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)

A fibrinogenase from Vipera lebetina venom was isolated by gel filtration in a Superose 12 column prep grade HR 16/50 and by ion-exchange in a Mono Q HR 5/5 column. The purified enzyme, which was obtained with a yield of 8 mg from 60 mg of crude venom, is a glycoprotein having an isoelectric point of 5.9 +/- 0.1 and a mol. wt of 26,000 +/- 1000 as estimated by SDS-PAGE. The biochemical characterization of the enzyme revealed that it hydrolyzes readily the B beta chain of fibrinogen and the A alpha chain as well as fibrin and casein. Over a pH range from 4 to 11 the enzyme was not inactivated by a 20 min treatment at 90 degrees C. The isolated fibrinogenase is inhibited by ethylenediamine tetraacetic acid, dithiothreitol and L-cysteine but not by phenylmethylsulfonyl fluoride. On the other hand, it is activated by Ca2+ and Mg2+. Purified fibrinogenase up to a dose of 100 micrograms/mouse shows no toxicity and has no hemorrhagic activity.
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PMID:Purification and characterization of a fibrinogenase from Vipera lebetina (desert adder) venom. 192 82

We have used the polymerase chain reaction (PCR) and differential oligonucleotide melting to screen for mutations in selected CpG dinucleotides in the factor VIII genes of haemophilia A patients. By this means we have identified and confirmed by sequencing a novel point mutation in codon 372 (CGC) of the factor VIII gene of a moderately severe CRM+ haemophiliac. The first C of this codon has been substituted by T resulting in the non-conservative substitution of cysteine for arginine at an essential thrombin cleavage site in factor VIII. Analysis of three intragenic restriction fragment length polymorphisms was uninformative in the patient's family. However, DNA analysis for the specific mutation shows one sister and the patient's mother to be carriers, and the other sister to be normal. This DNA analysis confirmed the results of phenotype analysis by factor VIII coagulant to von Willebrand factor antigen ratios for the females at risk. The two carrier females had low factor VIII coagulant activity and excess VIII antigen as predicted but the non-carrier sister also had anomalously high VIII antigen in her plasma. When feasible, mutation specific DNA analysis is able to resolve the difficulties posed by variable phenotype data and unknown level of mutation in sporadic haemophilia A.
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PMID:CRM+ haemophilia A due to a missense mutation (372----Cys) at the internal heavy chain thrombin cleavage site. 197 1

To determine the relationship between the expression of bone proteins and the formation of mineralized-tissue matrix, the biosynthesis of non-collagenous bone proteins was studied in cultures of fetal-rat calvarial cells, which form mineralized nodules of bone-like tissue in the presence of beta-glycerophosphate. The temporal pattern of protein synthesis in both mineralizing and non-mineralizing cultures was studied by metabolic labelling with [35S]methionine, 35SO4(2-) or 32PO4(3-) over a 5-day period. After a 24 h labelling period, the culture media were harvested and the cell layers extracted sequentially with aq. 0.5 M-NH3, followed by 4 M-guanidinium chloride (GdmCl), 0.5 M-EDTA and a second extraction with 4 M-GdmCl. Protein associated with collagenous bone matrix was analysed after digestion with bacterial collagenase. On the basis of [35S]methionine labelling, the major proteins extracted from the mineralizing matrix were secreted phosphoprotein-1 (SPP-1; osteopontin), bone sialoprotein (BSP) and a 14 kDa phosphoprotein. The presence of SPP-1 and BSP in the conditioned media of both mineralizing and non-mineralizing cultures and their incorporation into the mineralizing nodules indicated that these proteins associate with preformed mineral crystals. However, some BSP was also present in GdmCl extracts and, together with a 35 kDa sulphated protein, was released from a bacterial-collagenase digestion of the tissue residue in both non-mineralizing and mineralizing cultures. Two forms of sulphated SPP-1 were identified, a highly phosphorylated 44 kDa species being the predominant form in the mineralized matrix. The BSP was more highly sulphated but less phosphorylated than SPP-1. Bone SPARC (secreted protein, acid and rich in cysteine) protein (osteonectin) was present almost entirely in the conditioned media and did not incorporate 32PO4(3-) or 35SO4(2-). The SPP-1 and the 14 kDa protein were susceptible to thrombin digestion, the 44 kDa SPP-1 being specifically cleaved into 28 and 26 kDa fragments. The fragments were labelled uniformly with [35S]methionine, but the 28 kDa fragment incorporated more 35SO4(2-), but less 32PO4(3-), than the 26 kDa fragment. These studies demonstrate that SPP-1 and BSP are the major osteoblast-derived bone proteins to bind to the bone mineral. That BSP also binds to the collagenous bone matrix indicates a potential role for this protein in linking the hydroxyapatite with collagen.
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PMID:Biosynthesis of bone proteins [SPP-1 (secreted phosphoprotein-1, osteopontin), BSP (bone sialoprotein) and SPARC (osteonectin)] in association with mineralized-tissue formation by fetal-rat calvarial cells in culture. 200 15

