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 effects of guanine nucleotides on arachidonic acid (AA) release were studied in intact and saponin-permeabilized human platelets. While GTP[S] itself caused a stimulation of AA release in permeabilized cells, GTP[S], GDP[S], GTP, ATP and other nucleotides inhibited AA release in response to thrombin and other agonists in intact, as well as permeabilized platelets. Inhibition of agonist-stimulated AA release by nucleotides was partially attenuated by addition of ADP, and was abolished by prior stimulation of platelets to discharge the ADP-containing dense granules. These results suggest: (i) that released ADP plays an important contributory role in agonist-stimulated platelet AA release, and (ii) that guanine nucleotides can modulate platelet activation through an extracellular action which is distinct from their effects on G-proteins.
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PMID:Guanine nucleotides inhibit agonist-stimulated arachidonic acid release in both intact and saponin-permeabilized human platelets. 313 60

Steady-state binding of ADP to blood platelets and isolated membranes has not previously been obtained because of complications arising from metabolism of the ligand and dilution due to its secretion from storage granules. In the present studies, competition binding isotherms (n = 9) using paraformaldehyde-fixed platelets showed that [2-3 H]ADP bound to two sites with a small amount (approximately 5% of total) of nonspecific binding: 410,000 +/- 40,000 sites of low affinity (Kd 7.9 +/- 2.0 mumol/L) and 160,000 +/- 20,000 sites of high affinity (Kd 0.35 +/- 0.04 mumol/L) corresponding to the ADP concentration required for activation in fresh platelets (0.1-0.5 mumol/L). All agonists and antagonists examined were able to compete with ADP at the high-affinity site. The strong platelet agonists 2-methylthio ADP and 2-(3-aminopropylthio)ADP competed with ADP at the high-affinity site with dissociation constant values of 7 mumol/L and 200 mumol/L, respectively. The partial agonist 2',3'-dialdehyde ADP and the weak agonist GDP also competed at the high-affinity site with Kd values of 5 mumol/L and 49 mumol/L, respectively. The sequence of binding affinities of other adenine nucleotides at the high-affinity site corresponded to their relative activities as known antagonists of platelet activation by ADP; namely, ADP(Kd 0.35 mumol/L) approximately equal to ATP (Kd 0.45 mumol/L) much greater than AMP (Kd 360 mumol/L). Adenosine and 2-chloroadenosine did not compete with ADP. ADP binding to the high-affinity site was inhibited by p-mercuribenzene sulfonate (Ki 250 mumol/L) but only very weakly by 5'-p-fluorosulfonylbenzoyladenosine (Ki 1 mmol/L). All the above nucleotides also competed with ADP at the low-affinity sites but, because of the high concentrations of competing nucleotide required, dissociation constants at this site were obtained only for ATP (21 mumol/L), 2-MeS ADP (200 mumol/L) and 2',3'-dialdehyde ADP (270 mumol/L). 8-Bromo ADP competed strongly with ADP at the high-affinity site (Kd 0.40 mumol/L) but weakly if at all at the low-affinity site. 8-Bromo ADP inhibited platelet activation induced by ADP (EC50 approximately 100 mumol/L) but not by collagen, thrombin, or ionophore A23187.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Identification of high-affinity (Kd 0.35 mumol/L) and low-affinity (Kd 7.9 mumol/L) platelet binding sites for ADP and competition by ADP analogues. 333 92

The effect of guanosine 5'-[beta-thio]diphosphate (GDP[beta S]), reported to be an antagonist of GTP at the G-protein-binding site, on human platelet activation was examined. GDP[beta S] (0.3-3 mM) had significant inhibitory effects on platelet aggregation and 5-hydroxytryptamine (5HT) secretion induced by thrombin, collagen, the thromboxane mimetic U46619 and 1,2-dioctanoylglycerol (diC8) in intact platelets, as well as in saponin-permeabilized platelets. Similar inhibitory effects in intact platelets were also observed with ATP (over similar concentration ranges) and GDP and GTP (at 2- and 10-fold higher concentrations respectively). All four nucleotides also inhibited ADP-induced platelet aggregation in indomethacin-treated platelets under conditions where no 5HT secretion occurred. Inhibition of thrombin-induced aggregation and secretion by GDP[beta S] and ATP in intact platelets was accompanied by a reduction in the thrombin-induced rise in intracellular Ca2+ levels and 45 kDa-protein phosphorylation. The results suggest that at least some of the effects of GDP[beta S] may be unrelated to inhibition of G-protein-GTP interaction, but, instead, may be mediated via an extracellular site, common to all the nucleotides tested and perhaps via inhibition of the effects of endogenous/released ADP. The usefulness of GDP[beta S] as a tool in studying G-protein-GTP interactions in platelets is thus questionable.
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PMID:Inhibition of agonist-induced platelet aggregation, Ca2+ mobilization and granule secretion by guanosine 5'-[beta-thio]diphosphate and GDP in intact platelets. Evidence for an inhibitory mechanism unrelated to the inhibition of G-protein-GTP interaction. 335 12

