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)

Three different gamma chains have been identified in fibrinogen isolated from normal human plasma with apparent molecular weights of 50000 (gamma 50), 55000 (gamma 55) and 57500 (gamma 57.5), as shown by SDS-polyacrylamide gel electrophoresis. Plasma fibrinogen was separated by ion-exchange chromatography on DEAE-Sephacel into three populations of molecules, each differing in gamma-chain composition. The first peak contained 87% of the total fibrinogen and was composed of molecules containing only gamma 50 chains; the second peak included 3% of the fibrinogen and contained one gamma 50 and one gamma 55 chain, and the third peak had 10% of the total which contained one gamma 50 and one gamma 57.5 chain. Cross-linked fibrin obtained from fibrinogen with only gamma 50 chains contained gamma gamma dimers exclusively of Mr 100000, representing a uniform gamma 50 gamma 50 dimer composition. The gamma gamma dimers from purified fibrinogen of gamma 50 gamma 55 type had molecular weights of 111000, 105000 and 100000, while dimers from gamma 50 gamma 57.5 fibrinogen were of Mr 115000, 108000 and 100000. The relative proportions of gamma gamma dimers from each purified fibrinogen population were consistent with random crosslinking of the gamma monomers. The gamma-chain identity of the variants was established by their conversion to covalent dimeric species after clotting by thrombin in the presence of Factor XIII, their incorporation of the fluorescent lysine analog dansyl cadaverine, by the staining intensity of monomeric and dimeric forms with the periodic acid-Schiff reagent after electrophoretic separation, and by plasmic degradation of all monomeric and dimeric forms in a pattern that was characteristic for gamma chains. Individual gamma gamma dimers were purified by preparative gel electrophoresis, and tyrosine was demonstrated to be the amino-terminal residue of all three gamma chain types. We conclude that normal human fibrinogen can be separated into three populations of molecules due to molecular weight heterogeneity of their gamma chains.
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PMID:Structural and chromatographic heterogeneity of normal plasma fibrinogen associated with the presence of three gamma-chain types with distinct molecular weights. 640 5

A previously undescribed gamma chain variant of human fibrinogen has been identified by application of a sensitive sodium dodecyl sulfate-polyacrylamide gradient gel electrophoretic technique to separate the polypeptide chains. This variant, called gamma B, clots and cross-links as well as the major species (gamma A), is similarly degraded by plasmin, and has a molecular weight of 53,100 as compared to 50,100 for gamma A. Cross-linked dimers of Mr = 100,100 and 108,500 are identified after action of thrombin and Factor XIIIa, suggesting the formation of gamma A-gamma A and gamma B-gamma B dimers rather than gamma A-gamma B hybrid species. The gamma B chain dimers represent 16% of the total cross-linked gamma chain forms. Two early plasmic derivatives of gamma A and gamma B chains have been demonstrated to have lost fragments of Mr = approximately 4,000 and 5,000 without loss of 5-dimethylaminonaphthalene-1-sulfonyl cadaverine fluorescence. Since the difference in molecular weight of gamma A and gamma B chains is maintained during plasmic degradation of both monomer and dimeric fluorescent forms, this suggests that the additional sequence of amino acids in gamma B is located at or near the COOH-terminal end of this polypeptide chain.
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PMID:Demonstration of a large molecular weight variant of the gamma chain of normal human plasma fibrinogen. 644 3

The formation of a fibrin clot is initiated after the proteolytic cleavage of fibrinogen by thrombin. The enzyme removes fibrinopeptides A and B and generates fibrin monomer which spontaneously polymerizes. Polymerization appears to occur though the interaction of complementary binding sites on the NH2-terminal and COOH-terminal (Fragment D) regions of the molecule. A peptide has been isolated from the gamma chain remnant of fibrinogen Fragment D1 which has the ability to bind to the NH2-terminal region of fibrinogen as well as to inhibit fibrin monomer polymerization. The peptide reduces the maximum rate and extent of the polymerization of thrombin or batroxobin fibrin monomer and increases the lag time. The D1 peptide does not interact with disulfide knot, fibrinogen, or Fragment D1, but it binds to thrombin-treated disulfide knot with a Kd of 1.45 X 10(-6) M at approximately two binding sites per molecule of disulfide knot. Fibrin monomer formed either by thrombin or batroxobin binds approximately two molecules of D1 peptide per molecule of fibrin monomer, indicating that the complementary site is revealed by the loss of fibrinopeptide A. The NH2-terminal sequence (Thr-Arg-Trp) and COOH-terminal sequence (Ala-Gly-Asp-Val) of the D1 peptide were determined. Therefore the gamma 373-410 region of fibrinogen contains a polymerization site which is complementary to the thrombin-activated site on the NH2-terminal region of fibrinogen.
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PMID:Localization of a fibrin polymerization site. 645 30

