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)

The influence of various proteinases on GTP hydrolysis was studied in membranes of human platelets. Of the proteinases examined, trypsin, acrosin and a recently described trypsin-like proteinase from bovine sperm, but not chymotrypsin, increased GTP hydrolysis. Similar to what was described previously for hormone-like agents, the stimulation of GTP hydrolysis by the proteinases was only observed at low GTP concentrations, with apparent Km values of 0.2-0.3 microM-GTP. Stimulation of the high-affinity GTPase by the proteinases occurred without apparent lag phase and was constant over a long period of incubation. The proteinase inhibitors leupeptin and soya-bean trypsin inhibitor blocked the stimulation of GTP hydrolysis, but did not reverse the effect of the proteinases. Treatment of platelet membranes with N-ethylmaleimide, which eliminates Gi-protein (inhibitory guanine-nucleotide-binding protein)-related GTPase stimulation by adrenaline, decreased stimulation of GTP hydrolysis by the proteinases only partially. Activation of GTP hydrolysis by the proteinases was partially additive with that caused by adrenaline, whereas thrombin stimulation was not increased further. The data indicate that, similarly to the proteinase thrombin, trypsin and trypsin-like proteinases can activate GTP-hydrolysing protein(s) that exhibit high affinity for GTP in platelet membranes. It is suggested that the proteinases interact in platelet membranes with a receptor site similar to that used by thrombin and that the observed GTPase stimulation is a reflection of a proteinase-receptor interaction with a guanine-nucleotide-binding regulatory protein.
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PMID:Stimulation of high-affinity GTPase by trypsin and trypsin-like proteinases in membranes of human platelets. 283 22

Human recombinant interleukin 1 alpha (IL-1 alpha) and IL-1 beta stimulated prostaglandin E2 synthesis in 3T3 fibroblasts in a time- and concentration-dependent manner. Enhanced prostaglandin E2 synthesis after IL-1 treatment was apparent by 1 hr and continued to increase for at least 2 days. Half-maximal stimulation occurred at 0.5 pM IL-1 alpha or IL-1 beta, and both interleukins were equally effective, with maximal stimulation occurring in response to 5-10 pM IL-1. In contrast to IL-1, bradykinin stimulation of prostaglandin E2 synthesis is rapid; its effect is maximal by 5 min. In cells that had been pretreated with IL-1 for 24 hr, prostaglandin E2 synthesis in response to bradykinin was amplified more than 10-fold. IL-1 also amplified the receptor-mediated formation of prostaglandin E2 by bombesin and thrombin. The lymphokine did not affect bradykinin receptor number or affinity. IL-1 treatment induced phospholipase A2 and cyclooxygenase but not phospholipase C or prostaglandin E isomerase. It also enhanced bradykinin-stimulated GTPase activity, suggesting possible induction of the GTP-binding regulatory protein coupled to the bradykinin receptor. Thus, IL-1 enhanced receptor-mediated release of prostaglandin E2 in response to bradykinin, bombesin, and thrombin by increasing the cellular levels of phospholipase A2, cyclooxygenase, and GTP-binding regulatory protein(s).
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PMID:Interleukin 1 amplifies receptor-mediated activation of phospholipase A2 in 3T3 fibroblasts. 290 Oct 97

