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)

Tissue factor (TF) is the cell-surface transmembrane receptor that initiates both the extrinsic and intrinsic blood coagulation cascades. The abilities of TF to associate with Factor VIIa and Factor X in a ternary complex and to enable proteolytic activation of Factor X by Factor VIIa reside in the extracellular domain of TF. We describe the expression of the surface domain of TF (truncated TF, tTF) in both Saccharomyces cerevisiae and Escherichia coli and the biochemical and physical characterization of the recombinant proteins. Wild-type tTF and several glycosylation-site mutants were secreted efficiently by S. cerevisiae under the control of the yeast prepro-alpha-signal sequence; the T13A,N137D double mutant was the most homogeneous variant expressed in milligram quantities. Wild-type tTF was expressed in a non-native state in E. coli inclusion bodies as a fusion protein with a poly(His) leader. The fusion protein could be fully renatured and the leader removed by proteolysis with thrombin; the correct molecular mass (24,729 Da) of the purified protein was confirmed by electrospray mass spectrometry. Recombinant tTFs from yeast, E. coli and Chinese hamster ovary cells were identical in their abilities to bind Factor VIIa, to enhance the catalytic activity of Factor VIIa and to enhance the proteolytic activation of Factor X by Factor VIIa. Furthermore, CD, fluorescence emission and NMR spectra of the yeast and E. coli proteins indicated that these proteins are essentially identical structurally.
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PMID:Recombinant soluble human tissue factor secreted by Saccharomyces cerevisiae and refolded from Escherichia coli inclusion bodies: glycosylation of mutants, activity and physical characterization. 765 2

A functional thrombin receptor (TR) structurally related to other members of the seven-transmembrane receptor family has been isolated from diverse cellular types intimately involved in the regulation of the thrombotic response. This receptor recapitulates many of the previously identified sequelae of thrombin-mediated cell activation phenomenon, and requires proteolytic cleavage for downstream effector-response coupling events. Using two complementary approaches, we have now completed the chromosomal assignment of the human thrombin receptor gene. Discordancy analysis of polymerase chain reaction products from a human-rodent hybrid cell mapping panel assigned the sequence to human chromosome 5 with no observed discordancies. Cytogenetic localization using fluorescence in situ hybridization on human metaphase chromosomes specifically localized the human TR gene to region q13 of chromosome 5, confirming its presence as a single-locus gene in the human genome. The chromosomal localization of the human TR gene is at or contiguous with the proximal breakpoint site identified in the majority of patients with the 5q- syndrome (dysmegakaryocytopoiesis and refractory anemia).
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PMID:Chromosomal assignment of the human thrombin receptor gene: localization to region q13 of chromosome 5. 839 10

Synthetic peptides (TRAP or Thrombin Receptor Activating Peptide) corresponding to at least the first five aminoacids of the new N-terminal tail generated after thrombin proteolysis of its receptor are effective to mimic thrombin. We have studied two different TRAPs (SFLLR, and SFLLRN) in their effectiveness to induce the different platelet responses in comparison with thrombin. Using Indo-l/AM-labelled platelets, the maximum rise in cytoplasmic ionized calcium was lower with TRAPs than with thrombin. At threshold concentrations allowing maximal aggregation (50 microM SFLLR, 5 microM SFLLRN and 1 nM thrombin) the TRAPs-induced release reaction was about the same level as with thrombin, except when external calcium was removed by addition of 1 mM EDTA. In these conditions, the dense granule release induced by TRAPs was reduced by over 60%, that of lysosome release by 75%, compared to only 15% of reduction in the presence of thrombin. Thus calcium influx was more important for TRAPs-induced release than for thrombin-induced release. At strong concentrations giving maximal aggregation and release in the absence of secondary mediators (by pretreatment with ADP scavengers plus aspirin), SFLLRN mobilized less calcium, with a fast return towards the basal level and induced smaller lysosome release than did thrombin. The results further demonstrate the essential role of external calcium in triggering sustained and full platelet responses, and emphasize the major difference between TRAP and thrombin in mobilizing [Ca2+]i. Thus, apart from the proteolysis of the seven transmembrane receptor, another thrombin binding site or thrombin receptor interaction is required to obtain full and complete responses.
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PMID:Different abilities of thrombin receptor activating peptide and thrombin to induce platelet calcium rise and full release reaction. 860 17

