scholarly journals A diacylglycerol kinase inhibitor, R59022, potentiates secretion by and aggregation of thrombin-stimulated human platelets

1987 ◽  
Vol 243 (3) ◽  
pp. 809-813 ◽  
Author(s):  
D L Nunn ◽  
S P Watson
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phosphatidic Acid ◽  
Kinase Inhibitor ◽  
Inositol Phosphates ◽  
Protein A ◽  
Diacylglycerol Kinase ◽  
Human Platelets ◽  
Kinase C ◽  
Kinase Phosphorylation

The diacylglycerol kinase inhibitor R59022 (10 microM) potentiates secretion and aggregation responses in human platelets challenged with sub-maximal concentrations of thrombin. Potentiation correlates closely with increased formation of diacylglycerol, increased phosphorylation of a 40 kDa protein, a known substrate for protein kinase C, and with decreased formation of phosphatidic acid, the product of diacylglycerol kinase. Phosphorylation of myosin light chains, formation of inositol phosphates and the mobilization of Ca2+ by thrombin are not affected by R59022 (10 microM). These data support a role for protein kinase C in platelet aggregation and secretion, and provide further evidence that endogenous diacylglycerols bring about the activation of this enzyme. These data also add further argument against a role for phosphatidic acid in platelet activation.

scholarly journals The action of the protein kinase C inhibitor, staurosporine, on human platelets. Evidence against a regulatory role for protein kinase C in the formation of inositol trisphosphate by thrombin

1988 ◽  
Vol 249 (2) ◽  
pp. 345-350 ◽  
Author(s):  
S P Watson ◽  
J McNally ◽  
L J Shipman ◽  
P P Godfrey
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phosphatidic Acid ◽  
Phorbol Esters ◽  
Inositol Phosphates ◽  
Protein A ◽  
Polymyxin B ◽  
Human Platelets ◽  
Phosphoinositide Metabolism ◽  
Kinase C

The ability of several putative inhibitors of protein kinase C (PKC) to block dioctanoylglycerol (DC8)-induced phosphorylation of a 47 kDa protein (a recognized substrate for PKC) in human platelets was investigated. Staurosporine (1 microM) caused complete inhibition of phosphorylation, whereas the other reagents were either inactive (polymyxin B) or gave only partial inhibition (C-1, H-7, tamoxifen). Staurosporine (1 microM) fully inhibited the phosphorylation of the 47 kDa protein in platelets challenged with thrombin, but also inhibited the phosphorylation of a 20 kDa protein which is a substrate for myosin light-chain kinase. The inhibition of both kinases by staurosporine was associated with the inhibition of thrombin-induced secretion of ATP and 5-hydroxytryptamine and a slowing of the aggregation response; staurosporine, however, had no effect on the formation of phosphatidic acid and inositol phosphates induced by thrombin. Staurosporine also reversed the inhibitory action of phorbol esters on thrombin-induced formation of phosphatidic acid. These data are consistent with a role for these two kinases in secretion and aggregation (although there must be additional control signals, since aggregation was only slowed, not inhibited), but suggest that neither kinase is involved in the regulation of phosphoinositide metabolism. This latter conclusion contradicts previous observations that the activation of PKC by phorbol esters or membrane-permeable diacylglycerols alters the apparent activity of both phospholipase C and inositol trisphosphatase. Possible explanations for this discrepancy are discussed.

A ROLE OF DIACYLGLYCEROL KINASE IN STIMULUS-SECRET I ON COUPLING OF HUMAN PLATELETS -DISSOCIATION OF SEROTONIN SECRETION FROM Ca2+ MOBILIZATION-

1987 ◽  
Author(s):  
T TOHMATSU ◽  
S NAKASHIMA ◽  
H HATTORI ◽  
A SUGANUMA ◽  
Y NOZAWA
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Kinase Inhibitor ◽  
Second Messenger ◽  
Diacylglycerol Kinase ◽  
Human Platelets ◽  
Kinase C ◽  
Serotonin Secretion ◽  
Tightly Coupled

