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)

Factor XIII is a blood protransglutaminase that is distributed in plasma and platelets. The extracellular and intracellular zymogenic forms differ in that the plasma zymogen contains A and B subunits, while the platelet zymogen has A subunits only. Both zymogens form the same enzyme. Erythrocytes, in contrast, contain a tissue transglutaminase that is distinct from Factor XIII. In this study other bone marrow-derived cells were examined for transglutaminase activity. Criteria that were used to differentiate Factor XIII proteins from erythrocyte transglutaminase included: (a) immunochemical and immunohistochemical identification with monospecific polyclonal and monoclonal antibodies to Factor XIII proteins, (b) requirement for thrombin cleavage to express activity, (c) pattern of fibrin cross-linking catalyzed by the enzyme, and (d) different electrophoretic mobilities in nondenaturing gel systems. By these criteria human peripheral blood monocytes, peritoneal macrophages, and monocytes maintained in culture contain an intracellular protransglutaminase that is the same as platelet Factor XIII. The monocyte-macrophage protein is thrombin-sensitive, and under appropriate conditions there is no enzyme expression without activation of the zymogen. Both the monocyte-macrophage zymogen and enzyme have the same electrophoretic mobilities as platelet Factor XIII zymogen and enzyme. Antibody to A protein reacts with the monocyte-macrophage protein. B protein is not associated with this intracellular zymogen. By immunoperoxidase staining monocyte-macrophage protein seems to be localized in the cytoplasm, similar to the known cytoplasmic distribution of platelet and megakaryocyte Factor XIII. These procedures were also used to study populations of human granulocytes and lymphocytes, and protransglutaminase activity was not observed in these cells.
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PMID:Identification of intracellular factor XIII in human monocytes and macrophages. 286 85

The present study examines the effects of water soluble extracts of gas-phase cigarette smoke on intracellular transglutaminase activities of intact, murine, bone marrow-derived macrophages maintained in culture. Western blotting of cell lysates utilizing noncross-reactive antisera indicate that mouse bone marrow-derived macrophages contain both tissue-type transglutaminase and factor XIII-associated transglutaminase. This finding is also supported by data indicating that the intracellular transglutaminase activity of these cells contains thrombin-dependent and -independent components. Macrophages incubated with cigarette smoke solutions for 15 minutes at 37 degrees C display a dose-dependent decrease (maximum inhibition = 55%, p less than .001) in tissue-type (thrombin-independent) transglutaminase activity, as compared to control cells incubated with phosphate-buffered saline. Factor XIII (zymogen) is not inactivated following incubation of macrophages with smoke extracts. Smoke exposure under the conditions employed has no effect on either cell viability or adherence. Incubation with 2 microM retinoic acid for 24 hours leads to a modest (2-fold) induction of tissue transglutaminase, but does not induce factor XIII; in contrast, incubation with 10% homologous serum for 24 hours results in a decrease in factor XIII, but does not affect tissue transglutaminase. These data indicate that: bone marrow-derived macrophages contain factor XIII as well as tissue-type transglutaminase; and gas-phase cigarette smoke can inactivate tissue transglutaminase within viable murine bone marrow-derived macrophages, but cannot inactivate zymogenic factor XIII.
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PMID:Characterization of two distinct transglutaminases of murine bone marrow-derived macrophages: effects of exposure of viable cells to cigarette smoke on enzyme activity. 288 87

Efforts to develop an improved assay for plasma and tissue transglutaminase have led us to a convenient, sensitive microtiter plate assay for coagulation factor XIII using human fibrinogen as an immobilized substrate. Factor XIII was activated in the presence of calcium, thrombin, and immobilized fibrinogen and then assayed by adding biotinylcadaverine. The reaction was terminated by adding EDTA and the level of incorporated biotin was measured with streptavidin-beta-galactosidase. In this assay, the analytical range for human platelet factor XIII was 0.01-100 ng and 1-100 ng for guinea pig liver transglutaminase. Fibrinogen-coated plates gave more than 100-fold increase in sensitivity compared with N,N-dimethylcasein-coated microtiter plates. The intraassay coefficient of variation was less than 5% (n = 12) and interassay less than 6% (n = 4). The sensitivity of this assay reduced the volumes of plasma samples required and consequently eliminated the need to remove fibrinogen from such test samples. As expected, factor XIII activity could be inhibited by putrescine and antibodies against factor XIII as well as by a monoclonal antibody that bound to the carboxyl terminus of human fibrin gamma-chains. The assay provided a sensitive, simple, and rapid method for measuring factor XIII.
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PMID:A microtiter assay for factor XIII using fibrinogen and biotinylcadaverine as substrates. 769 7

