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)

Single-stranded DNA molecules containing a 15-nucleotide consensus sequence have been reported to inhibit thrombin activity. The mechanism of the inhibition was studied using a consensus 15-mer oligonucleotide and two recombinant mutant thrombins: the anion-binding exosite mutant thrombin R70E, and thrombin K154A, in which the mutation was located in a surface loop outside of the exosite. The consensus 15-mer oligonucleotide inhibited both fibrinogen-clotting and platelet-activation activities of plasma-derived thrombin, recombinant wild type thrombin, and mutant thrombin K154A in a sequence-specific and dose-dependent manner, whereas it did not inhibit either activity of mutant thrombin R70E. The 15-mer oligonucleotide also inhibited thrombomodulin-dependent protein C activation by plasma-derived thrombin. In competition equilibrium binding experiments, binding of 125I-labeled diisopropyl phosphoryl-thrombin to thrombomodulin was completely inhibited by the consensus 15-mer oligonucleotide with a Kd value of 2.68 +/- 0.16 nM. These results suggest that Arg-70 in the anion-binding exosite of thrombin is a key determinant for interaction with specific single-stranded DNA molecules, and that binding of single-stranded DNA molecules to the exosite prevents the interaction of thrombin with fibrinogen, the platelet thrombin receptor, and thrombomodulin.
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PMID:Localization of the single-stranded DNA binding site in the thrombin anion-binding exosite. 133 57

We have examined the action of the thrombin receptor-derived polypeptide, S42FLLRNPNDKYEPF55 (TRP 42-55), in rat and guinea pig aortic rings and helical arterial strips, and we have compared the actions of the peptide with those of thrombin. In rat preparations, both TRP 42-55 and thrombin caused a concentration-dependent endothelium-dependent relaxation that was blocked by N omega-nitro-L-arginine methyl ester; the relaxation response of the intact rat aortic strip preparation to concentrations of the peptide in the range 30-60 micrograms/mL (17-34 microM) was equivalent to the response to 0.03-0.1 U/mL of thrombin (about 0.3-0.9 nM), yielding a potency ratio (TRP 42-55:thrombin) of about 38,000:1. In contrast with the complete desensitization of thrombin-treated rat aortic preparations to a second administration of the enzyme, the rat aortic tissue was not desensitized by repeated exposures to TRP 42-55 and remained responsive to the peptide even after treatment of the tissue by thrombin. In contrast with the rat aortic tissue, in either intact or endothelium-free guinea pig aortic preparations both TRP 42-55 and thrombin caused a concentration-dependent endothelium-independent contraction. The contractile action of 60 micrograms/mL of receptor peptide (34 microM) in guinea pig aortic strip preparations was equivalent to the contractile action of 0.1-0.3 U/mL thrombin (0.9-3 nM), yielding a potency ratio of about 17,000:1. In guinea pig aortic preparations with an intact endothelium that were precontracted with noradrenaline, neither thrombin nor TRP42-55 caused relaxation, whereas substance P did so.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Vascular actions of thrombin receptor peptide. 133 53

Peptides derived from the recently identified thrombin receptor were tested for their ability to induce platelet aggregation in platelet-rich plasma. The 14 amino acid peptide identified as the new N-terminus after thrombin cleavage (T-14) and an 11 amino acid peptide (T-11) lacking the 3 C-terminal amino acids of T-14 were studied. Both induced platelet aggregation at micromolar concentrations, with T-11 about twice as potent as T-14. Induction of platelet aggregation by these two peptides showed an unusual pH dependence, being more potent at pH 7.2 than at pH 8.1; thrombin-induced aggregation showed a reverse pH dependence. Proton NMR studies of T-11 demonstrated that the chemical shift of the C-alpha proton of the N-terminal serine had a pH dependence that mirrored the aggregation potency. Acetylating the N-terminus of T-11 resulted in loss of aggregating activity, and this peptide did not show the pH-dependence change in chemical shift. The T-14 and T-11 peptides lost aggregating activity when incubated in plasma due to cleavage of the N-terminal serine by an enzyme identified as aminopeptidase M based on its pattern of inhibition and the ability of purified aminopeptidase M (EC3.4.11.2) to cleave the T-11 peptide. Endothelial cell aminopeptidase M was also able to cleave T-11. Inhibiting aminopeptidase M with amastatin enhanced aggregation induced by T-11 but not thrombin. These studies suggest that ionization of the N-terminus of the T-11 and T-14 peptides may be important in initiating platelet aggregation.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Thrombin receptor activating peptides: importance of the N-terminal serine and its ionization state as judged by pH dependence, nuclear magnetic resonance spectroscopy, and cleavage by aminopeptidase M. 135 6

