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

---

Query: EC:3.4.21.5 (thrombin)
33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Studies have been carried out to investigate aspects of the structure of thrombomodulin, an endothelial cell glycoprotein that binds thrombin and accelerates both the thrombin-dependent activation of protein C and the inhibition of antithrombin III. We have determined the shape of SolulinTM, a soluble recombinant form of human thrombomodulin missing the transmembrane and cytoplasmic domains, by electron microscopy of preparations rotary-shadowed with tungsten. Solulin appears to be an elongated molecule about 20 nm long that has a large nodule at one end and a smaller nodule near the other end from which extends a thin strand. About half of the molecules form bipolar dimers apparently via interactions between these thin strands. Electron microscopy of complexes formed between Solulin and human alpha-thrombin revealed that a single thrombin molecule appears to bind to the smaller nodule of Solulin, suggesting that this region contains the epidermal growth factor-like domains 5 and 6. Epidermal growth factor-like domains 1-4 comprise the connector between the small and large nodule, which is the lectin-like domain; the thin strand at the other end of the molecule is the carbohydrate-rich region. With chondroitin sulfate-containing soluble thrombomodulin produced from either human melanoma cells Bowes or Chinese hamster ovary cells, a higher percentage of molecules bound thrombin and, in some cases, two thrombin molecules were attached to one soluble thrombomodulin in approximately the same region. These structural studies provide insight into the structure of thrombomodulin and its interactions with thrombin as well as aspects of the mechanisms of its actions.
...
PMID:The shape of thrombomodulin and interactions with thrombin as determined by electron microscopy. 894 Jan 62

Thrombomodulin (TM) is a multidomain protein that serves as a cofactor in a major natural anticoagulant system. To further characterize the structure-function of TM, we have transfected COS cells with different truncated forms of TM. In the first form, COS cells expressing TM that lacks the putative signal peptide (17 residues); the lectin-like, hydrophobic N-terminal domain (226 residues); and 12 residues of the first epidermal growth factor (EGF)-like repeat (COSdel.238 cells) were found to function normally with respect to TM transport to the cell surface and thrombin-dependent protein C activation. However, in contrast to wild-type TM, as visually studied by immunofluorescence and immunogold electron microscopy, the COSdel.238 cells did not constitutively internalize anti-TM-TM or thrombin-TM complexes. To identify the region responsible for mediating the endocytic process, deletant forms of TM lacking either the lectin-like region (residues 2-155) or the hydrophobic region of the N-terminal domain (residues 161-202) were expressed in COS cells (COSdel.2-155 and COSdel.161-202, respectively). Protein C cofactor activity was maintained in both cells. Although the COSdel.161-202 cells behaved similarly to wild-type TM-transfected cells, visual studies showed a lack of constitutive internalization of thrombin-TM or anti-TM-TM complexes in the COSdel.2-155 cells. We conclude that the lectin-like domain of human TM serves to regulate cell surface expression of TM via the endocytic route and therefore may also play a major physiologic role in controlling intracellular and extracellular accumulation of thrombin in a variety of biologic systems.
...
PMID:The amino terminal lectin-like domain of thrombomodulin is required for constitutive endocytosis. 900 69

Stimulation of HEL megakaryocytic cells by Fc gammaRIIA crosslinking is associated with tyrosine phosphorylation of syk and phospholipase C gamma2 (PLCgamma2) and is accompanied by formation of inositol phosphates and release of intracellular Ca2+. These responses are inhibited by the kinase inhibitors, staurosporine and ST271. In contrast, the G-protein receptor agonist, thrombin induces formation of inositol phosphates and release of intracellular calcium without an increase in tyrosine phosphorylation. The plant lectin wheat germ agglutinin (WGA) stimulates tyrosine phosphorylation of syk and PLCgamma2 but surprisingly does not stimulate formation of inositol phosphates and induce release of intracellular Ca2+. WGA also inhibited formation of inositol phosphates and release of intracellular Ca2+ by Fc gammaRIIA crosslinking and thrombin-stimulation. A similar inhibitory effect of WGA was observed against elevation of Ca2+ by the same two stimuli in MEG-01 megakaryotic cells. The results demonstrate a novel pathway of inhibition of PLC on crosslinking of cell surface proteins that is not present in platelets.
...
PMID:A novel inhibitory action of wheat germ agglutinin on phospholipase C in HEL and MEG-01 cell lines. 909 96