The inhibition by guanosine 5'-[beta-thio]diphosphate (GDP beta S) of phospholipase C was compared in intact and saponin-permeabilized human platelets in order to assess whether effects of GDP beta S on phospholipase C activation unrelated to guanine nucleotide binding function were occurring. GDP beta S exhibited no effect on phospholipase C activity, monitored by phosphatidic acid formation, in intact platelets that were unstimulated or stimulated with 0.5 U/ml thrombin or 20 nM ONO-11113 (a stable thromboxane A2 analogue). However, GDP beta S did cause a marked decrease in the activity of phospholipase C in saponin-permeabilized platelets. Thus GDP beta S is a viable tool for studying the role of G-proteins in transducing receptor-mediated activation of phospholipase C in platelets.
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PMID:Inhibition of GDP beta S of agonist-activated phospholipase C in human platelets requires cell permeabilization. 337 92

We have assessed the binding of [alpha-32P]GTP to platelet proteins from cytosolic and membrane fractions. Proteins were separated by NaDodSO4/PAGE and electrophoretically transferred to nitrocellulose. Incubation of the nitrocellulose blots with [alpha-32P]GTP indicated the presence of specific and distinct GTP-binding proteins in cytosol and membranes. Binding was prevented by 10-100 nM GTP and by 100 nM guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]) or GDP; binding was unaffected by 1 nM-1 microM ATP. One main GTP-binding protein (29.5 kDa) was detected in the membrane fraction, while three others (29, 27, and 21 kDa) were detected in the soluble fraction. Two cytosolic GTP-binding proteins (29 and 27 kDa) were degraded by trypsin; another cytosolic protein (21 kDa) and the membrane-bound protein (29.5 kDa) were resistant to the action of trypsin. Treatment of intact platelets with trypsin or thrombin, followed by lysis and fractionation, did not affect the binding of [alpha-32P]GTP to the membrane-bound protein. GTP[gamma S] still stimulated phospholipase C in permeabilized platelets already preincubated with trypsin. This suggests that trypsin-resistant GTP-binding proteins might regulate phospholipase C stimulated by GTP[gamma S].
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PMID:Specific binding of [alpha-32P]GTP to cytosolic and membrane-bound proteins of human platelets correlates with the activation of phospholipase C. 347 Jul 89

Exposure of human platelets to 10 discharges from a 4.5 microF capacitor charged at 3 kV permitted isolation of a stable preparation of permeabilized platelets that, after equilibration with Ca2+ buffers (pCa less than 6) for 15 min at O degrees C, secreted 5-hydroxytryptamine (5-HT) at 25 degrees C. Thrombin enhanced the sensitivity to Ca2+ of the secretion of 5-HT by about 10-fold, whereas Arg -vasopressin and the prostaglandin endoperoxide analogue, U-46619, increased sensitivity to Ca2+ by 3 to 4-fold. This action of thrombin was associated with stimulation of diacylglycerol formation, a marked increase in phosphorylation of protein P47 and a smaller increase in phosphorylation of the P-light chain of myosin. Thrombin exerted these effects at a [Ca2+ free] of 0.1 microM, suggesting that the receptor-activated breakdown of platelet phosphoinositides to diacylglycerol may not require prior Ca2+ mobilization in intact platelets. In both the presence and absence of thrombin, a higher [Ca2+ free] was required for optimal secretion than for maximal phosphorylation of P47 and myosin light-chain, indicating that Ca2+ and possibly diacylglycerol have roles in the secretory mechanism additional to activation of the enzymes that phosphorylate these proteins. Stable GTP analogues such as guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S), and to a lesser extent GTP itself, enhanced the Ca2+ sensitivity of the secretion of 5-HT from permeabilized platelets. Moreover, GTP potentiated the stimulatory action of thrombin. These effects of GTP gamma S and GTP were associated with increased diacylglycerol formation and were inhibited by guanosine-5'-O-(2-thiodiphosphate) (GDP beta S) suggesting that a GTP-binding protein may play a role in the receptor-activated breakdown of phosphoinositides. However, as GDP beta S did not inhibit the potentiation of secretion caused by thrombin alone, a GTP-independent pathway of platelet activation may also exist.
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PMID:Receptor-induced diacylglycerol formation in permeabilized platelets; possible role for a GTP-binding protein. 609 73