Thrombin is thought to activate platelets through multiple signaling pathways. Recently a new thrombin receptor was identified (Vu et al., Cell 64:1057-1068, 1991) that recognizes alpha-thrombin's anion-binding exosite. Thrombin cleaves this receptor generating a new N-terminal ("tethered-ligand") that activates the receptor. We report here that this receptor is involved in alpha-thrombin inhibition of platelet adenylate cyclase, a process thought mediated by thrombin's high-affinity pathway. In gel-filtered human platelets, iloprost-stimulated cAMP levels were lowered by alpha- and zeta-thrombin addition and, to a much lesser extent, by gamma-thrombin. The alpha- and zeta-thrombin mediated decreases in cAMP were prevented by the thrombin anion-binding exosite inhibitor, BMS 180742, implying that binding to thrombin's anion-binding exosite was required. The iloprost-stimulated increase in cAMP was also reversed (in a concentration-dependent fashion) by a peptide mimicking the new N-terminal of the "tethered-ligand" thrombin receptor (SFLLRNPNDKYEPF). In broken cell preparations, platelet adenylate cyclase activity was also inhibited by SFLLRNPNDKYEPF (but not by a similar peptide used as a control, FSLLRNPNDKYEPF). These results support the hypothesis that thrombin inhibition of platelet adenylate cyclase activity is mediated, at least in part, via the "tethered-ligand" receptor. Moreover, this data is consistent with the "tethered-ligand" receptor mediating the high affinity actions of alpha-thrombin.
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PMID:Involvement of the "tethered-ligand" receptor in thrombin inhibition of platelet adenylate cyclase. 137 79

To better define thrombin-receptor interactions, we synthesized human thrombin peptides and identified binding-domain peptides that bind thrombin receptors and activate mitogenic signals (Glenn, K.C., G.H. Frost, J.S. Bergmann, and D.H. Carney. 1988. Pept. Res. 1:65-73). Treatment of full dermal dorsal incisions with a single topical application of thrombin receptor-activating peptide (TRAP-508) or human alpha-thrombin in saline enhances 7-d incisional breaking strength in normal rats up to 82% or 55% over saline-treated controls, respectively. Control wounds require approximately 11.5 d to achieve breaking strength equivalent to TRAP-treated wounds at day 7. Thus, a single application of TRAP accelerates healing, shifting the time course forward by up to 4.5 d. Histological comparisons at day 7 show more type I collagen, less evidence of prolonged inflammation, and an increase in number and maturity of capillaries in TRAP- and thrombin-treated incisions. Angiograms also show 50-65% more functional vascularization going across thrombin- and TRAP-treated surgical incisions. Thus, alpha-thrombin and thrombin peptides, such as those released following injury, appear to initiate or enhance signals required for neovascularization and wound healing. The ability to accelerate normal wound healing events with synthetic peptides representing receptor binding domains of human thrombin may offer new options for management of wound healing in man.
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PMID:Enhancement of incisional wound healing and neovascularization in normal rats by thrombin and synthetic thrombin receptor-activating peptides. 137 40

Thrombin cleaves its receptor at arginine-41, resulting in the generation of a new receptor NH2-terminus with the sequence SFLLRNPNDKYEPF. This peptide (TRP-14) may signal a variety of thrombin's responses. We examined the effects of TRP-14 in inducing endothelial cell hyperadhesivity and neutrophil (PMN) adhesion to endothelial cell monolayers. Human umbilical vein endothelial cells (HUVECs) challenged with TRP-14 (10(-4) to 10(-5) M) produced concentration-dependent increases in endothelial adhesivity to PMN. In contrast, position 1 to 2 inverted peptide (FSLLRNPNDKYEPF) did not induce the response. The adhesion response was transient; that is, PMN adhesion increased within 15 minutes and decreased by 75 minutes after TRP-14 challenge of HUVECs. The transient endothelial adhesiveness paralleled the time course of P-selectin expression. TRP-14-induced release of P-selectin from intracellular stores may be a critical determinant of the response since treatment of endothelial cells with anti-P-selectin monoclonal antibody (mAb) G1 prevented the increase in PMN adhesion. Control nonneutralizing anti-P-selectin mAb S12 and mAb RR1/1 directed against intercellular adhesion molecule-1 (ICAM-1) on HUVECs were ineffective. The results indicate that the "tethered ligand" of the thrombin receptor created by the proteolytic action of thrombin on its receptor (i.e., TRP-14) signals increased endothelial adhesiveness by a P-selectin-dependent mechanism. Thrombin-induced PMN adhesion may involve formation of a new NH2-terminus of the endothelial thrombin receptor with the sequence SFLLRNPNDKYEPF followed by activation of endothelial second messenger pathways and the transient expression of P-selectin.
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PMID:Thrombin receptor 14-amino acid peptide mediates endothelial hyperadhesivity and neutrophil adhesion by P-selectin-dependent mechanism. 138 Dec 92