Factor C is an endotoxin-sensitive, intracellular serine protease zymogen which initiates the coagulation cascade system in the limulus hemolymph. We have determined the entire amino acid sequence of factor C using recombinant DNA technique. The zymogen consisted of 994 amino acid residues with a calculated molecular mass of 109,648 Da. Most interestingly, factor C has five repeating units ("Sushi" domain or short consensus repeat) of about 60 amino acid residues each, which have been found in many proteins participating in the mammalian complement system. In addition to a typical serine protease domain in the carboxyl-terminal portion, characteristic segments with an epidermal growth factor-like, a lectin-like, a cysteine-rich, and a proline-rich domain were also found, revealing a unique mosaic protein structure. The serine protease domain was most analogous to human thrombin. Factor C was identified to localize in large granules in the cell, indicating that it is released from the cell by lipopolysaccharide stimulation. Furthermore, we identified a transcript possibly derived by alternative splicing of factor C mRNA, which encodes a protein sharing the amino-terminal portion of factor C. We suggest that factor C, a newly discovered type of serine protease zymogen, is a "coagulation-complement factor" which may play important roles in both hemostasis and host defense mechanisms.
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PMID:Limulus factor C. An endotoxin-sensitive serine protease zymogen with a mosaic structure of complement-like, epidermal growth factor-like, and lectin-like domains. 200 2

A cDNA corresponding to almost the entire coding region of the mRNA for the beta chain of chicken fibrinogen was sequenced. At the protein level, significant homology to the beta subunits of other vertebrate fibrinogens was found, with the highest degree of amino acid identity localized in the C-terminal region. In general, features conserved in the fibrinogens from other species also characterize the chicken sequence, including the cysteine motifs bordering an alpha-helical permissive region of fixed length and a single glycosylation site in the C-terminal region. However, the site of thrombin-catalyzed cleavage, which in other species consists of an Arg-Gly peptide bond, is instead an Arg-Ala bond in the chicken beta chain. The Ala was confirmed directly from a sequencing analysis of the purified beta chain of chicken fibrin. This finding may explain the observed slow clotting time of chicken fibrinogen relative to that of other species.
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PMID:The beta chain of chicken fibrinogen contains an atypical thrombin cleavage site. 200 66

A new case of heterozygous dysfibrinogenemia characterized by the replacement of NH2-terminal amino acid of fibrin beta-chain was found in a 50-year-old man. Despite a prolonged thrombin time, the propositus' fibrinogen had a normal reptilase time with the normal release of fibrinopeptide A. Release of fibrinopeptide B by thrombin was strongly affected, but a very high concentration of thrombin almost completely released fibrinopeptide B with a normal elution pattern on reversed-phase high performance liquid chromatography (HPLC). Lysylendopeptidase-cleavage of purified B beta-chains analyzed on HPLC showed the decrease of one peptide compared with the normal and the appearance of an abnormal peptide peak. These peptides were treated with thrombin and further separated on HPLC. Amino acid sequence analysis of the abnormal peptide demonstrated that B beta glycine-15, NH2-terminus of the fibrin beta-chain, was replaced by cysteine. These findings will be of particular importance because they strongly support the hypothesis that the NH2-terminal portion of the fibrin beta-chain is involved in the polymerization reaction by thrombin. The propositus' daughter and two sisters had the same abnormal fibrinogen. This unique inherited abnormal fibrinogen was designated as fibrinogen Ise. During these studies, we found that a very high concentration of thrombin cleaves not only the A alpha Arg19-Val20 bond but also the COOH-terminal region of alpha-chains, which results in the generation of further degraded alpha-chains with apparent molecular weights of 44,000 or less.
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PMID:A new congenital abnormal fibrinogen Ise characterized by the replacement of B beta glycine-15 by cysteine. 201 36

