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)

We have devised an improved high pressure liquid chromatographic technique whereby serotonin, nucleosides, cyclic nucleotides, namely cAMP and cGMP, and 5'mono-, 5'di-, and 5'tri-nucleotides can be analyzed. The cyclic nucleotides have been measured in picomolar quantities. All nucleotides can be quantitated in a single step separation in 75 min using a 0.0015 M phosphoric acids vs. 1M pH 4.8 ammonium phosphate gradient. 5/10 ml of platelet-rich plasma furnishes an adequate sample for complete analysis. Nucleotide levels in platelets from 16 normal donors expressed in 10(11) platelets are as follows: cAMP, 6.32 (4.15) nanomoles and AMP, 0.32 (0.14); ADP, 2.48 (0.67); ATP 3.78 (0.68); GDP 0.38 (0.07) and GTP, 0.45 (0.07) micromoles. ADP and ATP values are lower than those previously published. However, the total nucleotide level approaches published values. Upon aggregation with thrombin, approximately 50% of ADP and 40% ATP is releaseed. Release is complete by 2 min. Thrombin is the most potent releasing agent with collagen and ADP occupying an intermediate role and epinephrine being the least effective. Upon aggregation cyclic AMP levels diminish along the other nucleotides. Patients with asthma showed depression of ADP, ATP, GDP and GTP levels.
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PMID:Measurement of nucleotide pools in platelets using high pressure liquid chromatography. 57

The uptake and binding of antimitotic alkaloid colchicine has been demonstrated in washed preparations of human pletelets. A silicone oil technique was adapted so that both uptake and binding of 14C-colchicine were examined in the same platelet preparations. The time dependence and amount of colchicine taken up and bound by different pletelet preparations during a 90 to 120 min incubation period were highly reproducible. Both colchicine uptake and binding by intact platelets, and colchicine binding by preparations of lysed platelets were specific and temperature dependent. Colchicine uptake was slowly reversible. Magnesium and GTP enhanced colchicine binding by lysed platelet preparations but calcium decreased binding. exposure of platelets to either cold (4 degrees C) or to thrombin, which disrupt platelet microtubules, produced significant increases in colchicine uptake and binding. The thrombin effect was maximal at 37 degrees C and resulted in a greater increase in uptake and binding than that produced by either cold treatment alone or, by cold treatment followed by incubation with thrombin at 37 degrees C. The amount of increase in uptake and binding produced by thrombin was independent of both thrombin (1--5 Units/10(9) platelets) and colchicine concentrations (1--50 X 10(-6) M). It is postulated that thrombin may initiate the formation, or make available, colchicine binding sites (microtubule subunits) within platelets.
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PMID:Colchicine uptake and binding by human platelets. 81 25

Thrombin stimulates multiple functions in cultured endothelial cells (EC), including an increase in cell surface adhesion sites for monocytes and the production of platelet-derived growth factor (PDGF). We have initiated studies to define the intracellular signaling pathways involved in these two thrombin-induced EC functions by focusing on the possible roles of the Na(+)-H+ antiporter and guanine nucleotide-binding proteins (G proteins). Amiloride suppressed thrombin-stimulated PDGF production by human aortic EC without affecting either basal PDGF production or overall protein synthesis. The steady-state mRNA levels of PDGF-A and PDGF-B chain were not reduced by amiloride. In replicate EC cultures, amiloride had no effect on thrombin-stimulated monocyte adhesion. In addition, thrombin induction of PDGF production, but not monocyte adhesion, was abrogated in the absence of extracellular sodium. Thrombin stimulation of both monocyte adhesion and PDGF production appeared to involve a pertussis toxin-insensitive G protein. Thrombin induced an increase in [35S]guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) binding to human EC membranes. GTP gamma S, in the presence of a suboptimal concentration of thrombin, caused maximal stimulation of both monocyte adhesion and PDGF production. The effect of GTP gamma S on PDGF production was at the level of transcription. These results indicate that the EC is capable of responding to a pluripotent agonist such as thrombin through multiple signaling pathways, which converge and diverge to achieve differential cellular responses.
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PMID:Thrombin stimulates PDGF production and monocyte adhesion through distinct intracellular pathways in human endothelial cells. 131 Feb 11

