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)

This study reports on the tolerance and the pharmacological activity of pentosan polysulfate (PPS) administered to healthy volunteers for 10 days. Three groups of 10 subjects received either one daily injections of 100 mg of PPS by I. M. route (group I), or two daily injection of 50 mg of PPS by I. M. or S. C. route (groups II and III, respectively). In each group two random subjects received a placebo for the 10 days; on day 0, each subject was injected by a placebo. Clinical tolerance was checked by a daily physical examination; biological tolerance was assessed comparing the results of the main biochemical and haematological constants measured before starting the treatment (day 0) and 12 or 24 h after the end of the treatment (day 11). The pharmacological activity was measured on serial samples taken before treatment and between 1 and 6 h after the drug injection on days 1, 3 and 10; the results were compared to those obtained on day 0. Clinical tolerance was good. The biological side effects concern the transaminase levels and the platelet counts. An increase above the upper normal limit was observed in 18/24 and 3/24 for alanine and aspartic transaminase respectively. The mean platelet reduction ranged between 24 and 34% according to the groups. The drug injection induced a slight Quick time (PT) prolongation, no significant alteration of factors II, VII-X, V levels and of thrombin clotting time. The activated partial thromboplastin time (APTT) was significantly prolonged and there was a weak but significant circulating anti-Xa activity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Tolerance and biological activity of pentosan polysulfate after intramuscular or subcutaneous administration for ten days in human volunteers. 242 78

A 5-Gla prothrombin, adsorbable onto barium oxalate and containing an admixture of one- and two-chain molecules, has been purified from the plasma of steers on a dicoumarol regimen for three years. Double-chain molecules were not detected in any of our six other variants containing zero to nine Gla residues. In this double-chain variant, the peptide bond between Arg52-Asn appears to be missing, as the preparation showed amino-terminal alanine and aspartic acid, and released both double- and single-chain F1 upon digestion with thrombin. The molecular masses of the F1 and its heavy chain were 24,000 and 19,000 daltons, respectively. The double-chain variant was similar to its single-chain counterpart [Malhotra, O.P. (1979) Thromb. Res. 14, 439-448] in that it (a) yielded thrombin equivalent to 30% of normal in three hr, (b) moved in the alpha 1 region in the absence of calcium ions and between the alpha 1 and alpha 2 macroglobulins in the presence of calcium ions, (c) contained five gamma-carboxyglutamyl residues (Gla), and (d) showed comparable antigenic activity with normal prothrombin.
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PMID:Purification and characterization of dicoumarol-induced prothrombins. Evidence of 5-Gla variant with two (or more) polypeptide chains. 244 45

Nonporous, microparticulate, monodisperse silicas with particle diameters between 0.7 and 2.1 microns are introduced as stationary phases in high-performance affinity chromatography. The immobilization of m-aminophenylboronic acid, p-aminobenzamidine, tri-L-alanine, and concanavalin A onto these silicas was successfully achieved using 3-isothiocyanatopropyl-triethoxysilane as an activation reagent. Immobilized phenylboronic acid was applied to the isolation of nucleosides, nucleotides, and glycoprotein hormones such as bovine follicotropin and human chorionic gonadotropin, while immobilized benzamidine was employed for the isolation of the serine proteases thrombin and trypsin, immobilized tri-L-alanine for the separation of pig pancreatic elastase and human leukocyte elastase, and immobilized concanavalin A for the isolation of horseradish peroxidase. In all affinity chromatographic systems studied, the nonporous monodisperse silicas showed improved chromatographic performance compared to results obtained with porous silica supports using identical activation and immobilization procedures. Furthermore, frontal analysis was used as a method to evaluate the influence of experimental parameters on biological activity and accessible ligand densities. Only minor changes in bioactivity were found with the nonporous affinity supports, where accessibilities were typically higher than ca. 60%. The immobilization of affinity ligands onto porous supports as used in this and associated papers thus represents a successful general procedure for the preparation of stable matrices with fast kinetics for use in high-performance affinity chromatography.
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PMID:High-performance liquid affinity chromatography with phenylboronic acid, benzamidine, tri-L-alanine, and concanavalin A immobilized on 3-isothiocyanatopropyltriethoxysilane-activated nonporous monodisperse silicas. 254 22

