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 primary phase of ADP-induced aggregation of human platelets does not involve appreciable formation of thromboxane A2 or release of granule contents; lack of formation of inositol trisphosphate has also been noted. Because these responses of platelets to ADP differ so markedly from their responses to other aggregating agents, the roles in ADP-induced aggregation of diacylglycerol, protein kinase C, increases in cytosolic [Ca2+], phosphorylation of pleckstrin (47 kDa) and phosphatases 1 and 2a were investigated. Washed human platelets, prelabelled with [14C]5-hydroxytryptamine and suspended in Tyrode solution (2 mM Ca2+, 1 mM Mg2+), were used for comparisons between the aggregation induced by 2-4 microM ADP, in the presence of fibrinogen, and that induced by 0.05 units/ml thrombin. The diacylglycerol kinase inhibitor 6-(2-[(4-fluorophenyl)phenyl-methylene]-1-piperidinylethyl)-7-meth yl-5H-thiazolo[3,2-a]-pyrimidin-5-one (R59022; 25 microM) had no, or only a slight, enhancing effect on ADP-induced aggregation, but potentiated thrombin-induced responses to a much greater extent. 1,2-Dihexanoyl-sn-glycerol or 1-oleoyl-2-acetyl-sn-glycerol (25 microM) added with or 30-90 s before ADP greatly potentiated aggregation without formation of thromboxane; staurosporine, an inhibitor of protein kinase C, reduced this potentiation. Staurosporine (25 nM) did not inhibit ADP-induced aggregation, although it strongly inhibited thrombin-induced aggregation and release of [14C]5-hydroxytryptamine. All these observations indicate little or no dependence of primary ADP-induced aggregation on the formation of diacylglycerol or on the activation of protein kinase C. At 2-4 microM, ADP did not significantly increase the phosphorylation of pleckstrin (studied with platelets prelabelled with [32P]orthophosphate), but 1,2-dihexanoyl-sn-glycerol- induced phosphorylation of pleckstrin was increased by ADP. Surprisingly, the diacylglycerols strongly inhibited the ADP-induced rise in cytosolic [Ca2+] concurrently with potentiation of ADP-induced aggregation; thus the extent of primary aggregation is independent of the level to which cytosolic [Ca2+] rises. Incubation of platelets with 1,2-dihexanoyl-sn-glycerol or 1-oleoyl-2-acetyl-sn-glycerol for several minutes reversed their potentiating effects on aggregation, and inhibition was observed. Incubation of platelets with okadaic acid, an inhibitor of phosphatases 1 and 2a, inhibited ADP- and thrombin-induced aggregation; although the reason for this effect is unknown, it is unlikely to involve inhibition of phospholipase C, since formation of diacylglycerol appears to have little involvement in the primary phase of ADP-induced aggregation.
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PMID:Activation of phospholipase C and protein kinase C has little involvement in ADP-induced primary aggregation of human platelets: effects of diacylglycerols, the diacylglycerols, the diacylglycerol kinase inhibitor R59022, staurosporine and okadaic acid. 838 48

The peptide YFLLRNP antagonizes the aggregation of human platelets when induced by low concentrations of alpha-thrombin or the thrombin receptor agonist peptide (SFLLRNP), demonstrating that it interacts specifically with the thrombin receptor. Platelets exposed to YFLLRNP show immediate shape change (pseudopod formation) and potentiation of the ADP and platelet-activating factor response, but no Ca2+ mobilization, P47 (pleckstrin) phosphorylation, secretion, or aggregation. Thus, YFLLRNP induces a state of partial activation of the platelets. Furthermore, with platelets prestimulated with adrenalin (10 microM), YFLLRNP induces aggregation, but no secretion, and only in the presence of added fibrinogen. We also found that prostacyclin inhibits the YFLLRNP-induced shape change; but EDTA, aspirin, and apyrase (ADP scavenger) do not. Thus, the thrombin receptor in platelets may communicate, independently of Ca2+ mobilization and P47 phosphorylation (protein kinase C activation), with intracellular signaling mechanisms that 1) modulate the cytoskeleton structure, 2) potentiate other platelet responses, and 3) stimulate coupling between the thrombin receptor and fibrinogen binding (the glycoprotein IIb-IIIa complex). YFLLRNP may be useful for differentiating between several possible activation states of the platelet thrombin receptor.
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PMID:A peptide ligand of the human thrombin receptor antagonizes alpha-thrombin and partially activates platelets. 839 Sep 90