1. Crude salivary gland extract of the giant Amazon leech, Haementeria ghilianii, contains an inhibitor of plasma factor XIIIa. 2. The inhibitory agent was purified to homogeneity by anion-exchange, cation-exchange, gel-filtration and reverse-phase chromatography to yield a single band on SDS/PAGE with an apparent molecular mass of 7.3 kDa. It has been named tridegin. 3. Micro-sequencing of proteolytic fragments showed tridegin to be a peptide of 66 amino acids. The sequence is unique with little similarity to other leech-derived proteins. 4. Inhibition of plasma factor XIIIa activity was confirmed by four independent methods: tridegin increased the solubility of fibrin clots in urea, inhibited ammonia produced from the incorporation of ethylamine into casein, inhibited the incorporation of 5'-(biotinamido)pentylamine into casein and prevented gamma-dimer formation in clotting fibrinogen. 5. The IC50 of tridegin (approx. 9.2 nM) is very close to the concentration of factor XIIIa used in the assay and in fact depends on its concentration. This is the most potent inhibitor of factor XIIIa yet described. 6. Tridegin also inhibits platelet factor XIIIa (factor XIIIAa) with a similar potency to that of the plasma enzyme. 7. Tridegin also inhibits tissue transglutaminase but with lower potency and independently of the enzyme concentration. 8. Tridegin appears to be specific for transglutaminases, since it has no effect on the coagulation times of human plasma, on thrombin or factor Xa. Moreover it has no effect on other thiol-containing enzymes and has no ability to digest fibrinogen or cleave the isopeptide substrate, L-gamma-glutamyl-4-nitroanilide.
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PMID:Tridegin, a new peptidic inhibitor of factor XIIIa, from the blood-sucking leech Haementeria ghilianii. 921 Apr 3

Human factor XIII (FXIII) and tissue transglutaminase (tTG) are homologous proteins. FXIII requires thrombin for activation and cross-links the gamma chains of fibrin(ogen) more efficiently than the Aalpha chains. On the other hand, tTG is thrombin-independent and forms predominantly Aalpha and Aalpha-gamma chain complexes. Previous work from this laboratory demonstrated that amino acid residues within exon 7 of FXIII were important for catalysis (Hettasch, J. M., and Greenberg, C. S. (1994) J. Biol. Chem. 269, 28309-28313). To determine to what extent the primary amino acid sequence within exon 7 defines substrate specificity, exon 7 of FXIII was replaced with the corresponding exon of tTG using gene splicing by overlap extension. Other work from this laboratory (Achyuthan, K. E., Slaughter, T. F., Santiago, M. A., Enghild, J. J., and Greenberg, C. S. (1993) J. Biol. Chem. 268, 21284-21292) using synthetic peptides identified two other domains that might play a role in substrate recognition (located in exons 3 and 5). Therefore, recombinant chimeras of FXIII/tTG were also created in which these two exons were exchanged. FXIII, tTG, and chimeras 3, 5, and 7 were expressed in Escherichia coli, purified, and the nature of the fibrin cross-linking pattern of these five proteins was determined by immunoblot analysis. FXIII preferentially formed the gamma-gamma dimer, whereas tTG formed Aalpha-gamma complexes. Chimera 7 formed Aalpha-gamma complexes that resembled the cross-linking pattern of tTG. This finding demonstrates that the primary amino acid sequence of exon 7 of tTG confers some of the specificity for the Aalpha and Aalpha-gamma cross-link pattern characteristic of tTG. Chimera 5 exhibited reduced cross-linking activity (50% of FXIII activity) but still retained preference for formation of the gamma-gamma dimer, whereas chimera 3 was not active. In conclusion, exchanging the primary amino acid sequence of the active site exon of human FXIII with that of human tTG modifies the enzyme such that the fibrin cross-linking pattern more closely resembles that of tTG (Aalpha and Aalpha-gamma complexes) instead of FXIII (gamma-gamma dimers).
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PMID:Analysis of factor XIII substrate specificity using recombinant human factor XIII and tissue transglutaminase chimeras. 931 26

C-CAM, a ubiquitously expressed cell adhesion molecule belonging to the carcinoembryonic antigen family, appears as two co-expressed isoforms, C-CAM-L and C-CAM-S, with different cytoplasmic domains, that can form homodimers in epithelial cells. In addition, C-CAM-L has been found in large molecular weight forms suggesting posttranslational, covalent modification. Here we have investigated the possibility that the cytoplasmic domain of C-CAM-L can act as a transglutaminase substrate. Glutathione S-transferase fusion proteins of the cytoplasmic domains of rat and mouse C-CAM-L as well as free cytoplasmic domains, released by thrombin cleavage from the fusion proteins, were converted into covalent dimers by tissue transglutaminase. These results demonstrate that the cytoplasmic domains of rat and mouse C-CAM-L are substrates for tissue transglutaminase, and lend support to the notion that higher molecular weight forms of C-CAM-L are formed by transglutaminase modification.
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PMID:The cell adhesion molecule C-CAM is a substrate for tissue transglutaminase. 954 Oct 24