The importance of PLC activation in cell proliferation is evident from the fact that the hydrolysis of PtdIns(4,5)P2 is one of the early events that follow the interaction of many growth factors and mitogens with their respective receptors. However, the importance of PLC activation is not restricted to proliferation; it is one of the most common transmembrane signaling events elicited by receptors that regulate many other cellular processes, including differentiation, metabolism, secretion, contraction, and sensory perception. It is also clear that cell proliferation signaling does not always require PLC, as indicated by the fact that growth factors such as insulin and CSF-1 do not appear to elicit the hydrolysis of PtdIns(4,5)P2, even though the intracellular domains of their receptors carry a PTK domain and the receptors show topologies very similar to those of the PLC-activating growth factors PDGF, EGF, and FGF. The growth factor-dependent activation of PLC is initiated by the formation of a complex between the receptor PTK and PLC-gamma; the formation of this complex is mediated by a specific interaction between a tyrosine phosphate residue on the intracellular domain of PTK and the SH2 domain of PLC-gamma. The receptor PTK subsequently phosphorylates PLC-gamma, of which two distinct isozymes, PLC-gamma 1 and PLC-gamma 2, have been identified. Proliferation of T cells and B cells in response to the aggregation of their respective cell surface receptors is also accompanied by the activation of PLC-gamma isozymes at an early stage. Unlike growth factor receptors, the T cell and B cell receptors lack intrinsic PTK activity but associate with several non-receptor PTKs of the Src and Syk families. Although the specific kinases are not known, one or more of these enzymes phosphorylate and activate PLC-gamma 1 and PLC-gamma 2. Transduction of growth signals by G protein-coupled receptors such as those for thrombin or bombesin also requires PtdIns(4,5)P2 hydrolysis, which, in this instance, is mediated by PLC-beta isozymes. The PLC-beta subfamily consists of four distinct members: PLC-beta 1, PLC-beta 2, PLC-beta 3, and PLC-beta 4. Agonist interaction with specific G protein-coupled receptors causes the dissociation of Gq proteins into G alpha and G beta gamma subunits and the exchange of GDP bound to G alpha for GTP. The resulting GTP-bound G alpha subunit then activates PLC-beta isoforms by binding to the carboxyl-terminal region of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Phosphoinositide-specific phospholipase C and mitogenic signaling. 749 69

Synthetic thrombin receptor peptides (TRPs), comprising the first 6-14 amino acids of the new N-terminus tethered ligand of the thrombin receptor that is generated by thrombin's proteolytic activity, were reported to activate platelets equally with thrombin itself and are considered to be full agonists [Vu et al. (1991) Cell 64, 1057-1068]. Using aspirin plus ADP-scavengers or the ADP-receptor antagonist adenosine 5'-[alpha-thio]triphosphate to prevent the secondary effects of the potent agonists that are normally released from stimulated platelets (i.e. ADP and thromboxane A2), we assessed the direct actions of thrombin and TRPs (i.e. TRP42-47 and TRP42-55). Compared with thrombin, under these conditions, TRPs: (1) failed to aggregate platelets completely; (2) produced less activation of glycoprotein (GP)IIb-IIIa; (3) did not cause association of GPIIb and pp60c-src with the cytoskeleton; and (4) caused less alpha-granule secretion, phosphorylation of cytoplasmic phospholipase A2, arachidonic acid release and phosphatidyl inositol (PtdOH) production. Furthermore, TRPs induced transient increases in protein phosphorylation mediated by protein kinase C and protein tyrosine phosphorylation, whereas these same responses to thrombin were greater and more sustained. Hirudin added after thrombin accelerated protein dephosphorylation, thereby mimicking the rate of spontaneous dephosphorylation seen after stimulation by TRPs. Platelets totally desensitized to very high concentrations of TRPs, by prior exposure to maximally effective concentrations of the peptides, remained responsive to alpha- and gamma-thrombins. Thrombin-stimulated PtdOH production in permeabilized platelets desensitized to TRPs was abolished by guanosine 5'-[beta-thio]diphosphate (GDP[beta S]), as in normal platelets. These results are discussed in terms of the allosteric Ternary Complex Model for G-protein linked receptors [Samama et al. (1993) J. Biol. Chem. 268, 4625-4636]. We conclude that: (1) TRPs are partial agonists for the thrombin receptor and produce incomplete receptor desensitization in keeping with their lower intrinsic activity; (2) thrombin's effects in platelets, even in TRP-desensitized platelets, are entirely mediated through the recently cloned G-protein linked receptor, and (3) thrombin's ability to produce sustained signals, compared with TRPs, may require the continued progressive proteolytic activation of naive thrombin receptors.
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PMID:Thrombin-receptor agonist peptides, in contrast to thrombin itself, are not full agonists for activation and signal transduction in human platelets in the absence of platelet-derived secondary mediators. 752 41

