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)

Octimibate inhibited ADP- and collagen-induced platelet aggregation in human, rabbit and rat platelet-rich plasma. Washed human platelets treated with octimibate had elevated cyclic AMP (cAMP) levels and cAMP-dependent protein kinase activity. When whole platelets were incubated with radiolabeled phosphate, octimibate produced an increase in the phosphorylation of platelet proteins with relative molecular weights of 22, 26, 50 and 80 kilodaltons. This pattern of protein phosphorylation is identical to that observed when the platelets were treated with forskolin, phosphodiesterase inhibitors or other compounds that elevate platelet cAMP levels. Octimibate also inhibited the rise in intracellular Ca++ caused by thrombin, as measured using Fura-2-loaded platelets, which is consistent with octimibate's ability to elevate platelet cAMP levels. When isolated platelet plasma membranes were treated with octimibate, adenylate cyclase activity was stimulated, reaching maximal activation at 1 microM octimibate. (The maximal activation of adenylate cyclase observed with octimibate is 70-75% of that observed with 10 microM PGE1.) This stimulation of platelet adenylate cyclase activity was enhanced by GTP. Octimibate competed for radiolabeled prostaglandin E1 and lloprost binding to isolated platelet membranes at submicromolar concentrations, but did not compete with radiolabeled prostaglandin D2 binding. These studies suggest that octimibate inhibits platelet aggregation by activating platelet adenylate cyclase through stimulation of platelet prostacyclin receptors.
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PMID:Octimibate inhibition of platelet aggregation: stimulation of adenylate cyclase through prostacyclin receptor activation. 217 92

Jakobs, Bauer & Watanabe [(1985) Eur. J. Biochem. 151, 425-430] reported that treatment of platelets with phorbol 12-myristate 13-acetate (PMA) prevented GTP- and agonist-induced inhibition of adenylate cyclase in membranes from the platelets. This was attributed to the phosphorylation of the inhibitory guanine nucleotide-binding protein (Gi) by protein kinase C. In the present study, the effects of PMA on cyclic [3H]AMP formation and protein phosphorylation were studied in intact human platelets labelled with [3H]adenine and [32P]Pi. Incubation mixtures contained indomethacin to block prostaglandin synthesis, phosphocreatine and creatine kinase to remove ADP released from the platelets, and 3-isobutyl-1-methylxanthine to inhibit cyclic AMP phosphodiesterases. Under these conditions, PMA partially inhibited the initial formation of cyclic [3H]AMP induced by prostaglandin E1 (PGE1), but later enhanced cyclic [3H]AMP accumulation by blocking the slow decrease in activation of adenylate cyclase that follows addition of PGE1. PMA had more marked and exclusively inhibitory effects on cyclic [3H]AMP formation induced by prostaglandin D2 and also inhibited the action of forskolin. Adrenaline, high thrombin concentrations and, in the absence of phosphocreatine and creatine kinase, ADP inhibited cyclic [3H]AMP formation induced by PGE1. The actions of adrenaline and thrombin were attenuated by PMA, but that of ADP was little affected, suggesting differences in the mechanisms by which these agonists inhibit adenylate cyclase. sn-1,2-Dioctanoylglycerol (diC8) had effects similar to those of PMA. The actions of increasing concentrations of PMA or diC8 on the modulation of cyclic [3H]AMP formation by PGE1 or adrenaline correlated with intracellular protein kinase C activity, as determined by 32P incorporation into the 47 kDa substrate of the enzyme. Parallel increases in phosphorylation of 20 kDa and 39-41 kDa proteins were also observed. Platelet-activating factor, [Arg8]vasopressin and low thrombin concentrations, all of which inhibit adenylate cyclase in isolated platelet membranes, did not affect cyclic [3H]AMP formation in intact platelets. However, the activation of protein kinase C by these agonists was insufficient to account for their failure to inhibit cyclic [3H]AMP formation. Moreover, high thrombin concentrations simultaneously activated protein kinase C and inhibited cyclic [3H]AMP formation. The results show that, in the intact platelet, the predominant effects of activation of protein kinase C on adenylate cyclase activity are inhibitory, suggesting actions additional to inactivation of Gi.
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PMID:Effects of activation of protein kinase C on the agonist-induced stimulation and inhibition of cyclic AMP formation in intact human platelets. 244 6