The effects of heparins and heparinoids were studied on adenylate cyclase and GTPase activities in human platelet membranes. Inhibition of adenylate cyclase by adrenaline and platelet activating factor was completely abolished by heparin at 1 microgram/ml. At similar concentration heparin blocked the stimulation of high affinity GTPase(s) by these hormonal factors. In contrast, heparin (up to 30 micrograms/ml) did not abolish adenylate cyclase inhibition and stimulation of GTP hydrolysis by thrombin in the absence of antithrombin III. In the presence of antithrombin III, thrombin action on adenylate cyclase was blocked by unfractionated and high molecular weight heparin at 0.1 microgram/ml. Low molecular weight heparins and pentosanpolysulfate were less or not effective. In contrast, all high and low molecular weight heparins tested were almost equally potent in inhibiting adrenaline-induced inhibition of adenylate cyclase in the absence of antithrombin III. The data indicate that heparins discriminate platelet activating factor and adrenaline-induced inhibition of adenylate cyclase from the inhibitory action of thrombin and delineate different structural requirements for the interaction of heparins with the adenylate cyclase system and antithrombin III.
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PMID:Heparin and heparinoids impair adrenaline and platelet-activating factor but not thrombin-induced inhibition of adenylate cyclase and stimulation of GTP hydrolysis in human platelet membranes. 296 81

Thrombin inhibits adenylate cyclase and stimulates GTP hydrolysis by high-affinity GTPase(s) in membranes of human platelets at almost identical concentrations. Both of these thrombin actions are similar to those observed with agonist-activated alpha 2-adrenoceptors coupling to the inhibitory guanine nucleotide-binding protein N1. However, stimulation of GTP hydrolysis caused by adrenaline (alpha 2-adrenoceptor agonist) and by thrombin at maximally effective concentrations was partially additive, whereas with regard to adenylate cyclase inhibition no additive response was observed. Furthermore, treatment of platelet membranes with pertussis toxin, which inactivates Ni and largely abolishes thrombin- and adrenaline-induced adenylate cyclase inhibition and adrenaline-induced GTPase stimulation, decreased the thrombin-induced stimulation of GTP hydrolysis by only about 30%. Additionally, the thiol reagent N-ethylmalemide (NEM) at rather low concentrations abolished thrombin- and adrenaline-induced stimulation of GTP hydrolysis was decreased by only 30-40% by treatment of platelet membranes with even high concentrations of NEM. Treatment with cholera toxin, which inhibits GTPase activity of the Ns (stimulatory guanine nucleotide-binding) protein, has no effect on thrombin-stimulated GTP hydrolysis. The data suggest that thrombin interaction with its receptor sites in platelet membranes leads to stimulation of two GTP-hydrolysing enzymes. One of these enzymes is apparently Ni and is also activated by agonist-activated alpha 2-adrenoceptors and is inactivated by pertussis toxin and NEM treatment. The other GTP-hydrolysing enzyme activated by thrombin may represent a guanine nucleotide-binding protein apparently involved in the coupling of thrombin receptors to the phosphoinositide phosphodiesterase.
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PMID:Evidence for two GTPases activated by thrombin in membranes of human platelets. 302 30

The thrombin-stimulated GTPase activity of human platelets was additive with respect to the GTPase stimulation effected by prostaglandin E1, but not with that stimulated by adrenaline, vasopressin and platelet-activating factor (PAF). Treatment of platelet membranes with pertussis toxin partially inhibited the thrombin-stimulated GTPase, but had no effect on the vasopressin-stimulated GTPase activity, whereas cholera toxin treatment had no effect on either of these stimulated GTPase activities. Thrombin, adrenaline and PAF, but not vasopressin, inhibited the adenylate cyclase activity of isolated plasma membranes through the action of Ni only, this being inhibited by pertussis toxin. It is suggested that thrombin exerts effects through both the inhibitory guanine nucleotide regulatory protein Ni and through the putative guanine nucleotide regulatory protein, Np, involved in regulating receptor-stimulated inositol phospholipid metabolism. However, vasopressin appears to exert its effects solely through the putative Np.
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PMID:Thrombin, unlike vasopressin, appears to stimulate two distinct guanine nucleotide regulatory proteins in human platelets. 309 63

