Platelet-activating Factor (PAF)-dependent Biochemical, Morphologic, and Physiologic Responses of Human Platelets: Demonstration of Translocation of Protein Kinase C Associated with Protein Phosphorylation

Platelets are released into the peripheral circulation from the bone marrow where they arise as fragments of megakaryocyte cytoplasm. To characterize the effects of platelet agonists on megakaryocytes, we examined calcium signaling and desensitization to thrombin, the thromboxane A2 (TxA2) mimetic (15S)-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5Z,13E-dienoic acid (U46619), and platelet-activating factor (PAF) in cultured CHRF-288-11 megakaryocytic cells. Initially, we compared agonist-stimulated calcium transients in fura-2-loaded CHRF-288-11 cells and human platelets. The 50% effective concentration values for the agonists to increase free cytosolic calcium were as follows: thrombin (0.11 +/- 0.02 U/ml in CHRF, 0.19 +/- 0.03 U/ml in platelets), U46619 (147 +/- 33 nM in CHRF, 157 +/- 5 nM in platelets), and PAF [15 +/- 2 nM in CHRF, 16 +/- 2 nM in platelets (n = 4 each)]. CHRF-288-11 thrombin, TxA2, and PAF receptors were demonstrated to be coupled to phospholipase C because each of the agonists stimulated phosphatidylinositol hydrolysis in myo-[3H]inositol-loaded CHRF-288-11 cells and pharmacological inhibition of phospholipase C-blunted agonist-stimulated calcium signaling. CHRF-288-11 cells exposed to the three agonists for 1 h showed different patterns and extent of homologous and heterologous desensitization. Protein kinase C activation appeared to be necessary but not sufficient for desensitization because 1) activation of protein kinase C with phorbol 12-myristate 13-acetate inhibited the calcium responses to all three agonists, 2) inhibition of protein kinase C with staurosporine attenuated subsequent desensitization to each agonist, and 3) each agonist increased protein kinase C activity in CHRF-288-11 cell homogenates.

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Effects of activation of protein kinase C on the agonist-induced stimulation and inhibition of cyclic AMP formation in intact human platelets

Biochemical Journal ◽

10.1042/bj2430667 ◽

1987 ◽

Vol 243 (3) ◽

pp. 667-678 ◽

Cited By ~ 29

Author(s):

K A Williams ◽

W Murphy ◽

R J Haslam

Keyword(s):

Creatine Kinase ◽

Protein Kinase C ◽

Protein Kinase ◽

Cyclic Amp ◽

Adenylate Cyclase ◽

Platelet Activating Factor ◽

Human Platelets ◽

Prostaglandin D2 ◽

Guanine Nucleotide Binding Protein ◽

Kinase C

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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Protein kinase C and cyclic AMP regulate reversible exposure of binding sites for fibrinogen on the glycoprotein IIB-IIIA complex of human platelets

Biochemical Journal ◽

10.1042/bj2730115 ◽

1991 ◽

Vol 273 (1) ◽

pp. 115-120 ◽

Cited By ~ 62

Author(s):

G van Willigen ◽

J W N Akkerman

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Cyclic Amp ◽

Binding Sites ◽

Platelet Activating Factor ◽

Human Platelets ◽

Common Mechanism ◽

Tight Coupling ◽

Kinase C ◽

Fibrinogen Binding

Platelet aggregation is mediated via binding of fibrinogen to sites on the membrane glycoprotein IIB-IIIA complex which become exposed when the cells are stimulated. We report here evidence of a dynamic and reversible exposure of binding sites for fibrinogen. In the absence of fibrinogen, exposed sites (B*) gradually lose their capacity to bind fibrinogen and close (Bo). On stimulation with platelet-activating factor (PAF, 500 nM) at 22 degrees C, closing of B* is enhanced by agents that raise cyclic AMP levels (10 ng of prostaglandin I2/ml; 5 mM-theophylline), inhibit protein kinase C (PKC; 25 microM-sphingosine; 1 microM-staurosporine), or disrupt the energy supply (30 mM-2-deoxy-D-glucose + 1 mM-CN-), or by raising the temperature to 37 degrees C. Conversely, activation of PKC 1 microM-1,2-dioctanoyl-sn-glycerol; 55 nM-phorbol 12-myristate 13-acetate) and an increase in intracellular [Ca2+] (100 nM-ionomycin + extracellular Ca2+) oppose the disappearance of B*. Phosphorylation of the 47 kDa protein illustrates the tight coupling between PKC and B* under all conditions tested, except when the cyclic AMP level is raised, and B* is converted to Bo without affecting PKC activity. Although the increase in PKC activity is much smaller with ADP or even absent upon stimulation with adrenaline, the control of B* is equally sensitive to modulation of cyclic AMP and PKC activity. We conclude that PAF, ADP and adrenaline regulate exposure of fibrinogen binding sites through a common mechanism consisting of two independent pathways, one dominated by PKC and the other by an as yet unidentified cyclic AMP-sensitive step.