Binding of the synthetic peptide AAKDSDWPFAS-DEDWNYKAPSGAR, a fibrinogen alpha 27-50 analog, to thrombin was studied by inhibition assays and affinity chromatography. Peptide alpha 27-50 corresponds to a segment of human fibrinogen downstream from the thrombin cleavage site, with cysteine residues at positions 28, 36, 45 and 49 replaced by alanine. The peptide inhibited clotting of fibrinogen with an inhibition constant of 190-400 microM. Cleavage of fibrinopeptides A and B was inhibited by the peptide and the peptide was competitive with fibrinogen for thrombin. Inhibition of the small substrate tosyl-Gly-Pro-Arg-p-nitroaniline was not observed indicating that the peptide did not block the active site of the enzyme. Peptide alpha 27-50 that was covalently linked to Sepharose bound active site-inhibited thrombin at low ionic strength and was eluted at higher salt concentration. The peptide was not cleaved on overnight exposure to thrombin as determined by reverse phase HPLC. In summary, the peptide bound to, but was not a substrate for thrombin. These results suggest that this region of fibrinogen contributes to binding of thrombin.
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PMID:A synthetic analog of fibrinogen alpha 27-50 is an inhibitor of thrombin. 205 3

The molecular defect responsible for moderate and severe hemophilia A has been identified for two unrelated patients with the CRM-positive form of this disorder (factor VIII activity of 0.02 and 0.05 U/mL with factor VIII antigen of 0.87 and 2.20 U/mL). In both cases, the immunopurified dysfunctional factor VIII protein is abnormal, in that the 80 Kd light chain is not cleaved by thrombin at arginine-1689. The basis for this failure was identified by polymerase chain reaction amplification of exon 14 of the variant factor VIII genes and direct sequencing of the amplified products. In both cases, a single base substitution (C to T) was identified that produces an arginine to cysteine substitution at amino acid residue 1689. These data identify the molecular defects of the two identical factor VIII variant proteins. The dysfunctional factor VIII has been designated "Factor VIII-East Hartford," the residence of the patient in whom the defect was first identified.
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PMID:Characterization of a thrombin cleavage site mutation (Arg 1689 to Cys) in the factor VIII gene of two unrelated patients with cross-reacting material-positive hemophilia A. 210 66

We have purified factor VIII from a patient with moderately severe hemophilia A (FVIII, 4 U/dL; FVIII:Ag, 110 U/dL) and subjected the protein to Western blot analysis after time course activation with thrombin. The cross reacting material-positive (CRM+) FVIII has the normal distribution of heavy and light chains before thrombin activation, and, after incubation with the enzyme, appropriate cleavages are made at positions 740 and 1689. However, the normal thrombin cleavage at position 372 in the heavy chain of this molecule does not occur. This result is consistent with the demonstration in the patient's leukocyte DNA of a C to T transition in codon 372, leading to the substitution of a cysteine for an arginine residue at the heavy chain internal cleavage site. The severely impaired functional activity of this molecule confirms that the heavy chain of FVIII must be proteolysed in order to effect full cofactor activation in vivo. However, a threefold activation was detected when this protein was incubated with thrombin. No evidence of thrombin-mediated cleavage at position 336 in the heavy chain was detected, in contrast to the variant recombinant B domainless-molecule, FVIII 372-Ile, described by Pittman and Kaufman (Proc Natl Acad Sci USA 85:2429, 1988). Using gel permeation studies of the FVIII/von Willebrand factor (vWF) complex before and after thrombin activation, we have demonstrated that the 40 Kd A2 domain of wild type FVIII dissociates from vWF after cleavage by the enzyme. In contrast, incomplete dissociation was detected in the case of FVIII 372-Cys. We conclude that the functional defect in FVIII 372-Cys is a consequence of the resistance to proteolysis of the internal scissile bond in the heavy chain.
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PMID:Purification and characterization of factor VIII 372-Cys: a hypofunctional cofactor from a patient with moderately severe hemophilia A. 210 44

A new congenital variant of fibrinogen, from which only half the normal amount of fibrinopeptide A can be released by thrombin, was found in three members of a family having no major bleeding or thrombotic tendency. Following carboxamidomethylation of the reduced fibrinogen chains, an abnormal peptide was cleaved by thrombin from the amino terminus of the A alpha-chain (A* alpha 1-19) and isolated by reversed phase high-performance liquid chromatography. Amino acid analysis indicated the presence of carboxymethyl cysteine in this A alpha-chain fragment which in normal fibrinogen is devoid of cysteine. We conclude that fibrinogen Geneva is another fibrinogen variant with the substitution A alpha 16 Arg----Cys.
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PMID:Fibrinogen Geneva, a new case of A alpha 16 Arg----Cys dysfibrinogenaemia. 213 Sep 25


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