Thrombin stimulation of 1321N1 astrocytoma cells leads to Ras-dependent AP-1-mediated transcriptional activation and to DNA replication. In contrast to what has been observed in most cell systems, in 1321N1 cells these responses are pertussis toxin-insensitive. The pertussis toxin-insensitive G-protein G12 has been implicated in cell growth and transformation in different cell systems. We have examined the potential role of this protein in AP-1-mediated transcriptional activation and DNA synthesis in 1321N1 cells. Transient expression of an activated (GTPase-deficient) mutant of G alpha 12 increased AP-1-dependent gene expression. This response was inhibited by co-expression of a dominant negative Ala-15 Ras protein. To determine whether the pertussis toxin-insensitive G12 protein is involved in the thrombin-stimulated DNA synthesis, an inhibitory antibody against the C-terminal sequence of G alpha 12 subunit was microinjected into 1321N1 cells. Microinjection of the anti-G alpha 12 resulted in a concentration-dependent inhibition of thrombin-stimulated DNA synthesis. In contrast, microinjection of nonimmune IgG or an antibody directed against the C terminus of G alpha o did not reduce the mitogenic response to thrombin. Furthermore, microinjection of the anti-G alpha 12 antibody had no effect on fibroblast growth factor-stimulated DNA synthesis. These results demonstrate a specific role for G alpha 12 in the mitogenic response to thrombin in human astroglial cells.
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PMID:G12 requirement for thrombin-stimulated gene expression and DNA synthesis in 1321N1 astrocytoma cells. 765 24

A thrombin receptor has been described that is activated by thrombin cleavage generating a new N-terminus. The newly exposed SFLLR-containing "tethered-ligand" then activates the receptor. In these studies, we used 3-mercapto-propionyl-Phe-Cha-Cha-Arg-Lys-Pro-Asn- Asp-Lys-amide (Mpapeptide) as a thrombin receptor antagonist. This compound was capable of preventing both thrombin- and SFLLR-peptide-induced platelet aggregation with little effect on collagen-induced platelet aggregation. It also prevented thrombin- and SFLLRNP-induced calcium mobilization with little effect on thromboxane receptor-activated platelet Ca2+ mobilization. Platelet membrane GTPase could be activated by peptides that activated the thrombin receptor, and the thrombin receptor antagonist also prevented receptor-stimulated GTPase activity. Platelet phospholipase A2 (PLA2) activity (measured as the release of radiolabeled arachidonic acid) and Na+/H+ exchange activation were stimulated by alpha-thrombin and by SFLLR-containing peptides. Activation of both processes with low concentrations of thrombin required thrombin's anion-binding exosite, as they were not activated by similar concentrations of gamma-thrombin, and the alpha- and zeta-thrombin activation was blocked by peptides mimicking the C-terminal region of hirudin. Stimulation of PLA2 and Na+/H+ exchange by both thrombin and SFLLR-containing peptides was inhibited by the thrombin receptor antagonist Mpa-peptide. These results support the hypothesis that thrombin stimulation of PLA2 activity and Na+/H+ exchange occurs via activation of the thrombin tethered-ligand receptor. Moreover, these data are consistent with the tethered-ligand receptor mediating most actions elicited by low concentrations of alpha-thrombin involved in human platelet activation.
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PMID:Inhibition of thrombin and SFLLR-peptide stimulation of platelet aggregation, phospholipase A2 and Na+/H+ exchange by a thrombin receptor antagonist. 787 57