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Comparison of the effects of phorbol 12-myristate 13-acetate and prostaglandin E1 on calcium regulation in human platelets

Biochemical Journal ◽

10.1042/bj2490487 ◽

1988 ◽

Vol 249 (2) ◽

pp. 487-493 ◽

Cited By ~ 14

Author(s):

K Yoshida ◽

F Stark ◽

V T Nachmias

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Cyclic Amp ◽

Protein Phosphorylation ◽

Prostaglandin E1 ◽

Physiological Role ◽

Calcium Transient ◽

Human Platelets ◽

Calcium Sequestration ◽

Kinase C

We compared the effects of phorbol 12-myristate 13-acetate (PMA) with those of prostaglandin E1 (PGE1) on the calcium transient in intact platelets and on 45Ca2+ uptake in saponin-treated platelets and microsomal fractions to determine the roles of protein kinase C and cyclic AMP in calcium sequestration. In intact platelets, PMA, like PGE1, stimulated the return of the calcium transient to resting values after a thrombin stimulus, but only the PGE1 effect was reversed by adrenaline. Both PMA and PGE1, when added before saponin, stimulated ATP-dependent 45Ca2+ uptake into the permeabilized platelets. Thrombin also stimulated 45Ca2+ uptake into saponin-treated platelets. Uptake of 45Ca2+ was increased in microsomal preparations from platelets pretreated with PMA or PGE1. PMA did not increase the cyclic AMP content of control or thrombin-treated platelets, and it induced a pattern of protein phosphorylation in 32P-labelled platelets different from that with PGE1. In correlation with the increased uptake of calcium in the saponin-treated preparation, we measured a rapid translocation of protein kinase C from supernatant to cell fraction after the addition of PMA. Our results suggest that activation of protein kinase C enhances calcium sequestration independently of an effect on cyclic AMP content in platelets. This activation could play a physiological role in the regulation of the calcium transient.

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Calcium mobilisation controls tyrosine protein phosphorylation independently of the activation of protein kinase C in human platelets

FEBS Letters ◽

10.1016/0014-5793(94)00414-5 ◽

1994 ◽

Vol 345 (1) ◽

pp. 87-91 ◽

Cited By ~ 20

Author(s):

Hervé Falet ◽

Francine Rendu

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Protein Phosphorylation ◽

Human Platelets ◽

Kinase C

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1,2-DIOCTANOYLGLYCERIOL (diC8) BUT NOT 1-OLEOYL 2-ACETYLGLYCEROL (OAG) INHIBITS AGONIST-INDUCED PLATELET RESPONSES

10.1055/s-0038-1644506 ◽

1987 ◽

Author(s):

S K Joseph ◽

S Krlshnamurthi ◽

Y Patel ◽

V V Kakkar

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Protein Phosphorylation ◽

Time Course ◽

Fold Increase ◽

Inhibitory Effect ◽

Human Platelets ◽

Single Cycle ◽

Kinase C ◽

Significant Inhibitory Effect

Inhibition of agonist-induced platelet responses by phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PrkC), has been reported. We have examined the effects of the diacylglycerol (DG) analogues OAG and diCg, both PrkC activators, as well as PMA on intracellular Ca2+ ([Ca2+]i) mobilisation and 5-hydroxytryptamine (5HT) release induced by thrombin (T), collagen (Coll.) and the thromboxane (Tx) mimetic U46619. All studies were performed using washed human platelets pre-labelled with either quin-2 or [14C]-5HT and maximal concentrations of agonists. Neither diCg or PMA elevated [Ca2+]i above resting levels though both agents induced a small (10-15%) secretory response. In response to 0.2U/ml T or 0.6uM U46619, but not 20μg/ml Coll., [Ca2+]i increased from resting levels of lOOnM to 758±108nM and 712±58nM respectively. Addition of diCg (60μM) or PMA (16nM) 10 sec before or after T or U46619 reduced the control responses by 10-15% and 30-80% respectively. In contrast, [14C]-5HT secretion in response to T and Coll. was unaffected by diCg or PMA and in the case of U46619 was potentiated 1.4-1.6 fold over control levels. With longer pre-incubation times (5 min) [Ca2+]i mobilisation was further reduced and an inhibitory effect (10-40%) on agonist-induced secretion was evident. Unlike diCg or PMA, OAG (63μM) had no significant inhibitory effect on agonist-induced [Ca2+]i mobilisation and [14C]-5HT secretion even with long pre-incubation times (5 min). However, like diCg and PMA, OAG potentiated U46619-induced secretion with a 10 sec incubation though it induced no secretion itself. The inability of OAG to inhibit may be related to its lesser potency as a PrkC activator. Over a 10 sec-5 min period OAG caused significantly less 40Kd protein phosphorylation ( < 2-fold increase in [32P]-labelling), compared to diCg and PMA (4-6-fold increase). Our results suggest that diCg may be a better tool as an activator of PrkC and DG mimic than OAG. Further, the time course of inhibition of agonist-induced [Ca2+]i mobilisation by diCg suggests that this effect may constitute a physiologically relevant phenomenon mediated by DG within a single cycle of agonist-induced events.

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