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)

Fibrinogen fragment D1 was converted to fragment D3 by plasmic digestion. This conversion eliminates the ability of the fragment to interact with thrombin-exposed sites on fibrin monomer. Peptides released during this plasmic digestion were assayed for the presence of a polymerization site by affinity chromatography on fibrin monomer-Sepharose. We found that a 33-residue peptide, corresponding to gamma-chain Thr-374 to Lys-406, binds to immobilized fibrin monomer. This peptide is a shorter variant of a previously isolated 38-residue peptide (gamma-chain Thr-374 to Val-411) that contains a polymerization site [Olexa, S. A. & Budzynski, A. Z. (1981) J. Biol. Chem. 256, 3544-3549]. The peptide mixture derived from fragment D1 was digested further with Staphylococcus aureus protease V8, and a 23-residue peptide, gamma-chain Thr-374 to Glu-396, carrying a polymerization site, was isolated by affinity chromatography. This 23-residue peptide inhibits the polymerization of desA-fibrinogen. We conclude that a polymerization site complementary to the site exposed by removal of fibrinopeptide A is present in this segment. The localization of the polymerization site within the gamma-chain segment 374-396 implies that the polymerization site does not overlap with segments of the gamma-chain that are responsible for platelet aggregation and for Staphylococcus clumping (residues 400-411 and 397-411, respectively) or with the residues involved in factor XIIIa-catalyzed fibrin crosslinking (Gln-398 and Lys-406).
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PMID:Localization of a fibrin gamma-chain polymerization site within segment Thr-374 to Glu-396 of human fibrinogen. 659 97

High-performance liquid chromatography was used to analyze the kinetics of the thrombin-catalyzed release of the activation peptide from the factor XIII zymogen (fibrin-stabilizing factor). The specificity constant (kcat/Km) for this reaction, measured at factor XIII concentrations much below Km, was (0.13-0.16) X 10(6) M-1 s-1 at pH 7.4, mu = 0.15, and 37 degrees C. Separate estimates, obtained from the dependence of the initial rates of release of the activation peptide on the concentration of factor XIII, gave values of 10 (+/- 3) s-1 for kcat and 84 (+/- 30) microM for Km, in terms of ab protomers of the zymogen. The thrombin-mediated release of the activation peptide was dramatically enhanced in the presence of fibrinogen. Furthermore, the time course of release, in relation to that of fibrinopeptide A, suggested that some des-A-fibrinogen species (e.g., alpha 2B beta 2 gamma 2) may be the true activator for promoting the cleavage of the Arg-36 peptide bonds in the a subunits of factor XIII. This observation suggests that generation of factor XIIIa and its substrate (fibrin) is coordinated so that thrombin-mediated zymogen activation proceeds efficiently only after the process of clotting has been initiated by the removal of fibrinopeptide A from fibrinogen.
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PMID:Promotion of thrombin-catalyzed activation of factor XIII by fibrinogen. 666 34

Human plasma fibrinogen is produced by liver parenchymal cells. Such molecules contain two classes of gamma-chains (gamma A, gamma'), which differ with respect to their COOH-terminal sequences. When fibrin is crosslinked in the presence of factor XIIIa and Ca2+, three types of gamma-dimer are formed (gamma A-gamma A; gamma A-gamma'; gamma'-gamma'). A separate fibrinogen pool is located in platelet alpha-granules. We analyzed this fibrinogen to determine whether gamma'-chains were present to the same extent (7%) that they are found in plasma fibrinogen. Electrophoretic analysis (Laemmli system) of reduced samples of the clot that formed subsequent to release of fibrinogen from thrombin-stimulated washed platelets, revealed a single crosslinked gamma-dimer band in the gamma A-gamma A position. Material collected into EDTA-containing buffer and subsequently crosslinked in the presence of added factor XIII and Ca2+ also revealed a gamma A-gamma A dimer band. This finding was further investigated by Western blotting of reduced gel specimens that had been reacted with an anti-gamma-chain antibody followed by 125I-labeled protein A. A single type of gamma-chain, gamma A, was present in the fibrinogen from a Triton X-100 or 10 M urea platelet lysate, or in noncrosslinked fibrin obtained from thrombin-treated platelets. Crosslinked reduced fibrin from thrombin-treated platelets or that prepared from the Triton lysate revealed a single type of gamma-dimer, gamma A-gamma A. We conclude that there are no gamma'-chains (less than 1%) in platelet fibrinogen. This structural difference from hepatic fibrinogen probably results from differences in the processing and/or regulation of the fibrinogen gamma-chain gene in megakaryocytes.
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PMID:Human platelet fibrinogen gamma chain structure. 671

A 73-year-old female was found to have prolonged thrombin and reptilase times in the immediate post-operative period. These abnormalities were not corrected by the addition of normal plasma. They were subsequently shown to be due to an IgG immunoglobulin which inhibited fibrin monomer polymerization. The IgG immunoglobulin activity could be neutralized completely by prior incubation with either patient or normal fibrinogen, uncrosslinked fibrin monomers or IgG antisera. No inhibitory effect on thrombin activity, fibrinopeptide A release or on the fibrin cross-linking reaction of factor XIIIa could be detected. Purified patient fibrinogen was functionally normal as demonstrated by normal fibrinogen-fibrin polymerization and fibrinopeptide A release. No underlying cause for this phenomenon was found. The presence of the inhibitor was associated with excessive blood loss during the post-operative period.
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PMID:Idiopathic autoantibody that inhibits fibrin monomer polymerization. 684 27