Diacylglycerol (DG) kinase catalyzes the reaction: DG + ATP phosphatidic acid + ADP and it is widely distributed in animal tissues. The enzyme seems to play a pivotal role in removing a second messenger, DG, which activates protein kinase C. DG kinase inhibitor, R 59 022 (6-[2-[4-[(4-fluorophenyl)pheny1- methylene] -1-piperidinyl] ethyl] -7-methyl-5H-thiazolo [3,2-a] -pirimidin-5-one) has recently been developed. In order to gain further insight into the role of DG in the secretory response, effects of the DG kinase inhibitor on secretory responses and Ca2+ mobilization were investigated in human platelets.The addition of the DG kinase inhibitor (10 μM) potentiated thrombin-induced accumulation of [3H]radioactivity of DG in platelets loaded with [3H] arachidonate. Thrombin-induced release of [3H] arachidonic acid and its metabolites was not affected by the inhibitor. The inhibitor did not cause significant secretion of [14C] serotonin by itself. However, the pretreatment with this agent potentiated the level of secretion in thrombin-stimulated platelets. When l-oleoyl-2-acetylglycerol(OAG) was added to [32pjpi-iabeled platelets in the presence of the DG kinase inhibitor, the formation of [32P] l-oleoyl-2-acetylphosphatidic acid was greatly prevented. The pretreatment with the inhibitor also potentiated OAG-induced serotonin secretion. With the view that Ca2+ is thought to be another important second messenger, we investigated the effect of the DG kinase inhibitor on Ca2+ mobilization. Two types of Ca2+ indicators, Quin2 and aequorin were used to measure cytosolic free Ca2+ concentration ([Ca2+]i). The inhibitor alone did not affect [Ca2+]i. Interestingly, thrombin-induced increase in [Ca2+]i was suppressed by the pretreatment with this agent both in the Quin2-loaded and aequorin-loaded platelets.These results indicate that diacylglycerol kinase may operate as an attenuator in the signal transduction system involving protein kinase C and that Ca2+ mobilization may not be tightly coupled to serotonin secretion.

scholarly journals Ethanol inhibits thrombin-induced secretion by human platelets at a site distinct from phospholipase C or protein kinase C

1990 ◽  
Vol 269 (2) ◽  
pp. 489-497 ◽  
Author(s):  
C Benistant ◽  
R Rubin
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Phorbol Ester ◽  
Inositol Phosphates ◽  
Shape Change ◽  
Protein A ◽  
Human Platelets ◽  
Kinase C ◽  
A Site

Ethanol is known to inhibit the activation of platelets in response to several physiological agonists, but the mechanism of this action is unclear. The addition of physiologically relevant concentrations of ethanol (25-150 mM) to suspensions of washed human platelets resulted in the inhibition of thrombin-induced secretion of 5-hydroxy[14C]tryptamine. Indomethacin was included in the incubation buffer to prevent feedback amplification by arachidonic acid metabolites. Ethanol had no effect on the activation of phospholipase C by thrombin, as determined by the formation of inositol phosphates and the mobilization of intracellular Ca2+. Moreover, ethanol did not interfere with the thrombin-induced formation of diacylglycerol or phosphatidic acid. Stimulation of platelets with phorbol ester (5-50 nM) resulted in 5-hydroxy[14C]tryptamine release comparable with those with threshold doses of thrombin. However, ethanol did not inhibit phorbol-ester-induced secretion. Ethanol also did not interfere with thrombin- or phorbol-ester-induced phosphorylation of myosin light chain (20 kDa) or a 47 kDa protein, a known substrate for protein kinase C. By electron microscopy, ethanol had no effect on thrombin-induced shape change and pseudopod formation, but prevented granule centralization and fusion. The results indicate that ethanol does not inhibit platelet secretion by interfering with the activation of phosphoinositide-specific phospholipase C or protein kinase C by thrombin. Rather, the data demonstrate an inhibition of a Ca2(+)-mediated event such as granule centralization.

scholarly journals 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

1993 ◽  
Vol 290 (3) ◽  
pp. 849-856 ◽  
Author(s):  
M A Packham ◽  
A A Livne ◽  
D H Ruben ◽  
M L Rand
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Okadaic Acid ◽  
Kinase Inhibitor ◽  
Diacylglycerol Kinase ◽  
Human Platelets ◽  
Primary Phase ◽  
Kinase C ◽  
Induced Aggregation

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.

scholarly journals Association of diacylglycerol kinase ζ with protein kinase C α

2003 ◽  
Vol 160 (6) ◽  
pp. 929-937 ◽  
Author(s):  
Bai Luo ◽  
Stephen M. Prescott ◽  
Matthew K. Topham
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phosphatidic Acid ◽  
Binding Site ◽  
Catalytic Domain ◽  
Diacylglycerol Kinase ◽  
Kinase Substrate ◽  
Signaling Complex ◽  
Kinase C ◽  
Local Accumulation