Convulxin, a very potent aggregating protein from rattlesnake venom, was purified by a new procedure and its heterodimeric structure alpha 3 beta 3 was confirmed. The polypeptide N-terminal sequences of convulxin subunits were determined by Edman degradation. They are very similar and appear homologous to botrocetin from Bothrops jararaca venom and to rattlesnake lectin from Crotalus atrox venom, both being classified among the C-type lectin family. The binding of 125I-labelled convulxin to blood platelets has also been analysed under equilibrium conditions. These studies indicated that convulxin binds to platelets with a high affinity (Kd = 30 pM) on a small number of binding sites (1000 binding sites per cell). The high-affinity binding of convulxin appears specific to platelets, since it is not observed on other cell types such as neutrophils and erythrocytes. Also, the high-affinity binding of convulxin to membranes platelet is not inhibited by alpha-thrombin, fibrinogen, collagen, laminin binding inhibitor, RGDS peptide, adenosine diphosphate, platelet-activating factor-acether, serotonin or epinephrine. This, together with the recent observation that platelet activation by convulxin is partially mediated by phospholipase C and involves other mechanisms as well, indicates that convulxin may interact with a specific platelet acceptor (receptor) protein which has yet to be characterized.
...
PMID:Convulxin, a potent platelet-aggregating protein from Crotalus durissus terrificus venom, specifically binds to platelets. 927 71

Bothrojaracin is a potent and selective thrombin inhibitor that has been isolated from the venom of Bothrops jararaca. It does not interact with the catalytic site of the enzyme but binds to both anion-binding exosites 1 and 2 resulting in a potent inhibition of thrombin activity towards fibrinogen and platelets [Zingali, R. B., Jandrot-Perrus, M., Guillin, M. C. & Bon, C. (1993) Biochemistry 32, 10794-108021. Bothrojaracin is a 27-kDa protein composed of two disulfide-linked polypeptide chains, A and B, of 15 kDa and 13 kDa, respectively. The sequences of A and B chains determined by molecular cloning exhibit a high degree of identity with other snake venom lectin-like proteins. In contrast to other ligands that interact with thrombin exosite 1, the amino acid sequence of bothrojaracin does not contain an acidic sequence similar to the C-terminal tail of hirudin. Expression of functional bothrojaracin was achieved in COS cells upon transfection with two pcDNA3 vectors containing the complete cDNAs. Recombinant bothrojaracin, which was secreted into the medium, was able to bind to and inhibit thrombin. When expressed alone, the B chain formed inactive dimers that were secreted into the culture medium. In contrast, no bothrojaracin-related protein was detected in conditioned media from cells transfected with the A chain.
...
PMID:Molecular cloning and expression of bothrojaracin, a potent thrombin inhibitor from snake venom. 934 15

Thrombomodulin (TM), recognized as an essential vessel wall cofactor of the antithrombotic mechanism, is also expressed by a wide range of tumor cells. Tumor cell lines subcloned from four patients with malignant melanoma displayed a negative correlation between TM expression and cell proliferation in vitro and in vivo. Overexpression of wild-type TM decreased cell proliferation in vitro and tumor growth in vivo. TM mutants with altered protein C activation capacity lead to a similar effect. In contrast, transfection of melanoma cells with mutant TM constructs, in which a portion of the cytoplasmic or lectin domain was deleted, abrogated the antiproliferative effect associated with overexpression of wild-type TM. Experiments performed with either peptide agonists/antagonists of the thrombin receptor, with hirudin, or with inhibitors of thrombin-TM interaction did not alter the growth inhibitory effect of TM overexpression. These data suggest that TM exerts an effect on cell proliferation independent of thrombin and the thrombin receptor, possibly related to the binding of novel ligands to determinants in the lectin domain which might trigger signal transduction pathways dependent on the cytoplasmic domain.
...
PMID:Thrombomodulin modulates growth of tumor cells independent of its anticoagulant activity. 952 72