The aim of this study was to establish further the role of protein kinase C in aggregation and secretion of 5-hydroxytryptamine (5-HT) from human platelets by using the selective inhibitor Ro 31-8220. Ro 31-8220 (3 microM) inhibited completely phosphorylation of pleckstrin, the major protein kinase C substrate, induced by thrombin, A23187 or phorbol dibutyrate (PDBu). Myosin light-chain phosphorylation induced by PDBu was also inhibited completely, but that induced by thrombin or A23187 was only inhibited partially. As myosin light chain is a substrate for both myosin light-chain kinase and protein kinase C, these results suggest that Ro 31-8220 is inhibiting only the protein kinase C-induced phosphorylation and that Ro 31-8220 has a greater selectivity to protein kinase C than does its structural analogue staurosporine. The stimulation of secretion of 5-HT by maximally effective concentrations of thrombin and A23187 was decreased significantly by 3 microM Ro 31-8220, but not inhibited completely. These results indicate a major role for protein kinase C in the stimulation of secretion by agonist- and ionophore-induced activation. On its own, a maximal concentration of PDBu induced a small degree of secretion (3.3 +/- 1.0%), but potentiated markedly the response to a submaximal concentration of A23187 (300 nM) to a level greater than seen with a maximal concentration of A23187. A similar set of results was also seen with aggregation, but not with shape change. We interpret these results to mean that the signalling event for secretion and aggregation is Ca2+, and this is potentiated markedly by protein kinase C. In the case of secretion, it appears that it is the synergy which is the major determining factor in influencing the extent.
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PMID:Synergy between Ca2+ and protein kinase C is the major factor in determining the level of secretion from human platelets. 842 66

The role of protein kinase C (PKC) in platelet activation by thrombin was assessed using a PKC inhibitor Ro 31-7549/001 (R2) which, in vitro, shows more selectivity for PKC than other kinase inhibitors. During early (1.5 s) thrombin-induced platelet activation, when phosphorylation of 47 kDa protein (pleckstrin) and myosin light chain by PKC and myosin light chain kinase, respectively, are most readily differentiated, R2 suppressed phosphorylation of pleckstrin more effectively than myosin light chain. R2-inhibited dense granule secretion (measured 0-10 s using quenched-flow techniques) with a dose dependency similar to that for inhibition of pleckstrin phosphorylation, supporting a role for PKC in this process. R2, at 0.5 microM inhibited 47 kDa protein phosphorylation by more than 60%, but had only minimal effects on the kinetics (0-3s) of ADP-induced primary aggregation. At this same concentration, R2 potentiated the thrombin-induced rise in cytosolic calcium during early (0-15 s) activation as measured in the presence or absence of external calcium. These data support the hypothesis that activation of PKC during early platelet function helps regulate cytosolic calcium levels by limiting calcium release into the cytosol.
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PMID:The role of protein kinase C in the initial events of platelet activation by thrombin assessed with a selective inhibitor. 846 70

This work was carried out to decide whether a non-specific perturbation of the platelet membrane with exogenous amphiphiles affects protein phosphorylation in platelets, especially phosphorylation mediated by PKC. Effects of amphiphiles per se on protein phosphorylation were also recorded. (i) Sublytic concentrations of the differently charged model surfactants cetyltrimethylammonium bromide (CTAB), Zwittergent 3-16, sodium tetradecyl sulphate, and octaethyleneglycol hexadecyl ether, as well as chlorpromazine, and Triton X-100, did not affect the thrombin-induced, PKC-mediated phosphorylation of pleckstrin, whereas sphingosine blocked this phosphorylation. (ii) The sphingosine-mediated phosphorylation blockade is not related to a non-specific perturbation of the membrane, but can instead be attributed to specific properties of sphingosine. (iii) The amphiphiles, per se, had differential effects on protein phosphorylation at sublytic concentrations: a treatment with CTAB, Zwittergent 3-16, and sodium tetradecyl sulphate for 1 min led to phosphorylation of a 49-kDa protein, while treatment with sphingosine for 1 min led to a transient phosphorylation of the myosin light chain as well as a weak phosphorylation of pleckstrin.
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PMID:Perturbation of the platelet plasma membrane is not sufficient for inhibition of thrombin-induced PKC-activity. 848 83