Activation of PKC depends on the availability of DAG, a signaling lipid that is tightly and dynamically regulated. DAG kinase (DGK) terminates DAG signaling by converting it to phosphatidic acid. Here, we demonstrate that DGKζ inhibits PKCα activity and that DGK activity is required for this inhibition. We also show that DGKζ directly interacts with PKCα in a signaling complex and that the binding site in DGKζ is located within the catalytic domain. Because PKCα can phosphorylate the myristoylated alanine-rich C-kinase substrate (MARCKS) motif of DGKζ, we tested whether this modification could affect their interaction. Phosphorylation of this motif significantly attenuated coimmunoprecipitation of DGKζ and PKCα and abolished their colocalization in cells, indicating that it negatively regulates binding. Expression of a phosphorylation-mimicking DGKζ mutant that was unable to bind PKCα did not inhibit PKCα activity. Together, our results suggest that DGKζ spatially regulates PKCα activity by attenuating local accumulation of signaling DAG. This regulation is impaired by PKCα-mediated DGKζ phosphorylation.

scholarly journals Activation of human platelets by peroxovanadate is associated with tyrosine phosphorylation of phospholipase Cγ and formation of inositol phosphates

1993 ◽  
Vol 290 (2) ◽  
pp. 471-475 ◽  
Author(s):  
R A Blake ◽  
T R Walker ◽  
S P Watson
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Tyrosine Phosphorylation ◽  
Platelet Activation ◽  
Inositol Phosphates ◽  
Human Platelets ◽  
Functional Responses ◽  
Phosphorylation Of Proteins ◽  
Kinase C

Vanadate ions in the presence of H2O2 (peroxovanadate) induce a marked increase in the degree of tyrosine phosphorylation of proteins in human platelets. This increase preceded the onset of platelet shape change and aggregation, and is associated with activation of phospholipase C and increased [32P]phosphorylation of proteins of 47 kDa, a substrate for protein kinase C, and 20 kDa, a substrate for both myosin light-chain kinase and protein kinase C. The non-selective inhibitor of protein kinases, staurosporine, inhibits the increase in tyrosine phosphorylation of nearly all proteins and inhibits completely all other functional responses, suggesting that these events may be linked. In support of this, peroxovanadate stimulates tyrosine phosphorylation of phospholipase C gamma 1, suggesting that this may underlie its mechanism of platelet activation. Staurosporine also inhibited activation of phospholipase C by collagen, suggesting that tyrosine phosphorylation has an important role in the early stages of collagen-induced platelet activation.

Duality of plasmin effect on cytosolic free calcium in human platelets

1995 ◽  
Vol 268 (4) ◽  
pp. C958-C967 ◽  
Author(s):  
K. Nakamura ◽  
M. Kimura ◽  
J. W. Fenton ◽  
T. T. Andersen ◽  
A. Aviv
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Tyrosine Kinase ◽  
Kinase Inhibitor ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
Free Calcium ◽  
Protein Kinase C Inhibitors ◽  
Kinase C ◽  
Small Rise

Plasmin caused a modest and gradual increase in platelet cytosolic Ca2+, mediated through both Ca2+ mobilization and external Ca2+ entry. This response was associated with accelerated Ca2+ extrusion and protein tyrosine phosphorylation. Plasmin-enhanced external Ca2+ entry and Ca2+ extrusion (but not Ca2+ mobilization) were attenuated by the tyrosine kinase inhibitor, genistein. Plasmin inhibited the thrombin-evoked increase in cytosolic Ca2+ and also inhibited the Ca2+ response to the tethered peptide TRAP-6 of the thrombin receptor. Furthermore, plasmin inhibited the binding of 125I-labeled alpha-thrombin to platelets. The inhibitory effect of plasmin on the thrombin response shared some characteristics with the effect of protein kinase C stimulators but was not reversed by protein kinase C inhibitors. Plasmin did not change platelet cyclic nucleotides. These results suggest a dual effect of plasmin. Plasmin produces a small rise in platelet cytosolic Ca2+ and a tyrosine kinase-dependent enhancement of Ca2+ turnover (external Ca2+ influx and Ca2+ efflux). However, it also attenuates the thrombin-evoked cytosolic Ca2+ response by blocking Ca2+ mobilization and slowing the rate of external Ca2+ influx. The latter feature would result in a plasmin-induced inhibition of thrombogenesis.

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