The catalytic domain of the Saccharomyces cerevisiae SDC25 gene product, including the last 550 C-terminal residues (Sdc25p-C), was produced as an Escherichia coli recombinant protein fused with glutathione S-transferase. The highly purified (greater than 95%) stable fusion protein, obtained by affinity chromatography, was very active in enhancing the dissociation rate or the GDP/GTP exchange of the GDP complex of Ras2p or human H-ras p21. This activity was further increased (three times) by glutathione S-transferase cleavage with thrombin. The stimulation of the guanine nucleotide release by Sdc25p-C was stronger for Ras2p.GDP than Ras2p.GTP, an effect that was less pronounced in the case of the p21 complexes. The association rate of the Ras2p.GDP (GTP) complex was also enhanced by Sdc25p-C. Monovalent and divalent salts inhibit the nucleotide-releasing activity of Sdc25p-C. Retention phenomena occurring on gel-filtration chromatography hindered the use of highly purified Sdc25p-C to study the formation of stable complexes with Ras2p. For this purpose, Sdc25p-C was produced as a non-glutathione-S-transferase fusion protein via pTTQ19. Upon partial purification, this product yielded a 54-kDa truncated form of Sdc25p-C (truncated Sdc25p-C) showing the same specific activity as the 64-kDa Sdc25p-C protein. On gel filtration, truncated Sdc25p-C and nucleotide-free Ras2p (or p21) formed a stable 1:1 stoichiometric complex that was dissociated by increasing concentrations of GDP. The properties of this complex were analyzed by using the mutant [S24N]Ras2p, the homologue of [S17N]p21 known to induce a dominant negative phenotype, [R80D, N81D]Ras2p, a recessive negative mutant insensitive to the truncated form of Sdc25p-C in vitro. The complex with [S24N]Ras2p was greater than 100-fold less sensitive to the dissociating effect of GDP, whereas [R80D, N81D]Ras2p was unable to form a stable complex with truncated Sdc25p-C. These results strongly suggest that the residues R80 and N81 are situated in or closely associated with the Ras2p specific site binding Sdc25p.
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PMID:Properties of the catalytic domain of sdc25p, a yeast GDP/GTP exchange factor of Ras proteins. Complexation with wild-type Ras2p, [S24N]Ras2p and [R80D, N81D]Ras2p. 785 34

Integrin-mediated adhesion is known to stimulate production of phosphatidylinositol 4,5-bisphosphate (4,5-PIP2) and increase 4,5-PIP2 hydrolysis in response to platelet-derived growth factor (PDGF). We now show that treatment of cells with lovastatin, which inhibits modification of small GTP-binding proteins, reduced PIP2 levels and decreased calcium mobilization in response to PDGF and thrombin. In cell lysates, GTP gamma S stimulated PIP 5-kinase activity, and this effect was blocked by botulinum C3 exoenzyme, suggesting that Rho was responsible. GTP-bound recombinant Rho stimulated PIP 5-kinase activity, whereas GDP-Rho was much less potent and GTP-bound Rac was ineffective. Microinjected botulinum C3 exoenzyme caused diminished calcium mobilization in response to PDGF or thrombin. Conversely, microinjection of activated Rho reversed the decrease in calcium mobilization normally seen in nonadherent cells. These data demonstrate that Rho regulates 4,5-PIP2 synthesis and, indirectly, 4,5-PIP2 hydrolysis. They also raise the possibility that PIP2 synthesis could mediate the effects of Rho on the actin cytoskeleton.
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PMID:The small GTP-binding protein Rho regulates a phosphatidylinositol 4-phosphate 5-kinase in mammalian cells. 795 16


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