We have examined the histamine-releasing effects of 4 different stimuli on murine bone marrow-derived mast cells (BMMC). We obtained 10(9) BMMC from one femur of a CBA/J mouse when it was cultured in the presence of conditioned medium from WEHI-3 cells for 4 weeks. When these cells were sensitized with monoclonal anti-DNP IgE antibodies, DNP35HSA antigen, ionomycin, thrombin and ATP induced 70%, 70%, 40% and 60% histamine release from BMMC, respectively. Thrombin induced rapid degranulation, whereas the kinetics of histamine release by other stimuli reached a plateau after 5 min. ADP, AMP and GTP as well as ATP but not adenosine caused histamine release from BMMC, suggesting the presence of P2-purinoceptors on these cells.
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PMID:The stimuli releasing histamine from murine bone marrow-derived mast cells. 1. The presence of P2-purinoceptors. 248 81

We compared the mechanisms by which thrombin and platelet-derived growth factor (PDGF) activate phospholipase C in cultured vascular smooth muscle cells. Thrombin caused a transient (less than 5 min) increase in inositol trisphosphate (IP3) while PDGF caused a sustained (greater than 10 min) increase. Both pertussis toxin and phorbol 12-myristate 13-acetate (PMA) inhibited the thrombin-induced increase in IP3 but neither agent affected the PDGF-induced increase in IP3. To examine the role of GTP binding (G) proteins in the activation of phospholipase C by these two hormones, GTP analogues were introduced into saponin-permeabilized cells. In the absence of hormones, guanosine 5'-O-(3-thiotrisphosphate) (GTP gamma S) caused a progressive increase in IP3 release which was inhibited 55% by PMA (200 ng/ml). In the presence of thrombin, GTP gamma S caused synergistic increase in IP3 release. The synergism between GTP gamma S and thrombin was virtually eliminated by 10 min prior exposure to PMA (200 ng/ml). When PDGF was the hormonal agonist, GTP gamma S also caused synergistic increase in IP3 release and guanosine 5'-O-(2-thiodiphosphate) blunted PDGF-induced IP3 release. However, in contrast to thrombin, the synergism between GTP gamma S and PDGF was unaffected by PMA. Thus, thrombin and PDGF activate phospholipase C by signal transduction systems which differ in kinetic properties and in sensitivity to PMA and pertussis toxin. Despite these differences, both systems appear to involve GTP binding proteins at some step.
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PMID:Guanosine 5'-O-(3-thiotrisphosphate) potentiates both thrombin- and platelet-derived growth factor-induced inositol phosphate release in permeabilized vascular smooth muscle cells. Signaling mechanisms distinguished by sensitivity to pertussis toxin and phorbol esters. 249 71

In rabbit platelets, collagen (50 micrograms/ml)- or thrombin (0.5 U/ml)-induced diacylglycerol formation was dose-dependently prevented by phorbol 12-myristate 13-acetate (PMA, 2-50 nM). However, collagen-induced arachidonic acid liberation and lysophosphatidylcholine formation were rather enhanced by PMA, while the thrombin-induced liberation was not. We also demonstrated with saponin-permeabilized platelets that collagen (100 micrograms/ml)-induced arachidonic acid liberation was enhanced by GTP gamma S and inhibited by GDP beta S, both dose-dependently. Since these results lead us to consider that protein kinase C affects a guanine-nucleotide-binding protein (G-protein) to modulate phospholipase A2 and C, we investigated this dual effect of PMA on arachidonic acid liberation and diacylglycerol formation induced by G-protein activator. Addition of GTP gamma S (100 microM) to saponin-permeabilized platelets significantly induced these responses, and PMA (2-10 nM)-pretreatment before the cell permeabilization inhibited diacylglycerol formation and enhanced arachidonic acid liberation and lysophosphatidylcholine formation, dose-dependently. Likewise, PMA (20 nM) had differential effects on the similar NaF (20 mM)-induced responses in intact platelets. Contrarily, 10 nM PMA had no effect on diacylglycerol formation caused by an addition of high concentration of Ca2+ (1 mM) alone after the cell permeabilization, while it still had a potentiating effect on arachidonic acid liberation under the condition. These results suggest that protein kinase C may have a dual regulatory effect on the activation of phospholipase A2 (positive feedback) and phospholipase C (negative feedback), probably through influences on two distinct G-proteins associated separately with these two enzymes.
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PMID:Differential effects of phorbol 12-myristate 13-acetate on GTP gamma S-induced diacylglycerol formation and arachidonic acid liberation in saponin-permeabilized rabbit platelets. 249 43