Cathepsin G, an enzyme released by stimulated polymorphonuclear neutrophils, and thrombin are two human proteinases which potently trigger platelet activation. Unlike thrombin, the mechanisms by which cathepsin G initiates platelet activation have yet to be elucidated. The involvement of the phospholipase C (PLC)/protein kinase C (PKC) pathway in cathepsin G-induced activation was investigated and compared with stimulation by thrombin. Exposure of 5-[14C]hydroxytryptamine-labelled platelets to cathepsin G, in the presence of acetylsalicylic acid and phosphocreatine/creatine kinase, induced platelet aggregation and degranulation in a concentration-dependent manner (0.1-3.0 microM). Time-course studies (0-180 s) comparing equivalent concentrations of cathepsin G (3 microM) and thrombin (0.5 unit/ml) resulted in very similar transient hydrolysis of phosphatidylinositol 4,5-bisphosphate and steady accumulation of phosphatidic acid. In addition cathepsin G, like thrombin, initiated the production of inositol phosphates. The neutrophil-derived proteinase also induced phosphorylation of both the myosin light chain and pleckstrin, a substrate for PKC, to levels similar to those observed in platelets challenged with thrombin. Inhibition of PKC by GF 109203X, a specific inhibitor, suppressed platelet aggregation and degranulation to the same extent for both proteinases. Using fura 2-loaded platelets, the rise in the cytosolic free Ca2+ concentration induced by cathepsin G was shown to result, as for thrombin, from both mobilization of internal stores and Ca2+ entry across the plasma membrane. These findings provide evidence that cathepsin G stimulates the PLC/PKC pathway as potently as does thrombin, independently of thromboxane A2 formation and ADP release, and that this pathway is required for platelet functional responses.
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PMID:The phospholipase C/protein kinase C pathway is involved in cathepsin G-induced human platelet activation: comparison with thrombin. 857 71

Platelet-agonist interaction results in activation of glycoprotein (GP) IIb-IIIa complex and fibrinogen binding, a prerequisite for platelet aggregation. Fibrinogen binding exposes new antibody binding sites on GPIIb-IIIa (ligand-induced binding sites: LIBS). Signal transduction events, including pleckstrin phosphorylation by protein kinase C (PKC), are considered to regulate GPIIb-IIIa activation. We studied a 16-year-old white male with lifelong mucocutaneous bleeding manifestations and abnormal platelet aggregation and secretion in response to multiple agonists. Pleckstrin phosphorylation was diminished in response to platelet-activating factor (PAF; 4 and 400 nmol/L) and thrombin (0.05 U/mL). Binding of monoclonal antibodies (MoAbs) 10E5 and A2A9, which bind to both resting and activated GPIIb-IIIa, was normal. Binding of MoAb PAC1, which binds to only activated GPIIb-IIIa, was diminished upon activation with PAF, adenosine diphosphate (ADP), thrombin receptor agonist peptide (SFLLRN), A23187, and 1,2-dioctonylglycerol (DiC8). Signal transduction-dependent LIBS expression (studied using MoAb 62) induced by ADP, SFLLRN, and DiC8 and signal transduction-independent LIBS expression induced by RGDS peptide or disintegrin albolabrin were normal or minimally decreased, indicating the presence of intact ligand binding sites. We conclude that the patient's platelets have a defect in inside-out signal transduction-dependent GPIIb-IIIa activation due to an upstream defect in the signal transduction mechanisms rather than in the GPIIb-IIIa complex itself. Our findings extend the spectrum of congenital mechanisms leading to impaired aggregation from defects in GPIIb-IIIa per se to aberrations in signaling mechanisms.
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PMID:Abnormal inside-out signal transduction-dependent activation of glycoprotein IIb-IIIa in a patient with impaired pleckstrin phosphorylation. 860 26