In airway smooth muscle cells ligand binding to the seven-transmembrane endothelin and thrombin receptors stimulates cell growth. Rapid activation of the extracellular regulated kinase 2 and c-Jun NH2-terminal kinase groups of mitogen-activated protein kinases was also observed. The results demonstrate a novel mechanism of seven-transmembrane receptor signaling involving activation of the Jun kinase pathway. Receptor coupling to Jun kinase activation may involve heterotrimeric G proteins since the kinase was enzymatically activated in cells treated with aluminum fluoride. The activity of Raf-1, measured by immune complex kinase assay, revealed that platelet-derived growth factor and phorbol 12-myristate 13-acetate both stimulated Raf-1 activity, while thrombin and endothelin did not appreciably stimulate Raf-1. The data suggest that endothelin and thrombin stimulate Raf-1-independent mechanisms of mitogen-activated protein kinase activation. Endothelin- or thrombin-induced activation of mitogen-activated protein kinases was significantly inhibited by activation of cyclic AMP-dependent protein kinase by forskolin. Proliferation of airway smooth muscle cells, measured by incorporation of [3H]thymidine into DNA, was also greatly attenuated by forskolin.
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PMID:The seven-transmembrane-spanning receptors for endothelin and thrombin cause proliferation of airway smooth muscle cells and activation of the extracellular regulated kinase and c-Jun NH2-terminal kinase groups of mitogen-activated protein kinases. 862 41

Thrombin's cellular actions are mediated by a novel G-protein coupled transmembrane receptor. We infused SFLLRN, a peptide that directly activates thrombin receptors, into the left circumflex coronary artery (CFX) of anesthetized dogs to evaluate the cardiovascular effects of thrombin receptor activation in vivo. Intracoronary SFLLRN, 0.9, 9 and 90 nmol/min, produced transient, dose-related increases in CFX blood flow, followed by sustained decreases in CFX and left anterior descending (LAD) blood flow. SFLLRN also decreased positive and negative dP/dtmax, arterial pressure, cardiac output and heart rate. Peripheral vascular resistance transiently decreased and then increased. SFLLRN decreased systolic wall thickening (WT) and increased ST segment level within the CFX perfusion area. In contrast, WT was increased, and ST segment was unchanged in the LAD perfusion area. CFX flow, but not LAD flow, increased transiently above control after SFLLRN infusion. FSLLRN, a peptide that does not activate thrombin receptors, had no effect at 90 nmol/min. The response to intravenous SFLLRN was greatly attenuated when compared with intracoronary infusion, and regional changes in coronary flow and function were absent. Decreases in arterial pressure, heart rate, coronary blood flow, and positive and negative dP/dtmax, were inhibited after bilateral vagotomy. Moreover, arterial pressure and peripheral resistance increased in response to SFLLRN after vagotomy. Initial CFX flow increase, regional dysfunction, ST level changes and hyperemic response were comparable but attenuated after vagotomy. Ex vivo platelet function was not affected by SFLLRN up to 100 microM. We conclude that regional myocardial ischemia and cardiac dysfunction result from thrombin receptor-mediated local coronary vasoconstriction. Thus, thrombin generation at a site of vascular injury or thrombus may significantly affect vascular tone and myocardial perfusion.
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PMID:Cardiovascular actions of thrombin receptor activation in vivo. 896 61