Preincubation of human platelets with activators of protein kinase C such as phorbol 12-myristate 13-acetate (PMA) has been shown previously to attenuate the ability of agonists both to suppress formation of cAMP and to stimulate hydrolysis of phosphoinositides. In the present study, we have examined whether the attenuation caused by PMA can be attributed to the phosphorylation of the alpha subunit(s) of Gi, a GTP-binding regulatory protein implicated in several pathways of signal transduction. PMA was found to promote the phosphorylation of several proteins within saponin-permeabilized and intact platelets incubated with [gamma-32P]ATP and [32P]H3PO4, respectively. None of the phosphoproteins, however, was precipitated by either of two antisera containing antibodies differing in specificities for epitopes within Gi alpha, despite precipitation of a substantial fraction of the subunit itself. In contrast, other antisera, containing antibodies specific for the recently described Gz alpha or both Gz alpha and Gi alpha, precipitated a 40-kDa phosphoprotein. Phosphorylation of this protein occurred not only in response to PMA, but to thrombin and the thromboxane A2 analog U46619. These data suggest that activators of protein kinase C lead to the phosphorylation within platelets of a select population of G alpha subunits. The identified phosphoprotein is not Gi alpha, but is similar or identical to Gz alpha. Because Gz alpha does not contain the consensus site for ADP-ribosylation by the Bordetella pertussis toxin islet-activating protein, the data also suggest that effects of PMA on processes otherwise sensitive to this toxin are not exerted at the level of G proteins responsible for transduction.
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PMID:Thrombin and phorbol esters cause the selective phosphorylation of a G protein other than Gi in human platelets. 250 48

We have separated multiple small Mr GTP-binding proteins (G proteins) from bovine brain membranes by several column chromatographies and purified to near homogeneity four of them, including a novel Mr 24,000 G protein (smg p25A), a novel Mr 22,000 G protein (smg p21), the rho protein (rho p20), and the c-Ki-ras protein (c-Ki-ras p21). Among these small Mr G proteins, only smg p21 is phosphorylated stoichiometrically by cAMP-dependent protein kinase (protein kinase A), and c-Ki-ras p21 is phosphorylated to a small extent by protein kinase A in a cell-free system. None of smg p25A, rho p20, and other partially purified small Mr G proteins is phosphorylated by protein kinase A. Neither smg p21 nor other small Mr G proteins are phosphorylated by protein kinase C. About 1 mol of phosphate is maximally incorporated into 1 mol of smg p21 by protein kinase A. Only serine residue(s) are phosphorylated. The guanosine 5'-3-O-(thio) triphosphate (GTP gamma S)-bound and GDP-bound forms of smg p21 are phosphorylated with the same reaction velocity. The phosphorylation of smg p21 affects neither its GTP gamma S-binding nor GTPase activity. smg p21 is found in human platelets, and this human platelet smg p21 is also phosphorylated by protein kinase A at the same site(s) as bovine brain smg p21 in a cell-free system. When intact human platelets are stimulated by prostaglandin E1 known to elevate the cAMP level, four proteins with apparent Mr values of 240,000, 50,000, 24,000, and 22,000 are phosphorylated. These four proteins are also phosphorylated by the action of dibutyryl cAMP but not by the action of thrombin, Ca2+ ionophore A23187, or 12-O-tetradecanoylphorbol-13-acetate. Among the four proteins, the Mr 22,000 protein is identified as smg p21. The site(s) of phosphorylation of smg p21 by protein kinase A in a cell-free system are identical to that phosphorylated in response to prostaglandin E1 in intact platelets. These results indicate that among many small Mr G proteins, smg p21 is selectively phosphorylated by protein kinase A and that this G protein is also phosphorylated by this protein kinase in response to prostaglandin E1 in intact human platelets.
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PMID:Phosphorylation of smg p21, a ras p21-like GTP-binding protein, by cyclic AMP-dependent protein kinase in a cell-free system and in response to prostaglandin E1 in intact human platelets. 250 24

Incubation of rabbit platelets with thrombin resulted in rapid accumulations of inositol trisphosphate (IP3) in [3H]inositol-labeled platelets, increases of [3H]arachidonic acid [( 3H]AA) release, and [3H]serotonin secretion from the platelets prelabeled with these labeled compounds. The experiments using phospholipase A2 or C inhibitor suggested that not only phospholipase C but also phospholipase A2 activity plays an important role in serotonin secretion. We then studied the regulatory mechanisms of phospholipase A2 activity. Guanosine 5'-(3-O-thio)triphosphate (GTP gamma S), guanyl-5'-(beta,gamma-iminio)triphosphate), or AlF4- caused a significant liberation of AA in digitonin-permeabilized platelets but not in intact platelets. Thrombin-stimulated AA release was not observed in permeabilized platelets, whereas thrombin acted synergistically with GTP or GTP analogs to stimulate AA release. GTP analog-stimulated AA release was inhibited by guanosine 5'-(2-O-thio)diphosphate) and was also inhibited by decreased Mg2+ concentrations. Thrombin-induced, GTP-dependent AA release, but not IP3 formation, was diminished by 100 ng/ml of pertussis toxin, associated with ADP-ribosylation of membrane 41-kDa protein(s). Thrombin-stimulated AA release from intact platelets and GTP gamma S-stimulated release from permeabilized platelets were both markedly dependent on Ca2+. However, Ca2+ addition could not enhance AA release without GTP gamma S even when Ca2+ was increased up to 10(-4) M in permeabilized platelets. The results show that thrombin-stimulated AA release from rabbit platelets is mainly mediated by phospholipase A2 activity, not by phospholipase C activity, and that Ca2+ is an important factor to the activation of phospholipase A2 but is not the sole factor to the regulation. GTP-binding protein(s) is involved in receptor-mediated activation of phospholipase A2.
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PMID:Pertussis toxin-sensitive GTP-binding proteins may regulate phospholipase A2 in response to thrombin in rabbit platelets. 250 76