Recent studies have shown that mastoparan, an amphiphilic peptide derived from wasp venom, accelerates guanine nucleotide exchange and GTPase activity of purified GTP-binding proteins. In the present study we have examined the functional consequences of exposure of intact human platelets to mastoparan. Mastoparan promoted rapid (less than or equal to 1 min) dose-dependent increases in 5-hydroxy[14C]tryptamine and beta-thromboglobulin release from dense-granule and alpha-granule populations respectively. The exocytotic response did not result from a lytic effect of mastoparan and occurred in the complete absence of platelet shape change and aggregation. Liberation of [3H]arachidonate and increases in cytosolic [Ca2+] (detected with fura 2) were not observed in platelets stimulated with mastoparan. Similarly, in platelets preloaded with [3H]inositol during reversible electroporation, mastoparan did not cause the accumulation of [3H]inositol phosphates. Mastoparan-induced secretion was unaffected by preincubation with either the protein kinase C inhibitor staurosporine (10 nM-10 microM) or prostacyclin (PGI2; 100 ng/ml) and was not accompanied by phosphorylation of the 45 kDa protein kinase C substrate or the 20 kDa protein normally associated with platelet activation. The G-protein inhibitor guanosine 5'-[beta-thio]diphosphate (GDP[S]; 1 mM) attenuated the secretion induced by mastoparan in both intact and saponin-permeabilized platelets. Encapsulation of GDP[S] during reversible permeabilization inhibited mastoparan-induced secretion, providing evidence for an intracellular action of GDP[S]. In all these studies thrombin (0.05-0.2 unit/ml) elicited characteristic responses, and thrombin-induced secretion was inhibited by staurosporine, PGI2 and GDP[S]. Mastoparan also increased intra-platelet cyclic AMP in a dose-dependent manner. Mastoparan and PGI2 increased 32P incorporation into a protein of approx. 24 kDa, whereas phosphorylation of a 50 kDa substrate was only seen in PGI2-stimulated platelets. These results indicate that mastoparan promotes secretion by a mechanism which does not involve stimulation of phospholipase C and suggest that the secretory event may result either from a direct fusogenic action of mastoparan and/or from stimulation of the putative exocytosis-linked G-protein, Ge.
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PMID:Mastoparan promotes exocytosis and increases intracellular cyclic AMP in human platelets. Evidence for the existence of a Ge-like mechanism of secretion. 131 May 99

Membranes of cultured newborn rat cardiomyocytes contain enzymatic activities that regulate the formation and the breakdown of inositol 1,4,5-trisphosphate (1,4,5-IP3). GTP gamma S increased the rate of exogenous [3H]phosphatidyl 4,5-bisphosphate ([3H]PIP2) hydrolysis (EC50: 40 microM). This effect was dependent on the presence of deoxycholate and maximal at 2 mM deoxycholate. GTP gamma S increased the efficacy of phospholipase C (PLC) (by 2.3-fold), but did not alter the apparent affinity of the enzyme for PIP2. Other nucleotides, GDP beta S and ATP gamma S, and pyrophosphate also stimulated PIP2 hydrolysis, while AlF4- was ineffective. The effect of GTP gamma S was not inhibited by GDP beta S. The agonists norepinephrine and thrombin, which by themselves had no effect, did not potentiate the response to GTP gamma S. In contrast, 1,4,5-IP3 hydrolysis was decreased by GTP gamma S (EC50: 100 microM) as well as by other nucleotides and by pyrophosphate, but not by AlF4-. GDP beta S did not antagonize the GTP gamma S-induced inhibition of IP3 hydrolysis. These results suggest that GTP can stimulate the hydrolysis of exogenous PIP2 by an action on membrane-bound PLC at a site beyond the G protein activating PLC and inhibit the hydrolysis of 1,4,5-IP3 by a mechanism common to all nucleotides. Thus, GTP can regulate 1,4,5-IP3 metabolism by stimulating its formation and inhibiting its breakdown.
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PMID:Regulation of inositol 1,4,5-trisphosphate metabolism by guanine nucleotides in membranes of cultured newborn rat cardiomyocytes. 131 33