Fibronectin is a high molecular weight glycoprotein which is found in a soluble form in plasma (cold-insoluble globulin) and other body fluids and in an insoluble form in connective tissues and associated with basement membranes. Soluble fibronectin interacts with denatured collagen, heparin, fibrin, and Staphylococcus aureus, and is a substrate for thrombin, plasma and factor XIIIa. Fibronectin is thought to function as an adhesive and opsonic glycoprotein, supporting adhesion of cells including platelets, growth of connective tissue cells to fibrin clots, connective tissue formation, and in the phagocytic clearance by the reticuloendothelial system.
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PMID:Fibronectin (cold-insoluble globulin): role in defence. 702 90

Human fibrinogen was treated with thrombin in the presence of fibrinoligase and calcium ion at pH 8.5, ionic strength 0.45, and the ensuring polymerization was interrupted at various time intervals (t) both before and after the clotting time (tc) by solubilization with a solution of sodium dodecyl sulfate and urea. Aliquots of the solubilized protein were subjected to gel electrophoresis on polyacrylamide gels after disulfide reduction by dithiothreitol and on agarose gels without reduction. The degree of gamma-gamma ligation was determined from the former and the size distribution of ligated oligomers, for degree of polymerization x from 1 to 10, from the latter. The degree of gamma-gamma ligation was calculated independently from the size distribution with the assumption that every junction between two fibrin monomers remaining intact after solubilization is ligated, and this agreed well with the direct determination. The size distribution at t/tc = 1.3 to 1.6 differed somewhat from that calculated by the classical theory of linear polycondensation on the assumption that all reactive sites react with equal probability and rate. Analysis of the difference suggests that ligation of a fibrin digomer is not a random process; the probability of ligation of a given junction between two monomers increases with the oligomer length. The number-average degree of polymerization, xn, of ligated oligomers increases approximately linearly with time up to a value of 1.6.
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PMID:Kinetics of ligation of fibrin oligomers. 739 Oct 26

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

This study examined the rheological properties of fibrin gels formed by adding thrombin to plasma samples from 99 subjects with fibrinogen concentrations ranging from 1.45 to 4.14 g/l. A highly significant (r = 0.757; P < 0.001) inverse correlation was observed between plasma fibrinogen concentration and the extent of clot deformability as estimated from the final value of the storage modulus (G') of the fibrin gel when obtained by rheological analysis. A similarly significant correlation (r = 0.844; P < 0.001) was obtained using samples from 47 subjects in which fibrin cross-linking was blocked by addition of 0.1 mM iodoacetamide to inactivate factor XIIIa. The characteristics of the relationship between G' and fibrinogen concentration in the plasma samples was comparable with that observed when the fibrin gel was formed by adding thrombin to purified fibrinogen. These results suggest that the increased risk of myocardial infarction associated with an elevated plasma fibrinogen concentration may, in part, be explained on the basis of a decreased deformability of the fibrin clot formed.
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PMID:Changes in clot deformability--a possible explanation for the epidemiological association between plasma fibrinogen concentration and myocardial infarction. 786 77

Stabilization of a clot is dependent on fibrin cross-linking mediated by the transglutaminase, factor XIIIa (FXIIIa). In addition to fibrin stabilization, FXIIIa acts on a number of platelet-reactive proteins, including fibronectin and vitronectin, as well as the platelet proteins, glycoprotein (GP) IIb-IIIa, myosin, and actin. However, conditions inducing the platelet-activation dependent binding of FXIIIa have not been characterized nor have the sites mediating FXIIIa binding been identified. The generation of FXIIIa and consequent detection of FXIIIa on the platelet surface were compared with other thrombin-induced activation events; the rate at which FXIIIa bound to activated platelets was much slower than platelet degranulation or fibrin(ogen) binding. Whereas platelets could be rapidly induced to express a functional receptor for FXIIIa, the rate of FXIIIa binding to platelets is limited by the rate of conversion of FXIII to FXIIIa. Immunoprecipitation of radiolabeled platelets using polyclonal anti-FXIII A-chain antibody identified two proteins corresponding to GPIIb and GPIIIa. Preincubation of intact platelets with 7E3, a monoclonal antibody that blocks the fibrinogen binding site, or GRGDSP peptide inhibited FXIIIa binding by about 95% when measured by flow cytometry; FXIIIa binding to purified GPIIb-IIIa was also inhibited by 7E3. The binding of FXIIIa to purified GPIIb-IIIa was enhanced by the addition of fibrinogen, but not by that of fibronectin or thrombospondin, suggesting that FXIIIa also binds to fibrinogen associated with the complex. These observations suggest that activated platelets bearing FXIIIa may enhance stabilization of platelet-rich thrombi through surface-localized cross-linking events.
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PMID:Factor XIIIa binding to activated platelets is mediated through activation of glycoprotein IIb-IIIa. 790 63


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