphiphilic helix (Jacobo-Molina, A., Peterson, R., and Yang, D. C. H. (1989) J. Biol. Chem. 264, 16608-16612). Human aspartyl-tRNA synthetase (hDRS) and an N-terminal 32-residue truncated form of human aspartyl-tRNA synthetase (hDRS delta 32) were expressed in Escherichia coli under the control of the inducible tac promoter as glutathione-S-transferase (GST) fusion proteins linked through a thrombin cleavage site. The GST-hDRS fusion protein and the GST-hDRS delta 32 were purified by affinity chromatography on glutathione-agarose and were fully active in aspartylation of mammalian tRNA. After cleavage of GST from the fusion proteins by thrombin, hDRS and hDRS delta 32 were purified by affinity chromatography on tRNA-Sepharose. Both hDRS and hDRS delta 32 were present as a mixture of monomeric and dimeric forms. GST-hDRS formed high molecular weight aggregates while GST-hDRS delta 32 was a dimeric protein. Both hDRS and hDRS delta 32 bound to hydrophobic interaction gels such as aminohexyl-agarose. In the absence of propylene glycol, hDRS bound to amino-hexyl-agarose weaker than hDRS delta 32, but, in the presence of 50% propylene glycol, hDRS bound tighter than hDRS delta 32. Both hDRS and hDRS delta 32 were fully active in aspartylation of mammalian tRNA and ATP-PPi exchange. In comparison to the N-terminal truncated form, the full-length enzyme showed greater thermal stability and ATP-PPi exchange activity but lower aminoacylation activity. The catalytic constant of hDRS delta 32 for aminoacylation of tRNA was 2-fold higher than that of hDRS. The Michaelis-Menten constants for aspartic acid and tRNAAsp were 302 microM and 13 nM for hDRS, and 29 microM and 130 nM for hDRS delta 32, respectively. These results suggest that the newly evolved N-terminal peptide in hDRS may modulate the enzymatic activity, the stability, and the chromatographic behavior of hDRS. The structure and function of the N-terminal peptide in aspartyl-tRNA synthetase and in the synthetase complex will be discussed.
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PMID:Expression of human aspartyl-tRNA synthetase in Escherichia coli. Functional analysis of the N-terminal putative amphiphilic helix. 844 60

A thrombin-like enzyme, calobin, has been purified to homogeneity from the venom of Agkistrodon caliginosus by a procedure involving Bio-Gel P-100, Mono S, and Pro-RPC. The enzyme was identified as a monomer with a molecular weight of 34,000 on SDS-PAGE, and its isoelectric point was 6.2. Calobin acted on fibrinogen to form fibrin with a specific activity of 226 NIH equivalent units, and also exhibited arginine esterase activity. The enzyme predominantly cleaved the alpha-chain of fibrinogen with little degradation of the beta-chain. It contained abundant asparagine/aspartic acid residues, but very few tyrosine or methionine residues. The proteolytic activity of the enzyme with TAME as a substrate was higher than that of thrombin. However, it showed neither lysine esterase nor caseinolytic activity. The enzyme activity was strongly inhibited by PMSF, and moderately by benzamidine and soybean trypsin inhibitor, indicating it is a serine protease. On the other hand, the enzyme activity was not inhibited by hirudin or aprotinin. cDNA (1.6 kb) for calobin has been cloned from an A. caliginosus cDNA library. The cDNA sequence indicates that calobin is synthesized as a pre-zymogen of 262 amino acids, including a putative secretory signal peptide of 18 amino acids and a proposed zymogen peptide of 6 amino acid residues. The cDNA sequence encodes a 238-amino acid residue molecule exhibiting strong amino acid sequence homology to those of ancrod, batroxobin, and flavoxobin isolated from other snake venoms. Calobin contains 12 cysteine residues. As judged on alignment of the amino acid sequences of other thrombin-like enzymes (batroxobin, ancrod, and flavoxobin), calobin constitute the formation of six disulfide bridges. Amino acid residues, His43, Asp88, and Ser182, which are thought to be the catalytic active site are highly conserved. As calobin is a glycoprotein, its possible glycosylation site, Asn-X-Thr, is located at amino acid residues 81-83.
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PMID:Purification and molecular cloning of calobin, a thrombin-like enzyme from Agkistrodon caliginosus (Korean viper). 879 81

Lipoprotein lipase (LPL) is made by several cell types, including macrophages within the atherosclerotic lesion. LPL, a dimer of identical subunits, has high affinity for heparin and cell surface heparan sulfate proteoglycans (HSPGs). Several studies have shown that cell surface HSPGs can mediate cell binding to adhesion proteins. Here, we tested whether LPL, by virtue of its HSPG binding could mediate monocyte adhesion to surfaces. Monocyte binding to LPL-coated (1-25 micrograms/mL) tissue culture plates was 1.4- to 7-fold higher than that of albumin-treated plastic. Up to 3-fold more monocytes bound to the subendothelial matrix that had been pretreated with LPL. LPL also doubled the number of monocytes that bound to endothelial cells (ECs). Heparinase and heparitinase treatment of monocytes or incubation of monocytes with heparin decreased monocyte binding to LPL. Heparinase/heparitinase treatment of the matrix also abolished the LPL-mediated increase in monocyte binding. These results suggest that LPL dimers mediate monocyte binding by forming a "bridge" between matrix and monocyte surface HSPGs. Inhibition of LPL activity with tetrahydrolipstatin, a lipase active-site inhibitor, did not affect the LPL-mediated monocyte binding. To assess whether specific oligosaccharide sequences in HSPGs mediated monocyte binding to LPL, competition experiments were performed by using known HSPG binding proteins. Neither antithrombin nor thrombin inhibited monocyte binding to LPL. Next, we tested whether integrins were involved in monocyte binding to LPL. Surprisingly, monocyte binding to LPL-coated plastic and matrix was inhibited by approximately 35% via integrin-binding arginine-glycine-aspartic acid peptides. This result suggests that monocyte binding to LPL was mediated, in part, by monocyte cell surface integrins. In summary, our data show that LPL, which is present on ECs and in the subendothelial matrix, can augment monocyte adherence. This increase in monocyte-matrix interaction could promote macrophage accumulation within arteries.
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PMID:Lipoprotein lipase can function as a monocyte adhesion protein. 926 Dec 75