Tissue factor (TF) is a transmembrane receptor that serves as a cofactor for factor VIIa and initiates the extrinsic pathway of blood coagulation. Under normal physiological conditions, TF is expressed in extravascular and perivascular cells but not in vascular endothelial cells and monocytes. TF can be induced in these cells by inflammatory regulators and other stimulators, such as LPS, thrombin, oxidized lipoproteins, and certain growth factors. An earlier study showed that growing primary cultures of human umbilical vein endothelial cells (HUVECs) with endothelial cell growth supplement (ECGS) and heparin had impaired the ability of monolayers to express surface membrane TF activity after perturbation. The mechanism by which ECGS suppressed TF activity was not known. In the present study, we investigated the effect of recombinant acidic and basic fibroblast growth factors (aFGF and bFGF) on the induction of TF in a HUVEC cell line and a fibroblast cell line. Both aFGF and bFGF suppressed the phorbol myristate acetate-induced expression of TF in endothelial cells but not the serum-induced expression of TF in fibroblast cells. Diminished expression of the cell surface TF activity observed in endothelial cells grown with aFGF or bFGF was due to the accumulation of a lower number of TF mRNA transcripts. TF mRNA stability was not altered in HUVECs grown with aFGF or bFGF. Nuclear run-on experiments revealed that the transcription of TF and several other genes that play an important role in inflammation and angiogenesis was reduced in the endothelial cells that were cultured with aFGF or bFGF. The diminished expression of TF may be part of a generalized response of endothelial cells to FGF that facilitates migration of endothelial cells during angiogenesis.
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PMID:Acidic and basic fibroblast growth factors suppress transcriptional activation of tissue factor and other inflammatory genes in endothelial cells. 915 59

We and others have shown that both high and low molecular mass kininogens are able to inhibit the thrombin-induced aggregation of gel-filtered platelets, indicating that the locus for inhibition resides in the heavy chain. The inhibitory site is present in domain 3, confined to the C-terminal portion of the region encoded by exon 7 (K270-G292), and the minimal effective sequence is a heptapeptide (L271-A277; Kunapuli et al, J Biol Chem 271:11228, 1996). Kininogens inhibit thrombin binding to platelets and thus inhibit thrombin-induced aggregation. The molecular mechanism by which kininogens inhibit thrombin-induced aggregation of platelets is unknown. Thrombin has previously been shown to bind to two receptors on the platelet surface, glycoprotein (GP) Ib-IX-V complex and the hepta-spanning transmembrane receptor coupled to G protein(s). We now show that, unlike its effect on normal platelets, kininogen (2 micromol/L) did not inhibit the thrombin-induced aggregation of Bernard-Soulier platelets, which lack the GP Ib-IX-V complex, suggesting that kininogen interacts either directly or indirectly with that complex and restricts access by thrombin to this receptor. We further show that both recombinant K270-G292 polypeptide and the synthetic peptide L271-A277 derived from high molecular mass kininogen lower thrombin binding to platelets in a manner similar to monoclonal antibodies to or ligands (von Willebrand factor and echicetin) of GP Ib-IX. The anti-GP Ib-IX-V complex antibodies, TM-60 and SZ 2, can inhibit 125I-high molecular mass kininogen binding to platelets. Conversely, kininogen could block the binding of biotinylated TM-60 or of 125I-SZ 2. Kininogen inhibited the binding of biotinylated thrombin bound to a mouse fibroblast cell line transfected with the GP Ib-IX-V complex. These results indicated that kininogen binds to the GP Ib-IX-V complex modulating thrombin binding to platelets and the consequent platelet aggregation. Kininogen can thus serve as an important regulator of the early stages of platelet stimulation by thrombin.
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PMID:Human kininogens regulate thrombin binding to platelets through the glycoprotein Ib-IX-V complex. 926 68