In platelets, and in several other cell systems, pre-treatment with protein kinase C activators such as phorbol 12-myristate 13-acetate (PMA) results in the inhibition of receptor-mediated responses, suggesting that protein kinase C may play an important role in the termination of signal transduction. In the present study, we have attempted to locate the site of action of phorbol ester by comparing thrombin-induced (i.e. receptor-mediated) platelet activation with that induced by guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and NaF, two agents which by-pass the receptor and initiate platelet responses by directly modulating G-protein function. After a 10 s pre-treatment with PMA (16 nM), dense-granule secretion induced by thrombin (0.2 unit/ml), GTP[S] (40 microM) and NaF (30 mM) was potentiated, resulting in a greater than additive response to agent plus PMA. However, after a 5 min pre-treatment, thrombin-induced secretion alone was inhibited, whereas PMA plus GTP[S]/NaF-induced release remained greater than additive. [32P]Phosphatidate formation in response to all three agents, in contrast, was inhibited by 50-70% in PMA (5 min)-treated platelets. That secretion induced by these agents is a protein kinase C-dependent event was demonstrable by using staurosporine, a protein kinase C inhibitor which at concentrations of 1-10 nM inhibited (70-90%) PMA-induced as well as thrombin- and NaF-induced secretion and protein phosphorylation. In membranes from PMA-treated platelets, thrombin-stimulated GTPase activity was significantly enhanced compared with that in untreated membranes (59% versus 82% increase over basal activity). The results suggest that inhibition of receptor-mediated responses by PMA may be directed towards two sites relating to G-protein activation: (i) receptor-stimulated GTPase activity and (ii) G-protein-phospholipase C coupling. Furthermore, the lack of inhibition of NaF- and GTP[S]-induced secretion by PMA suggests that different mechanisms may be involved in thrombin-induced and G-protein-activator-induced secretion.
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PMID:Effect of phorbol ester treatment on receptor-mediated versus G-protein-activator-mediated responses in platelets. Evidence for a two-site action of phorbol ester at the level of G-protein function. 251 Jul 16

By using antibodies specific for alpha subunits of inhibitory GTP-binding proteins (Gi alpha polypeptides) to probe Western blots of whole platelet protein, we detected Gi alpha-2 as the predominant Gi alpha species present in platelets. The subcellular compartmentalization of distinct Gi alpha-2-immunoreactive polypeptides coupled to thrombin and alpha 2-adrenergic receptors was examined in Triton X-100 platelet lysates prepared by highspeed centrifugation. This treatment permitted separation of the Triton-insoluble membrane skeleton from Triton-soluble cell components. In cells treated with either alpha-thrombin or epinephrine, we observed that a greater proportion of Gi alpha-2 was localized in the Triton-soluble fraction than in the Triton-insoluble fraction. Pertussis toxin was found to catalyze ADP-ribosylation of Gi alpha-2 in whole platelets. In thrombin-stimulated cells, this activity was confined to the Triton-soluble fraction and was markedly lower than that of unstimulated cells. Epinephrine, on the other hand, promoted translocation of a portion of the pertussis toxin-sensitive Gi alpha-2 from the Triton-soluble fraction to the Triton-insoluble fraction. In addition, epinephrine stimulated translocation of a phosphorylated protein of approximately 38 kDa that was not ADP-ribosylated by pertussis toxin. This protein expressed immunoreactivity with the general Gi alpha antiserum AS/7 but not with the Gi alpha-2 antiserum LE/3. These findings suggest a role for specific localization of Gi alpha proteins in epinephrine-induced platelet responses.
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PMID:Epinephrine induces changes in the subcellular distribution of the inhibitory GTP-binding protein Gi alpha-2 and a 38-kDa phosphorylated protein in the human platelet. 253 14


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