Lysate from quiescent platelets promotes rapid hydrolysis of [gamma-32P]GTP bound to rap1B. Various platelet agonists, including platelet-activating factor, phorbol 12,13-dibutyrate, alpha-thrombin, epinephrine, ADP, and iloprost, that affect platelet metabolism by different signal transduction pathways were used to stimulate intact platelets and study their effects on rap1B.GAP-activated GTPase activity (GAP, GTPase-activating protein). Only epinephrine was found to dramatically decrease not only the rate but also the amount of hydrolysis of rap1B-bound GTP activated by rap1B.GAP. This effect was dose dependent and occurred rapidly. The suppression of GTPase activity was specific for rap1B.GAP in that ras.GAP- and rap2B.GAP-activated GTPase activity were not affected by epinephrine stimulation. This effect appears to be mediated by the alpha 2-adrenergic receptor, as evidenced by a similar suppression of GTPase activity by stimulating platelets with the synthetic alpha 2-adrenergic receptor agonist UK14304 (bromoxidine). Furthermore, the selective alpha 2-adrenergic receptor antagonist yohimbine blocked the suppression of GTPase activity expressed in epinephrine-stimulated cell lysates. No apparent changes in the patterns of protein expression or tyrosine phosphorylation were observed. Although the migration characteristics upon anion-exchange chromatography of rap1B.GAP and ras.GAP activities were unaffected by epinephrine stimulation, the specific activity of rap1B.GAP was noticeably decreased with 250 and 500 microM epinephrine. These results suggest a possible role for rap1B and rap1B.GAP in epinephrine-stimulated signal transduction.
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PMID:Epinephrine suppresses rap1B.GAP-activated GTPase activity in human platelets. 131 68

Platelet activity is increased in persons with insulin dependent diabetes mellitus (IDDM). Receptor-medicated phospholipase C (PLC) activation and hydrolysis of phosphatidylinositol bisphosphate (PIP2) accompanies platelet activation. Previous work from our laboratory has shown that PIP2 hydrolysis is decreased in platelets of persons with IDDM. PIP2 hydrolysis is mediated via a phosphoinositide(PI)-specific PLC. PI-PLC activity is regulated by guanine nucleotide(GTP)-binding proteins. We therefore examined the hypothesis that platelet aggregations and PI turnover in platelet from subjects with IDDM is linked to alterations in PI-specific PLC activity. We found thrombin induced platelet aggregation was increased in the IDDM group. Basal PI and PIP2-specific PLC activity was not statistically different for the two groups. Guanine-nucleotide stimulated PIP2-specific PLC activity was decreased in the IDDM platelets. The mechanism for the reduced PLC activity and its role in the platelet hyperaggregation requires further study.
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PMID:Reduced guanine nucleotide-stimulated polyphosphoinositide specific phospholipase C in platelet hyperaggregation in IDDM. 131 91

The findings described above illustrate how the src kinase can influence several new pathways of inositol phosphate metabolism, both at the membrane level with the production of novel D-3 phosphoinositides and the activation of PI-3 kinase, and at the cytosolic level by altering the expression of certain inositol polyphosphates, in particular Ins(1,4,5,6)P4. At present, it is difficult to speculate on the role these phenomena play in cellular transformation by src, since the functions of D-3 phosphoinositides and most inositol polyphosphates are unclear. There is evidence, however, that these new pathways of phosphoinositide metabolism occur in response to other types of cellular stimulations besides src transformation. Novel D-3 phosphoinositides are expressed in a variety of nonneoplastic cells, including human platelets treated with thrombin, smooth muscle cells and stimulated neutrophils. In addition, unusual InsP4 isomers such as D/L-Ins(1,4,5,6)P4 are found in chicken erythrocytes, murine macrophages, AR4-2J rat pancreatoma cells and adrenal glomerulosa cells, to name only a few. Recently, associations have been reported between PI-3 kinases and cytoskeletal elements in thrombin- stimulated platelets, and between activated ras proteins in rat liver epithelial cells. The latter discovery is particularly intriguing since GTP-binding proteins such as ras are known to influence cell shape and serve as downstream effector proteins in the signal transduction pathways of numerous growth factor receptors. Thus, one function of novel phosphoinositides and their metabolites may lie at the level of cytoskeletal and cell shape regulation. Clearly, additional roles for phosphoinositides exist in cells besides their traditional use as precursors for the generation of Ins(1,4,5)P3 and diacylglycerol.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Phosphoinositides and cell growth. 133 66