The alpha 2-adrenergic receptor-mediated stimulation of GTPase activity was investigated in human platelet membranes. The stimulatory effect of (-)-epinephrine was strictly dependent on Mg2+ and derived from a high-affinity GTPase activation. (-)-Epinephrine and (-)-norepinephrine stimulated GTPase activity in a concentration-dependent manner with EC50 values of 200 and 600 nM, respectively. These effects were stereospecific, since (+/-)-epinephrine, (+/-)-norepinephrine, and (+)-epinephrine were less potent in stimulating the enzyme activity with EC50 values of 4, 1 and 3 microM, respectively. Thrombin also stimulated GTPase activity concentration dependently with an EC50 value of 0.02 U/mL. The maximal effects of (-)-epinephrine, (-)-norepinephrine, and thrombin were not additive in any combination. Clonidine did not stimulate GTPase activity, whereas another synthetic alpha 2-adrenergic agonist, p-aminoclonidine, had the characteristics of a partial agonist. The rank order of potency for antagonists to inhibit the activation of GTPase by 1 microM (-)-epinephrine was yohimbine = rauwolscine > idazoxan = oxymetazoline = phentolamine = WB4101 = (+)-mianserin > (-)-mianserin > prazosin > (-)-propranolol. Negative logarithms of the IC50 values of these antagonists corresponded well with the negative logarithmic values of Ki(pKi) for the alpha 2A-adrenergic receptors determined by a receptor-binding technique in human platelets. These results indicate that epinephrine stimulates high-affinity GTPase activity of G proteins (putatively Gi2), which are also coupled with thrombin receptors, in a Mg(2+)-dependent and stereospecific manner, via alpha 2A-adrenergic receptor activation in human platelet membrane preparations.
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PMID:Pharmacological characterization of epinephrine-stimulated GTPase activity in human platelet membranes. 790 35

Staurosporine in the micromolar range raised inositol trisphosphate in intact human platelets to levels comparable to that mediated by thrombin. This response was inhibited by neomycin, a phospholipase C antagonist. Staurosporine alone induced a weak, transient rise in cytosolic free calcium levels ([Ca2+]i) from release of internal Ca2+ stores but potentiated the effect induced by thrombin. Therefore, it is unlikely that this alkaloid suppressed inositol trisphosphate mobilization of Ca2+. Additional studies show that staurosporine, 0.5-5 microM, stimulated GTPase activity in platelet membranes while 2 microM K252a and 20 microM H7 were inactive. Present results suggest that staurosporine may activate platelet phospholipase C at the level of G proteins or receptors.
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PMID:Staurosporine induces hydrolysis of phosphatidyl inositol 4,5-bisphosphate in human platelets. 816 13

Human platelet thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptors are linked to phosphoinositide-specific phospholipase C (PI-PLC) via a G protein tentatively identified as a member of the Gq class. In contrast, platelet thrombin receptors appear to activate PI-PLC via other unidentified G proteins. Platelets from most dogs are TXA2 insensitive (TXA2-); i.e., they do not aggregate irreversibly or secrete although they bind TXA2, but they respond normally to thrombin. In contrast, a minority of dogs have TXA2-sensitive (TXA2+) platelets that are responsive to TXA2. To determine the mechanism responsible for TXA2- platelets, we evaluated receptor activation of PI-PLC. Equilibrium binding of TXA2/PGH2 receptor agonists, [125I]BOP and [3H]U46619, and antagonist, [3H]SQ29,548, revealed comparable high-affinity binding to TXA2-, TXA2+, and human platelets. U46619-induced PI-PLC activation was impaired in TXA2- platelets as evidenced by reduced (a) phosphorylation of the 47-kD substrate of protein kinase C, (b) phosphatidic acid (PA) formation, (c) rise in cytosolic calcium concentration, and (d) inositol 1,4,5 trisphosphate (IP3) formation, while thrombin-induced PI-PLC activation was not impaired. GTPase activity stimulated by U46619, but not by thrombin, was markedly reduced in TXA2- platelets. Antisera to Gq class alpha subunits abolished U46619-induced GTPase activity in TXA2-, TXA2+, and human platelets. Direct G protein stimulation by GTP gamma S yielded significantly less PA and IP3 in TXA2- platelets. Immunotransfer blotting revealed comparable quantities of Gq class alpha-subunits in all three platelet types. Thus, TXA2- dog platelets have impaired PI-PLC activation in response to TXA2/PGH2 receptor agonists secondary to G protein dysfunction, presumably involving a member of the Gq class.
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PMID:Thromboxane-insensitive dog platelets have impaired activation of phospholipase C due to receptor-linked G protein dysfunction. 822 62


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