Thrombin, the key regulatory protein of hemostasis, is a potent stimulus for endothelial cell activation, a process implicated in a variety of ischemic, thrombotic, and inflammatory vascular disorders. Activation of the thrombin receptor requires a novel mechanism of receptor proteolysis generating a tethered receptor ligand. Synthetic peptides whose sequences are identical to this newly exposed receptor NH2-terminus reproduce thrombin effects on human and bovine endothelial cell activation. Receptor cleavage by catalytically active alpha-thrombin is tightly coupled to a PI-PLC, with resultant generation of IP3 and DAG, increases in [Ca2+]i, and translocation of PKC (Fig. 3). Both the increase in [Ca2+]i and PKC activation are required for thrombin-stimulated PLA2 and PLD activity, PGI2 synthesis, and barrier dysfunction, the latter occurring as the result of Ca2+ and PKC effects on specific cytoskeletal protein elements and other contractile proteins (Fig. 3). Further investigations are ongoing to identify more clearly not only the precise biochemical intermediates involved in the endothelial cell response to thrombin but also the specific protein kinase systems involved in thrombin-mediated signal transduction in vascular endothelium.
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PMID:Molecular mechanisms of thrombin-induced human and bovine endothelial cell activation. 140 26

Augmentation of thrombin-modulated chemotaxis and mitogenic activity within the early phase of soft tissue repair is now possible. Identification of high-affinity thrombin receptor binding domains within thrombin has enabled the synthesis of a family of peptides which interact with thrombin receptors and enhance in vitro mitogenesis. A single (5.0 micrograms/wound) application of the thrombin receptor-activating peptide (P517-30) significantly increased wound breaking strength from Day 5 (31% over controls) to Day 12. Two models of impaired healing created by radiotherapy (RT) were used to elucidate possible mechanisms of P517-30 action. Although P517-30 did not completely overcome the RT-induced healing impairments, it increased breaking strength under conditions of penetrating whole body RT-induced pancytopenia by 22% and of nonpenetrating surface RT-induced dermal cell damage by 42%. This suggests that P517-30 directly stimulates resident endothelial cells, fibroblasts, or other cells to overcome dermal and circulating monocytic deficits. These results suggest a method to accelerate wound healing with potential clinical applications and emphasize the activity of thrombin as a growth factor.
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PMID:Acceleration of soft tissue repair by a thrombin-derived oligopeptide. 140 99

Some, if not all, of the cellular actions of alpha-thrombin are now believed to be mediated by proteolytic cleavage of the cell surface thrombin receptor to yield a tethered ligand that initiates signal transduction via the receptor. We have investigated the actions of alpha-thrombin on the regulation of cytosolic free Ca2+ concentration ([Ca2+]i) and intracellular pH (pHi) in human osteoblast-like Saos-2 cells. After acidification with nigericin, thrombin induced an acute increase of [Ca2+]i and a rise in pHi. The action of thrombin on pHi was dependent on activation of the Na(+)-H+ antiporter. Thrombin elicited parallel concentration-dependent increases in both [Ca2+]i and pHi, and the rise in [Ca2+]i was a prerequisite for the increase in pHi. Preincubation of thrombin with the active site proteolytic inhibitor, BOC-D-Phe-L-Pro-D,L-Lys-CF3, prevented the alkalinization response to thrombin but had little or no effect on the thrombin-induced rise in [Ca2+]i. Hirudin, a natural inhibitor of thrombin, acts by tight binding to several discrete regions on the thrombin molecule. Preincubation of thrombin with hirudin completely blocked the rise in both [Ca2+]i and pHi. These results demonstrate that the thrombin-induced rise in [Ca2+]i alone is not sufficient to cause alkalinization in Saos-2 cells. More importantly, our findings reveal that not all of the cellular actions of thrombin can be explained by proteolytic cleavage of the thrombin receptor and suggest that different domains on the thrombin molecule may be required for eliciting signals that raise [Ca2+]i and pHi in Saos-2 cells.
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PMID:Regulation of pHi in Saos-2 cells by thrombin: roles of proteolytic activity and cytosolic calcium transients. 147 67

1. Three distinct lines of evidence indicate that proteinases are involved in the growth of cultured animal cells. 2. Endogenous growth-related proteinases have been identified, and exogenous proteinases can also stimulate cell proliferation, probably by different mechanisms. In some cases, higher concentrations of proteinases are cytotoxic. 3. Proteinase inhibitors, not surprisingly, inhibit cell growth, but can also be mitogenic at sub-inhibitory concentrations. 4. There must, therefore, be at least three major cellular processes in which proteinases or proteinase inhibitors can operate to exert a direct effect on cell proliferation. 5. Details of one action of an exogenous proteinase, typified by thrombin and the thrombin receptor, are becoming clear at the molecular level, but thrombin probably activates at least two intracellular signalling systems, as well as acting as a growth inhibitor in some situations. 6. Much remains to be investigated in other examples.
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PMID:Proteinases and proteinase inhibitors as modulators of animal cell growth. 147 61


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