The protein tyrosine phosphatase (PTPase) inhibitor pervanadate (vanadyl hydroperoxide) stimulated protein tyrosine phosphorylation 29-fold more than did thrombin in intact and saponin-permeabilized platelets. Increased tyrosine phosphorylation preceded, or was coincident with, a fall in PtdIns(4,5)P2 levels, production of PtdIns(3,4)P2 and phosphatidic acid, mobilization of intracellular Ca2+, stimulation of protein kinase C-dependent protein phosphorylation, secretion of dense and alpha-granules, increased actin polymerization, shape change and aggregation which required fibrinogen and was mediated by increased surface expression of GPIIb-IIIa. The tyrosine kinase inhibitor RG 50864 totally prevented induction of tyrosine phosphorylation by pervanadate, as well as all other responses measured; in contrast, the inactive structural analogue, tyrphostin #1, had no effect. Dense-granule secretion induced by pervanadate required protein kinase C activity; however, aggregation and alpha-granule secretion were independent of protein kinase C. In saponin-permeabilized platelets pervanadate and thrombin stimulated phospholipase C activity by GTP-independent and GTP-dependent mechanisms respectively. We conclude that PTPases are important regulators of signal transduction in platelets.
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PMID:Activation of signal transduction in platelets by the tyrosine phosphatase inhibitor pervanadate (vanadyl hydroperoxide). 153 May 76

Recently, we reported that in mouse mastocytoma P-815 cells the cytosol contains some factor(s) which promotes the release of GTP-activated Gi2 alpha from the membrane, and that thrombin induces the translocation of Gi2 alpha from the membrane to the cytosol (Takahashi, S., Negishi, M. and Ichikawa, A. (1991) J. Biol. Chem. 266, 5367-5370). Here we investigated the mechanism underlying the thrombin-induced translocation of Gi2 alpha in mastocytoma cells. Thrombin induced a rapid and transient increase in the intracellular Ca2+ concentration ([Ca2+]i) within 1 min, attenuated pertussis toxin-catalyzed ADP-ribosylation of Gi2 in the membrane, and caused the subsequent translocation of Gi2 alpha. Thrombin induced the translocation of protein kinase C from the cytosol to the membrane, and a protein kinase C inhibitor, staurosporine, completely inhibited the thrombin-induced translocation of Gi2 alpha. When cells were treated with thrombin, the ability of the cytosol to release Gi2 alpha from the membrane in the presence of GTP gamma S markedly increased. This stimulatory effect of thrombin on the ability of the cytosol was mimicked by 12-O-tetradecanoylphorbol 13-acetate (TPA), but not by the Ca2+ ionophore, ionomycin. The thrombin- and TPA-induced potentiation of the ability of the cytosol to release Gi2 alpha was completely abolished by staurosporine. Furthermore, phosphorylation of the cytosol by protein kinase C markedly potentiated the ability of the cytosol to release Gi2 alpha. These results together demonstrate that the thrombin-induced translocation of Gi2 alpha is due to enhancement of the ability of the cytosol to release Gi2 alpha via activation of protein kinase C.
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PMID:Involvement of protein kinase C in thrombin-induced translocation of Gi2 alpha from the membrane to the cytosol in mouse mastocytoma P-815 cells. 154 55


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