Platelet glycoproteins IIb and IIIa function as a fibrinogen receptor on the activated platelet. We have shown that these glycoproteins can be incorporated onto the surface of phosphatidylcholine vesicles with retention of fibrinogen and antibody binding properties and can permit Ca2+ transit across the phospholipid bilayer. In the current study we demonstrate that this apparent Ca2+ channel function is specifically inhibited by the synthetic analogue of the fibrinogen gamma COOH-terminal peptide, His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val (His-12-Val), but not by the adhesive protein sequence Arg-Gly-Asp-Ser (RGDS). Prior incubation of IIb-IIIa liposomes with RGDS prevented Ca2+ transit inhibition by 25 microM His-12-Val, analogous to RGDS inhibition of His-12-Val binding to platelets. His-12-Val inhibited a minor component of transmembrane Ca2+ influx into ADP and thrombin-activated human platelets but had no effect on steady-state platelet 45Ca flux. These data indicate that ligand binding may exert a regulatory influence on transmembrane Ca2+ influx into activated platelets. The difference in inhibitory potency of the peptides studied may be related to differences in conformational changes in the glycoprotein IIb-IIIa complex induced by His-12-Val and RGDS, steric considerations, or differences in interactions with glycoprotein IIb Ca2+ binding domains.
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PMID:Ligand inhibition of the platelet glycoprotein IIb-IIIa complex function as a calcium channel in liposomes. 254 26

To clarify the mechanism which causes the coagulation time of fetal fibrinogen to be longer than that of adult fibrinogen, the N-terminal amino acid residues of fetal fibrinogen were analyzed. The results showed that the amount of A alpha chain's N-terminal alanine residues in fetal fibrinogen was only 54% of that in adult fibrinogen. When the amount of A alpha chain's N-terminal residual alanine in adult fibrinogen was decreased to 57% of the normal level by digestion with aminopeptidase M, the adult fibrinogen yielded a prolonged thrombin time and a retarded release rate for fibrino-peptides A and B, both values approximating to those of fetal fibrinogen. The results suggest that the deficiency in alanine residue at the N-terminus of the A alpha chain is a major cause of the slow coagulation of fetal fibrinogen.
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PMID:A deficiency in A alpha chain's N-terminal alanine residue as a major cause of the slow coagulation of fetal fibrinogen. 257 Nov 97

The cloning of antithrombin III (ATIII) complementary deoxyribonucleic acids and the determination of the ATIII gene structure have permitted a systematic evaluation of the molecular basis for inherited ATIII deficiencies. Sixteen kindreds with the most common form of the deficiency, in which plasma ATIII antigen levels and activity are proportionately reduced, were studied. Two polymorphic deoxyribonucleic acid markers were used to resolve parental ATIII alleles and to trace their inheritance patterns. In 15 of 16 cases, the structure of the affected ATIII allele was indistinguishable from normal, suggesting that relatively small mutations, resulting in gene inactivation, are responsible for the lower ATIII levels in these affected families. In the remaining kindred, complete deletion of one ATIII allele was seen. Also investigated was the molecular basis for a qualitative form of ATIII deficiency in a French-Canadian family with normal levels of immunoreactive protein but only half the expected levels of serine protease inhibitor activity. Using polymorphic markers, the abnormal allele was identified, cloned, and partially sequenced from the propositus. A single G----A transition was seen in the first base of codon 382, resulting in an alanine----threonine substitution in the defective protein. This mutation, together with others in this vicinity, defines a minimal length for a fully functional thrombin-binding domain.
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PMID:Molecular genetics of inherited antithrombin III deficiencies. 257 68