We purified and characterized the mRNAs coding for each of the three subunits of Xenopus fibrinogen. Purification was accomplished by electrophoretic separation of liver polyadenylated RNA in a fully denaturing gel, followed by recovery of the RNA from the gel via transfer to an ion-exchange membrane. This procedure yielded fractions which were highly enriched for the mRNAs for each of the fibrinogen chains. The fibrinogen mRNAs were identified by two methods: (i) in vitro translation followed by subunit-specific cleavage with the proteases thrombin and batroxobin; and (ii) cross-hybridization with cDNA clones for individual subunits of rat fibrinogen. The results demonstrate that the A alpha and gamma chains of frog fibrinogen are each coded by a single mRNA species. The A alpha mRNA is ca. 3,100 nucleotides in length, which is nearly twice the minimum size required to code for the A alpha precursor polypeptide. The gamma chain mRNA comprises about 1,600 bases and includes only a small untranslated region. In contrast, the B beta subunit is synthesized from two mRNAs, one of which is 2,500 and the other 1,800 nucleotides long. The 2,500-base mRNA includes a large noncoding region, whereas the smaller one is near the minimum required size. The larger B beta mRNA is ca, fivefold more abundant that the smaller species.
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PMID:Xenopus fibrinogen: characterization of the mRNAs for the three subunits. 651 31

Lactoperoxidase catalyzed iodination has been used to probe for differences in surface orientation of tyrosine residues in the amino-terminal disulfide knot (N-DSK) domain of fibrinogen, in N-DSK and in Fb-N-DSK prepared from fibrin. The central region of N-DSK containing the beta chain Tyr 41 and gamma chain Tyr 18 and 32 are much more susceptible to iodination than when an integral part of the fibrinogen molecule. Cleavage of the N-DSK domain from fibrinogen "loosens" up the tertiary structure of N-DSK and allows iodination of its central region. The iodination pattern of comparable tyrosine residues did not change between N-DSK and Fb-N-DSK. This result implies that no significant change occurs in the tertiary structure of N-DSK upon thrombin activation. These results favor the concept that removal of the fibrinopeptides removes a steric hindrance or exposes a binding site for polymerization.
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PMID:Comparative study of the iodination of tyrosines in the amino terminal domain of fibrinogen and in N-DSK and fibrin-N-DSK. 663 42

In this paper we describe the purification and characterization of Xenopus plasma fibrinogen and the hormonal factors which regulate synthesis and secretion of fibrinogen in liver parenchymal cells in primary culture. As in other vertebrate species, Xenopus fibrinogen is composed of three nonidentical polypeptide chains, A alpha, B beta, and gamma. In contrast to mammalian fibrinogens, the B beta chain of Xenopus fibrinogen has a higher apparent molecular weight than the A alpha chain. The gamma chain has the lowest molecular weight in the frog protein, as in that of other species. The relatively large size of the frog B beta chain results from the unusually large size of the NH2-terminal B fibrinopeptide, which is released by thrombin cleavage of fibrinogen. Hormonal regulation of fibrinogen biosynthesis was examined using a primary cell culture system. Purified Xenopus liver parenchymal cells, maintained for several weeks in a defined culture medium, gradually decrease the synthesis and secretion of fibrinogen. Sustained production of this protein is dependent upon the addition of a glucocorticoid, dexamethasone, to the culture medium. Fibrinogen production is suppressed if an estrogen, estradiol-17 beta, is added to the culture medium together with dexamethasone and triiodothyronine. The Xenopus system provides new insight into the structure of fibrinogen, the evolution of this protein, and the hormonal factors which regulate its synthesis.
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PMID:Xenopus fibrinogen. Characterization of subunits and hormonal regulation of biosynthesis. 683 68

To further evaluate the role of sialic acid in the dysfibrinogenemia associated with liver disease, we studied the effect of removal of excess sialic acid residues from the fibrinogen of five patients with liver disease on the thrombin time and fibrin monomer aggregation. Patient fibrinogens containing 1.4-3.4 residues of sialic acid per molecule in excess of normal controls, with thrombin times 12-22 sec longer than normal and with abnormal fibrin monomer aggregation, were stripped of their excess sialic acid by incubation with Vibrio cholerae neuraminidase, followed by rapid removal of the enzyme by antineuraminidase antibody affinity chromatography. These partially desialylated patient fibrinogens, with a normal number of sialic acid residues remaining, exhibited normal thrombin times and normal fibrin monomer aggregation. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of reduced normal, patient, and partially desialylated patient (sialyl-3H)-fibrinogen exhibited 60% of the radioactivity in the B beta chain and 40% in the gamma chain. There was no radioactivity detectable in the A alpha chain. These studies provide additional evidence that the increased sialic acid content of the acquired dysfibrinogenemia of liver disease is responsible for its functional defect and that the excess sialic acid is distributed on the B beta chain and gamma chains of the fibrinogen.
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PMID:The role of sialic acid in the dysfibrinogenemia associated with liver disease: distribution of sialic acid on the constituent chains. 683 20