Signal transduction on platelet activation involves phosphoinositide-specific phospholipase C (PLC)-mediated hydrolysis of phosphatidylinositides and formation of inositol-1,4,5-triphosphate [I(1,4,5)P3], which mediates Ca2+ mobilization, and diacylglycerol (DG), which activates protein kinase C (PKC) to phosphorylate a 47-kD protein (Pleckstrin). We studied these events in two related patients previously reported (Blood 74:664, 1989) to have abnormal aggregation and 14C-serotonin secretion, and impaired intracellular Ca2+ mobilization in response to several agonists. Thrombin-induced I(1,4,5)P3 and phosphatidic acid formation were diminished. Pleckstrin phosphorylation was impaired on activation with thrombin, platelet-activating factor, and ionophore A23187, but was normal with PKC activator 1,2-dioctonyl-sn-glycerol (DiC8). Ca2+ mobilization induced by guanosine triphosphate (GTP) analog guanosine 5'-0-(3 thiotriphosphate) (GTP gamma S) was diminished. Pretreatment with either A23187 or DiC8 did not correct the impaired adenine diphosphate-induced secretion; however, upon stimulation with A23187 plus DiC8, pleckstrin phosphorylation and secretion were normal, indicating that both PKC activation and Ca2+ mobilization are essential for normal secretion. We conclude that these patients have a unique inherited platelet defect in formation of two key intracellular mediators [I(1,4,5)P3 and DG] and in the responses mediated by them due to a defect in postreceptor mechanisms of PLC activation.
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PMID:Human platelet signaling defect characterized by impaired production of inositol-1,4,5-triphosphate and phosphatidic acid and diminished Pleckstrin phosphorylation: evidence for defective phospholipase C activation. 878 23

The effects of cell-permeable C2 and C6-ceramides on human platelet responses were investigated. In thrombin-activated platelets, C6(5-30 microM) potentiated Ca2+ mobilization and Ca2+ influx, and decreased the rate of removal of Ca2+ from cytosol. The effect of C2 was not significant. Phorbol ester or calyculin A inhibition of thrombin-induced rises in platelet [Ca2+]i was attenuated by C6. Assays show that C6 either prolonged the generation, or retarded the metabolism of inositol trisphosphates. Previous studies indicate that protein kinase C (PKC) acts in a negative feedback manner by inhibiting phosphatidylinositol breakdown, accelerating inositol trisphosphate metabolism, and increasing Ca2+ pump activity. C6 may counter these PKC effects indirectly. The synthetic ceramides inhibited platelet aggregation weakly and had no effect on pleckstrin (p47) phosphorylation. Recently we reported that C2 but not C6 inhibits superoxide generation and store-regulated Ca2+ influx in neutrophils at similar concentrations. Cellular differences in ceramide metabolism or ceramide-sensitive enzymes and their substrates may account for the disparate results.
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PMID:C6-ceramide maintains elevated cytosolic calcium levels in activated platelets. 882 37

Platelet signal transduction involves not only reversible phosphorylation of proteins on both tyrosine and serine/threonine residues, but also mechanisms of cross-talk to coordinate different pathways. We have, therefore, investigated the effect of okadaic acid, a potent inhibitor of serine/threonine protein phosphatases type 1 and type 2A (PP1 and PP2A), to better understand the interplay that must exist between serine/threonine and tyrosine phosphorylations during platelet activation. Okadaic acid drastically inhibits thrombin-induced platelet aggregation, secretion, and thromboxane synthesis. The inhibition is accompanied by a marked increase in the phosphorylation of at least 5 proteins (230, 210, 74, 57, and 50 to 52 kDa). However, protein kinase C activity is not modified because thrombin-and phorbol-12-myristate-13-acetate-induced phosphorylation of pleckstrin is still occurring, although slightly decreased. Inhibition of platelet function and extent of the phosphorylation of the 5 substrates in the presence of okadaic acid are concentration and time dependent, suggesting a relation between the accumulation of one or more phosphoproteins and the inhibitory effect of okadaic acid. Okadaic acid inhibits thrombin-induced tyrosine phosphorylation in a concentration-dependent manner. According to Brautigan and Pinault, the inhibition of protein phosphatases in kidney cells resulted in the activation of a 55-kDa-tyrosine phosphatase and the tyrosine phosphatase activity was synergistically increased when okadaic acid acted in concert with prostaglandin I2 (PGI2). Interestingly, in agreement with these results, the okadaic acid-induced phosphorylation of the 50-kDa substrate, which occurs without a cyclic adenosine monophosphate increase in platelets, has the same molecular weight as the platelet membrane tyrosine phosphatase isolated by Dawicki and Steiner. Furthermore, we also found that thrombin-induced tyrosine phosphorylation was markedly inhibited in the presence of low concentrations of both okadaic acid and PGI2, therefore explaining the synergistic inhibition of platelet aggregation and secretion. The results greatly support the notion of a cross-talk between stimulation of serine/threonine kinases (in response to inhibition of serine/threonine PP) and inhibition of tyrosine phosphorylations and emphasize the role of the 50-kDa substrate in regulating platelet activation.
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PMID:Serine/threonine dephosphorylation may be involved in tyrosine phosphorylation: a new mode of signal transduction in platelets. 894 16


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