The platelet-specific integrin alphaIIb beta3 achieves a high affinity binding state in response to extracellular agonists such as thrombin, ADP, or collagen. During this activation, the receptor undergoes a number of conformational changes. To characterize the different conformations of alphaIIb beta3, we expressed recombinant alphaIIb beta3 in human embryonic kidney (HEK) 293 cells. Antigenic and peptide recognition specificities of the full-length recombinant receptor resembled those of the native receptor in platelets. We used an array of peptidic and nonpeptidic arginine-glycine-aspartic acid (RGD) mimics that specifically bind to human platelet alphaIIb beta3 to determine the affinity state of the receptor. Some of these RGD mimics were previously shown to clearly discriminate between resting and activated alphaIIb beta3. Solution-phase binding of these RGD mimics to the recombinant cells suggested that in HEK 293 cells the full-length alphaIIb beta3 is expressed in a "transitional" activation state. This observation was confirmed by the binding of the activation-specific, monoclonal anti-alphaIIb beta3 antibody PAC1 to cells expressing the full-length recombinant alphaIIb beta3. Deletion of the entire cytoplasmic domain of the beta subunit was sufficient to convert the receptor in HEK 293 cells to a fully active form, as found in activated platelets. In addition, the full-length receptor was capable of mediating agonist-independent aggregation of cells in the presence of fibrinogen. Thus, by using RGD mimics, we have identified a functional transitional activation state of alphaIIb beta3 that is capable of mediating fibrinogen-dependent cell aggregation.
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PMID:Arginine-glycine-aspartic acid mimics can identify a transitional activation state of recombinant alphaIIb beta3 in human embryonic kidney 293 cells. 927 45

We have adapted the ultrasonic interferometry technique (Echo-Cell), which was initially designed to study red blood cell aggregation and agglutination, to the detection of human platelet microaggregates. The experimental parameter chosen was the slope of the signal over the first 5 minutes of sedimentation. We compared our new method with the conventional aggregometry for the measurement of aggregates after thrombin-, collagen-, and epinephrine-induced platelet activation. Under these conditions we demonstrated the particular sensibility of the present method in detecting small platelet aggregates induced in the first phase of aggregation and formed by low concentrations of agonists. Furthermore, as an illustration of this method, we showed an inhibition of the formation of thrombin-induced platelet aggregates in a concentration-dependent manner by the well known antagonist arginine-glycine-aspartic acid-serine with a median inhibitory concentration of 0.4 micromol/L, which is 30 times lower than the median inhibitory concentration found by aggregometry.
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PMID:Measurement of human platelet microaggregates by a new method: ultrasonic interferometry. 927 63

A 64-year-old white male was referred for evaluation of prolonged prothrombin time (PT) and activated partial thromboplastin time (aPTT) obtained before elective surgery with initial PT and PTT results of 14.9 and 38.4 seconds, respectively, which corrected to normal in 1:1 mixes with normal plasma. Functional prothrombin assay indicated a level of 51% with thromboplastin as an activator. The prothrombin antigen was 102%. This discordance in the functional and immunologic prothrombin levels was evidence for dysprothrombinemia. Western blotting showed that thrombin was formed at a normal rate in diluted plasma consistent with a mutation within the thrombin portion of prothrombin. DNA was isolated from leukocytes and the thrombin exons were amplified by polymerase chain reaction, cloned, and sequenced. For exon 13, eight clones were sequenced with four clones showing a point mutation in the codon for Arg517, which would result in substitution by Gln. Arg517 is part of the Arg-Gly-Asp(RGD) sequence in thrombin and contributes to an ion cluster with aspartic acid residues 552 and 554. Mutation at this residue most probably distorts the structure of the Na+ binding site in thrombin. This is the first report indicating the critical role of Arg517 in the normal physiological interaction of thrombin with fibrinogen. This dysprothrombin is designated Prothrombin Greenville.
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PMID:Prothrombin Greenville, Arg517-->Gln, identified in an individual heterozygous for dysprothrombinemia. 949 Jun 87

A murine monoclonal antibody to the zymogen form of human protein C that blocks protein C activation by thrombin-thrombomodulin both in vitro and in vivo has been humanized using the consensus and resurfacing methods. While the binding of the parent murine antibody to protein C is calcium-dependent (1.5-2-fold decrease in binding without calcium), the humanized antibody exhibited a significant increase in its calcium-dependence (5-fold decrease in binding without calcium). Two exposed human framework residues in the variable light domain of the humanized antibody, aspartic acid L1 and glutamine L3, are responsible for the increase in calcium-dependence.
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PMID:Humanization of an antibody against human protein C and calcium-dependence involving framework residues. 968 Jan 94


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