Thrombomodulin is a cofactor protein on vascular endothelial cells that inhibits the procoagulant functions of thrombin and enhances thrombin-catalyzed activation of anticoagulant protein C. Thrombomodulin also accelerates the proteolytic activation of a plasma procarboxypeptidase referred to as thrombin-activable fibrinolysis inhibitor (TAFI). In this study, we describe structures on recombinant membrane-bound thrombomodulin that are required for human TAFI activation. Deletion of the N-terminal lectin-like domain and epidermal growth factor (EGF)-like domains 1 and 2 had no effect on TAFI or protein C activation, whereas deletions including EGF-like domain 3 selectively abolished thrombomodulin cofactor activity for TAFI activation. Provided that thrombomodulin EGF-like domain 3 was present, TAFI competitively inhibited protein C activation catalyzed by the thrombin-thrombomodulin complex. A thrombomodulin construct lacking EGF-like domain 3 functioned normally as a cofactor for protein C activation but was insensitive to inhibition by TAFI. Thus, the anticoagulant and antifibrinolytic cofactor activities of thrombomodulin have distinct structural requirements: protein C binding to the thrombin-thrombomodulin complex requires EGF-like domain 4, whereas TAFI binding also requires EGF-like domain 3.
...
PMID:Activation of thrombin-activable fibrinolysis inhibitor requires epidermal growth factor-like domain 3 of thrombomodulin and is inhibited competitively by protein C. 957 59

Human platelets afford a suitable and physiologically relevant model to study receptor-dependent cell aggregation and ensuing biosignaling reactions. Since cell surface glycoconjugates can serve as ligands in recognitive protein--carbohydrate interactions, it is of interest to investigate the reactivity of such epitopes for a plant lectin and the elicited intracellular responses. Therefore, the galactose-specific lectin (Viscum album agglutinin, VAA) was employed as a tool for this purpose. It was found that VAA induced platelet aggregation at a concentration of 2.5 microgram/ml using 2.5. 108 cells/ml, composed of the formation of both lactose-sensitive (Lac+) and lactose-resistant (Lac-) intercellular contacts. Lac- aggregates were formed only by metabolically active platelets of about 70% of the samples from the group of studied volunteers. The requirement of metabolic activity for formation of these contacts which no longer depend on lectin--ligand recognition was underscored by the lack of their appearance in the presence of metabolic inhibitors such as nordihydroguaiaretic acid, trifluoperazine, N-ethylmaleimide and menadione. With respect to biosignaling, the effective aggregation of platelets did not affect the basal level of Ca2+ in cells and reduced the rate of the menadione-dependent generation of H2O2. In parallel series platelet aggregation induced by bovine thrombin (0.03 U/ml) triggered an increase in the cytoplasmic Ca2+ level and an enhancement of the H2O2 generation. Overall, these results imply metabolically controlled post-binding reactions which strengthen the lectin-induced cell association and demonstrate differential responses with respect to the Ca2+ level and H2O2-generation between lectin- or thrombin-mediated aggregation of human platelets.
...
PMID:Formation of lactose-resistant aggregates of human platelets induced by the mistletoe lectin and differential signaling responses to cell contact formation by the lectin or thrombin. 963 85