Thrombin formation is increased at the sites of vascular injury. Previous studies by our group and other groups indicated that the generation of plasminogen activator inhibitor-1 (PAI-1), the major physiological inhibitor for plasminogen activators, from cultured vascular smooth muscle cells (SMC) is elicited by thrombin. The present study demonstrates that the thrombin receptor, pertussis toxin-sensitive G protein, genistein-sensitive tyrosine kinase, phospholipase C, and protein kinase C may be involved in thrombin-induced PAI-1 production in cultured baboon aortic SMC. Forskolin and 8-bromo-cyclic AMP inhibited thrombin-induced PAI-1 production in cultured SMC. Treatment with hirulog-1, a synthetic thrombin receptor inhibitor, suppressed thrombin-induced PAI-1 generation at mRNA and protein levels in SMC. The results of the present study suggest that transmembrane receptor and multiple signal transduction systems are involved in thrombin-induced increase in PAI-1 transcription in vascular SMC. The production of PAI-1 stimulated by thrombin in vascular SMC may be pharmacologically modulated by thrombin receptor inhibitor.
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PMID:Transcellular signaling and pharmacological modulation of thrombin-induced production of plasminogen activator inhibitor-1 in vascular smooth muscle cells. 957 36

Although activation of one seven-transmembrane receptor can influence the response of a separate seven-transmembrane receptor, e. g., the phenomenon of synergism, the underlying mechanism(s) for this signaling process is unclear. The present study investigated communication between two receptors that exhibit classical synergism, e.g., human platelet thrombin and thromboxane A2 receptors. Activation of thrombin receptors caused an increase in ligand affinity of thromboxane A2 receptors. This effect (i) was shown to be specific, since a similar increase in ligand affinity was not caused by ADP or A23187; (ii) did not require cytosolic components, e.g., kinases, proteases, phosphatases, etc., because it occurred in isolated platelet membranes; (iii) was G protein-mediated because it was blocked by an Galphaq C terminus antibody; and (iv) was associated with a net increase in Galphaq coupling to thromboxane A2 receptors. Collectively, these data provide evidence that seven-transmembrane receptors that share a common Galpha subunit can communicate with each other via a redistribution of their G proteins. Thus, activation of thrombin receptors increases Galphaq association with thromboxane A2 receptors thereby shifting them to a higher affinity state. This signaling phenomenon, which modulates receptor-ligand affinity, may serve as a molecular mechanism for cellular adaptive processes such as synergism.
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PMID:A molecular mechanism for signaling between seven-transmembrane receptors: evidence for a redistribution of G proteins. 972 9

Laser scanning confocal fluorescence microscopy techniques were used to study the localization of von Willebrand factor (vWf; Factor VIII-related antigen) and thrombomodulin (transmembrane receptor for thrombin) in the microvascular endothelial cells in the normal human lung. Tissues were obtained from lobectomy specimens resected for solitary nodules (7 adenocarcinomas and 4 hamartomas) from 11 patients. The plasma membranes of the capillary endothelial cells in the alveolar zones (A-zones) showed red linear fluorescence for thrombomodulin. However, their cytoplasm was mostly unreactive for vWf. The microvessels which were located in the connective tissue (C-zones), including peribronchial, and subpleural areas and large vascular walls, consistently demonstrated band-like green fluorescence for vWf in their cytoplasm, and their plasma membranes usually lacked reactivity for thrombomodulin. Only a limited number of peribronchial capillaries measuring <10 microm in diameter showed a mosaic-like appearance, in which red fluorescence along the plasma membranes was found together with green fluorescence in the subjacent cytoplasm. In the juxtaalveolar (J-zones) microvessels located along the borders between A- and C-zones, and measuring up to 40 microm in diameter, the endothelial cells showed a mosaic-like pattern of distribution of the two antigens. However, the localization of thrombomodulin in the J-zone microvessels was separate and independent from that of vWf. The thrombomodulin-reactive cells were directly connected to the alveolar capillary endothelial cells. Heterogeneous patterns of distribution of thrombomodulin and vWf suggest that topographic differences of endothelial function occur to maintain a balance of coagulation and anticoagulation in the normal human lung.
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PMID:Heterogeneous distribution of thrombomodulin and von Willebrand factor in endothelial cells in the human pulmonary microvessels. 1075 1


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