A monoclonal antibody (C6.7) has been generated against the calcium-replete form of human platelet thrombospondin (TSP). C6.7 is specific for TSP as determined by both competitive radioimmunoassay and immunoprecipitation. This antibody inhibits both thrombin- and A23187-induced aggregation of gel-filtered platelets in a concentration-dependent manner without affecting the secretion of serotonin. The epitope on TSP recognized by C6.7 has been localized to an 18-kDa fragment that is present in mild chymotryptic digests of TSP. This fragment is disulfide-linked to a 120- to 140-kDa fragment in unreduced digests, and both reduction and denaturation are required to separate the 18-kDa peptide from the larger fragments. A 25-kDa heparin binding domain is also present in the chymotryptic digest. However, the 18-kDa peptide is distinct from the heparin binding domain. The amino acid sequence at the NH2 terminus of the 18-kDa fragment is Asp-Thr-Asn-Pro-Thr-Arg-Ala-Gln-Gly-Tyr-.
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PMID:A monoclonal antibody against human thrombospondin inhibits platelet aggregation. 258 13

A novel protein concatenated dimer structure was generated during the folding/oxidation of inclusion bodies of recombinant bovine somatotropin synthesized in Escherichia coli. The structure of this dimeric molecule was determined by peptide mapping with trypsin, and limited proteolysis by thrombin. Peptide mapping demonstrated that the two disulfide pairs in bovine somatotropin dimer were identical to those in monomer. Limited proteolysis with thrombin resulted in the cleavage of only a single peptide bond between arginine-132 and alanine-133 in bovine somatotropin dimer. This single peptide bond cleavage was sufficient to convert this dimer to a monomeric molecular weight species as analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and HPLC. Since the single cleaved peptide bond is present in the large disulfide loop of bovine somatotropin, these data demonstrate that the dimeric molecule exists as a novel concatenated structure through the interlocking of the disulfide loops of this protein.
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PMID:A novel concatenated dimer of recombinant bovine somatotropin. 269 95

Thrombin Quick II is one of two dysfunctional forms of thrombin derived from the previously described congenital dysprothrombin prothrombin Quick. Thrombin Quick II does not clot fibrinogen, hydrolyze p-nitroanilide substrates of thrombin, or bind N2-[5-(dimethylamino)naphthalene-1-sulfonyl]arginine N,N-(3-ethyl-1,5-pentanediyl)amide, a high-affinity competitive inhibitor of thrombin. To determine the structural alteration in thrombin Quick II, the reduced, carboxymethylated protein was hydrolyzed by a lysyl endopeptidase. A peptide not present in a parallel thrombin hydrolysate was identified by reverse-phase chromatography. The peptide was purified by rechromatography and subjected to Edman degradation which showed that Gly-558 of human prothrombin had been replaced by Val. This corresponds to a point mutation of the Gly codon GGC to GUC. This Gly residue, which is highly conserved in the chymotrypsin family of serine proteases, forms part of the substrate binding pocket for bulky aromatic and basic side chains in chymotrypsin and trypsin, respectively. However, in porcine elastase 1, the corresponding residue is threonine. Consistent with the identified structural alteration, thrombin Quick II incorporates [3H]diisopropyl fluorophosphate stoichiometrically and hydrolyzes the elastase substrate succinyl-Ala-Ala-Pro-Leu-p-nitroanilide with a relative kcat/KM of 0.14 when compared to thrombin. This results from a 3-fold increase in KM and a 2.5-fold decrease in kcat for thrombin Quick II when compared to thrombin acting on the same substrate. These results and those of other investigators studying mutant trypsins support the conclusion that the catalytic activity of serine proteases is very sensitive to structural alterations in the primary substrate binding pocket.
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PMID:Substitution of valine for glycine-558 in the congenital dysthrombin thrombin Quick II alters primary substrate specificity. 271 46

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


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