In the present study, we described the interaction of succinylated human serum albumin (Suc-HSA), a negatively charged anti-HIV-1 active protein, with HIV-1 gp120 and in detail with the third variable domain of gp120 (V3 loop). To this end, different assay formats were tested in which gp120- and V3-related peptides were presented in various configurations in order to investigate the effect of the conformational structure of the V3 loop on the interaction with negatively charged albumins. When gp120 presented via a lectin was used, it was observed that Suc-HSA bound to native gp120. The binding site appeared to be located at or near the thrombin digestion site (GPGRAF sequence) in the V3 loop of gp120, since the cleavage of the loop resulted in decreased binding of Suc-HSA. In addition, Suc-HSA was able to protect the V3 region of gp120 from cleaving with thrombin. In contrast, significant binding of Suc-HSA to V3 loop or gp120 peptides was not observed when both were presented in a fluid phase system, suggesting the involvement of a monovalent-low affinity binding of Suc-HSA. Using overlapping peptides delineating the whole V3 loop immobilized to CNBr-Sepharose, we noticed that the interaction of the V3 loop with Suc-HSA was predominantly induced by electrostatic interactions between positively charged linearized peptide fragments and Suc-HSA and was positively influenced by the presence of hydrophobic amino in the V3 loop fragments as well. Moreover, the highest affinity site was located at sites near the GPGRAF sequence. These observations add to the evidence, collected earlier, that Suc-HSA interferes at the level of virus entry, independent of interaction with the CD4 receptor. Since the recently discovered chemokine receptors are negatively charged, we can hypothesize that Suc-HSA is able to prevent the positively charged V3 loop from interacting with these types of receptors, thereby inhibiting virus entry.
...
PMID:Mechanism of anti-HIV activity of succinylated human serum albumin. 1008 22

Tyrosine phosphorylation of the non-receptor tyrosine kinases pp72syk and pp125FAK and of the gamma2 isoform of phospholipase C (PLCgamma2) in human platelets stimulated with the lectin Concanavalin A was investigated. Concanavalin A induced the rapid tyrosine phosphorylation of pp72syk and PLCgamma2 with a similar kinetics, while tyrosine phosphorylation of pp125FAK occurred in a later phase of platelet activation. When compared with other platelet agonists, Concanavalin A revealed to be at least as potent as collagen in inducing tyrosine phosphorylation of PLCgamma2 and pp125FAK, while tyrosine phosphorylation of pp72syk induced by the lectin was much stronger than that induced by thrombin or collagen. Concanavalin A-induced tyrosine phosphorylation of pp72syk, PLCgamma2 and pp125FAK was not dependent on platelet aggregation as it occurred normally even in the absence of sample stirring and when fibrinogen binding to integrin alphaIIb-beta3 was inhibited by the peptide RGDS. Tyrosine phosphorylation of pp72syk, PLCgamma2 and pp125FAK required the binding of the lectin to the platelet surface, but was not observed in platelets treated with succinyl-Concanavalin A, a derivative of the lectin that interacts with the same receptors but does not promote clustering of membrane glycoproteins. Moreover, the aggregation-independent tyrosine phosphorylation of pp125FAK and pp72syk induced by Concanavalin A required the expression of integrin alphaIIb-beta3 on the platelet surface as it was strongly inhibited in platelets from patients affected by Glanzmann thrombasthenia. By contrast, tyrosine phosphorylation of PLCalpha2 occurred normally also in thrombasthenic platelets stimulated with Concanavalin A. These results demonstrate that, even in the absence of aggregation, the clustering of integrin alphaIIb-beta3 induced by Concanavalin A on the platelet surface directly promotes tyrosine phosphorylation of pp72syk and pp125FAK and provide further evidence that the oligomerization of the fibrinogen receptor promoted by its natural ligand during platelet aggregation may be responsible for the tyrosine phosphorylation of these proteins induced by physiological agonists.
...
PMID:Clustering of integrin alphaIIb-beta3 differently regulates tyrosine phosphorylation of pp72syk, PLCgamma2 and pp125FAK in concanavalin A-stimulated platelets. 1034 3


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---