Transglutaminases (TGases) form cross-links between glutamine and lysine side-chains of polypeptides in a Ca2+-dependent reaction. The structural basis of the Ca2+-effect is poorly defined. 43Ca NMR, surface polarity analysis combined with multiple sequence alignment and the construction of a new homology model of human tissue transglutaminase (tTGase) were used to obtain structural information about Ca2+ binding properties of factor XIII-A2, tTGase and TGase 3 (each of human origin). 43Ca NMR provided higher average dissociation constants titrating on a wide Ca2+-concentration scale than previous studies with equilibrium dialysis performed in shorter ranges. These results suggest the existence of low affinity Ca2+ binding sites on both FXIII-A and tTGase in addition to high affinity ones in accordance with our surface polarity analysis identifying high numbers of negatively charged clusters. Upon increasing the salt concentration or activating with thrombin, FXIII-A2 partially lost its original Ca2+ affinity; the NMR data suggested different mechanisms for the two activation processes. The NMR provided structural evidence of GTP-induced conformational changes on the tTGase molecule diminishing all of its Ca2+ binding sites. NMR data on the Ca2+ binding properties of the TGase 3 are presented here; it binds Ca2+ the most tightly, which is weakened after its proteolytic activation. The investigated TGases seem to have very symmetric Ca2+ binding sites and no EF-hand motifs.
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PMID:Calcium binding of transglutaminases: a 43Ca NMR study combined with surface polarity analysis. 1156 52

Atherosclerosis is characterized by thickening of the vessel wall, smooth muscle cell proliferation, macrophage infiltration, and deposition of a fibrin network. Transglutaminases are a family of enzymes catalyzing the formation of stable covalent cross-links between proteins. Here, we show that tissue transglutaminase (tTG) synthesis by human umbilical vein endothelial cells is upregulated by thrombin, the serine protease that causes fibrin formation and many cellular inflammatory effects. Thrombin upregulated tTG 2-fold at the mRNA and protein level. Cellular cross-linking activity was increased to an even greater extent; antibody to tTG neutralized the increased activity. The effect on tTG expression required active thrombin and was mediated mainly through protease-activated receptor-1, a thrombin receptor. Increased tTG antigen and activity were evident in human umbilical vein endothelial cells and extracellular matrix in situ. Thrombin treatment also led to a cellular redistribution of tTG. Normal vessel wall stained positively for tTG in the smooth muscle cells and in the subendothelium. The intensity of staining increased in vessel walls with plaque, where there was a striking increase in tTG in the smooth muscle cells immediately below the plaque. These studies indicate a role for tTG in the stabilization of atherosclerotic plaques and suggest that its local expression can be controlled by thrombin.
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PMID:Thrombin upregulates tissue transglutaminase in endothelial cells: a potential role for tissue transglutaminase in stability of atherosclerotic plaque. 1159 46

The beta-thymosins constitute a family of highly conserved and extremely water-soluble 5 kDa polypeptides. Thymosin beta4 is the most abundant member; it is expressed in most cell types and is regarded as the main intracellular G-actin sequestering peptide. There is increasing evidence for extracellular functions of thymosin beta4. For example, thymosin beta4 increases the rate of attachment and spreading of endothelial cells on matrix components and stimulates the migration of human umbilical vein endothelial cells. Here we show that thymosin beta4 can be cross-linked to proteins such as fibrin and collagen by tissue transglutaminase. Thymosin beta4 is not cross-linked to many other proteins and its cross-linking to fibrin is competed by another family member, thymosin beta10. After activation of human platelets with thrombin, thymosin beta4 is released and cross-linked to fibrin in a time- and calcium-dependent manner. We suggest that thymosin beta4 cross-linking is mediated by factor XIIIa, a transglutaminase that is coreleased from stimulated platelets. This provides a mechanism to increase the local concentration of thymosin beta4 near sites of clots and tissue damage, where it may contribute to wound healing, angiogenesis and inflammatory responses.
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PMID:Thymosin beta4 is released from human blood platelets and attached by factor XIIIa (transglutaminase) to fibrin and collagen. 1197 33

TGF-beta is a cytokine with varied properties and pleiotropic activity. It is released in an inactive form. To exhibit its biological activity, it requires binding to extracellular matrix proteins and, after that, proteolytic elimination of LAP (Latent Associated Protein) and LTBP (Latent TGF-beta Binding Protein). The process involves, among others, tissue transglutaminase, thrombin and plasmin. By stimulation of specific receptors, it influences transcription of some genes and translation of formed mRNA. Locally, it demonstrates proinflammatory properties whereas systemically, it has primarily a potent immunosuppressive effect. TGF-beta, by affecting proliferation, differentiation and migration of cells, as well as stimulation of extracellular matrix protein production, plays an important role in tissue regeneration and remodeling, but also in fibrosis. TGF-beta is also indispensable to maintain immune homeostasis of the organism. Reduced TGF-beta activity is considered to be responsible for development of autoimmune disorders in the course of several pathologic conditions. This cytokine plays an important role in the pathogenesis of chronic inflammatory processes taking place, among others, in inflammatory bowel diseases (IBD) and chronic hepatitis B and C. The paper presents a review of literature concerning diagnostic and prognostic value of TGF-beta level determinations in blood and tissue bioptates of patients with chronic non-specific enteritis and chronic hepatitis B and C.
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PMID:TGF-beta (transforming growth factor-beta) in chronic inflammatory conditions - a new diagnostic and prognostic marker? 1211 14


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