The conformation of fibrinogen in solution has been investigated by steady-state fluorescence polarization measurements. Factor XIIIa has been employed to enzymatically incorporate 1-6 mol of dansylcadaverine/mol of fibrinogen into a specific glutamine residue near the carboxy terminus of the gamma chain and up to two sites on the alpha chain. The fluorescence emission maximum of the labeled protein is shifted to 495 nm (from 538 nm for the fluorophore in solution) and the intensity substantially enhanced, indicating the covalently linked dansyl groups residue in a hydrophobic environment in the interior of the protein. This covalent modification does not interfere with the formation of fibrin, following thrombin activation. Steady-state fluorescence polarization measurements were carried out as a function of temperature and in high viscosity solvents. The fluorescent lifetime of dansylcadaverine-fibrinogen was determined by a phase shift technique. Analysis of the data by the Perrin-Weber treatment yields a rotational relaxation time of 160 ns, considerably faster than any realistic hydrodynamic model of fibrinogen would predict. The results are discussed in terms of segmental flexibility.
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PMID:Steady-state fluorescence polarization of dansylcadaverine-fibrinogen: evidence for flexibility. 708 49

Fibrinogen (340 kDa) is a plasma protein that plays an important role in the final stages of blood clotting. Human fibrinogen is a dimer with each half-molecule composed of three different polypeptides (A alpha, 67 kDa; B beta, 57 kDa; gamma, 47 kDa). To understand the mechanism of fibrinogen chain assembly and secretion and to obtain a system capable of producing substantial amounts of fibrinogen for structure-function studies, we developed a recombinant system capable of secreting fibrinogen. An expression vector (pYES2) was constructed with individual fibrinogen chain cDNAs under the control of a Gal-1 promoter fused with mating factor F alpha 1 prepro secretion signal (SS) cascade. In addition, other constructs were prepared with combinations of cDNAs encoding two chains or all three chains in tandem. Each chain was under the control of the Gal-1 promoter. These constructs were used to transform Saccharomyces cerevisiae (INVSC1; Mat alpha his3-delta 1 leu2 trp1-289 ura3-52) in selective media. Single colonies from transformed yeast cells were grown in synthetic media with 4% raffinose to a density of 1 x 10(8) cells/ml and induced with 2% galactose for 16 h. Yeast cells expressing all three chains contained fibrinogen precursors and nascent fibrinogen and secreted about 30 micrograms/ml of fibrinogen into the culture medium. The B beta and gamma chains, but not A alpha, were glycosylated. Glycosylation of B beta and gamma chains was inhibited by treatment of transformed yeast cells with tunicamycin. Intracellular B beta and gamma chains, but not the A alpha chains in secreted fibrinogen, were cleaved by endoglycosidase H. Carbohydrate analysis indicated that secreted recombinant fibrinogen contained N-linked asialo-galactosylated biantennary oligosaccharide. Recombinant fibrinogen yielded the characteristic plasmin digestion products, fragments D and E, that were immunologically indistinct from the same fragments obtained from plasma fibrinogen. The recombinant fibrinogen was shown to be biologically active in that it could form a thrombin-induced clot, which, in the presence of factor XIIIa, could undergo gamma chain dimerization and A alpha chain polymer formation.
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PMID:Secretion of biologically active recombinant fibrinogen by yeast. 1245 84

Intermolecular end-to-middle domain pairing between a thrombin-exposed 'A' polymerization site in the central 'E' domain of fibrin, and a constitutive complementary 'a' site in each outer 'D' domain ('D:E'), is necessary but not alone sufficient for normal fibrin assembly, as judged from previous studies of a congenital dysfibrinogen, Tokyo II (gamma 275 arg-->cys), which showed defective fibrin clot assembly and a normal D:E interaction (Matsuda, M., M. Baba, K. Morimoto, and C. Nakamikawa, 1983. J. Clin. Invest. 72:1034-1041). In addition to the 'a' polymerization site, two other constitutive intermolecular association sites on fibrinogen D domains have been defined: between gamma chain regions containing the carboxy-terminal factor XIIIa crosslinking site ('gamma XL:gamma XL'); and between sites located at the outer ends of each molecule ('D:D') (Mosesson, M. W., K. R. Siebenlist, J. F. Hainfeld, and J. S. Wall, manuscript submitted for publication). We evaluated the function of these sites in Tokyo II fibrinogen, and confirmed that there was a normal fibrin D:E interaction, as determined from a normal fibrin crosslinking rate in the presence of factor XIIIa. We also found a normal gamma XL: gamma XL interaction, as assessed by a normal fibrinogen crosslinking rate. Judging from electron microscopic images, factor XIIIa-crosslinked Tokyo II fibrinogen failed to form elongated double-stranded fibrils like normal fibrinogen. Instead, it formed aggregated disordered collections of molecules, with occasional short fibrillar segments. In addition, Tokyo II fibrin formed an abnormal, extensively branched clot network containing many tapered terminating fibers. These findings indicate that the Tokyo II fibrinogen defect results in a functionally abnormal D:D self-association site, and that a normal D:D site interaction is required, in addition to D:E, for normal fibrin or fibrinogen assembly.
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PMID:The role of fibrinogen D domain intermolecular association sites in the polymerization of fibrin and fibrinogen Tokyo II (gamma 275 Arg-->Cys). 763 41


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