Differential megakaryocytic desensitization to platelet agonists

1992 ◽  
Vol 263 (4) ◽  
pp. C864-C872 ◽  
Author(s):  
G. W. Dorn ◽  
M. G. Davis
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Calcium Signaling ◽  
Phospholipase C ◽  
Platelet Activating Factor ◽  
Dienoic Acid ◽  
Cytosolic Calcium ◽  
Peripheral Circulation ◽  
Human Platelets ◽  
Kinase C

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.

scholarly journals Protein kinase C promotes restoration of calcium homeostasis to platelet activating factor-stimulated human neutrophils by inhibition of phospholipase C

2009 ◽  
Vol 6 (1) ◽  
pp. 29 ◽  
Author(s):  
Gregory R Tintinger ◽  
Annette J Theron ◽  
Helen C Steel ◽  
Riana Cockeran ◽  
Lynette Pretorius ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Calcium Homeostasis ◽  
Platelet Activating Factor ◽  
Human Neutrophils ◽  
Kinase C

Stimulation of transient elevations in cytosolic Ca2+ is related to inhibition of Pi transport in OK cells

1990 ◽  
Vol 259 (3) ◽  
pp. F485-F493 ◽  
Author(s):  
A. Miyauchi ◽  
V. Dobre ◽  
M. Rickmeyer ◽  
J. Cole ◽  
L. Forte ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Parathyroid Hormone ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Cytosolic Calcium ◽  
Wild Type ◽  
Kinase C ◽  
Pi Transport ◽  
Sodium Dependent ◽  
Stimulation Of

Stimulation of changes in cytosolic free calcium by parathyroid hormone was determined in three opossum kidney (OK) cell types, OK wild-type, OKP clone, and OKH clone. All three types of OK cells express parathyroid hormone (PTH)-sensitive adenylate cyclase and adenosine 3',5'-cyclic monophosphate (cAMP) production. However, only the OK wild-type and the OKP clone respond to PTH with inhibition of sodium-dependent Pi transport and transient increase in cytosolic calcium. Characterization of the increases in cytosolic calcium in the wild-type and OKP clones revealed they were due in part to stimulation of Ca2+ release from intracellular stores, probably by inositol 1,4,5-trisphosphate (IP3), which was stimulated by PTH. PTH-stimulated Ca2+ transients were also inhibited by protein kinase C activation. These data are compatible with PTH receptor-mediated phospholipase C activation and its feedback inhibition by protein kinase C. The OKH cells demonstrated a slow increase in cytosolic calcium when stimulated by cyclic nucleotides but no evidence for PTH stimulation of Ca2+ release from intracellular stores. Thus the absence of an inhibitory response of sodium-dependent Pi transport to PTH in the OKH cells is associated with the absence of the rapid transient elevations of cytosolic Ca2+ such as those produced by IP3 production. These data suggest an important cooperative role for cAMP and the phospholipase C-stimulated Ca2(+)-protein kinase C message system in the regulation of Pi transport.

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.

scholarly journals Stimulus-response coupling in human platelets activated by monoclonal antibodies to the CD9 antigen, a 24 kDa surface-membrane glycoprotein

1990 ◽  
Vol 266 (2) ◽  
pp. 527-535 ◽  
Author(s):  
R C Carroll ◽  
R E Worthington ◽  
C Boucheix
Keyword(s):  
Monoclonal Antibody ◽  
Protein Kinase C ◽  
Platelet Aggregation ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Surface Membrane ◽  
Human Platelets ◽  
Membrane Glycoprotein ◽  
Kinase C ◽  
Autocrine Stimulation

The CD9 molecule is a 24 kDa surface-membrane glycoprotein present on platelets and a variety of haematopoetic and non-haematopoetic tissues. In the present study we utilized specific inhibitors of thromboxane A2 (TxA2) formation (aspirin), protein kinase C [H-7 [1-(5-isoquinolinesulphonyl)-2-methylpiperazine]] and autocrine stimulation by secreted ADP (apyrase) to modify platelet activation by a monoclonal antibody ALB-6 to the CD9 antigen. This activation is only partially inhibited by aspirin alone but, in combination with either H-7 or apyrase, more than 50% inhibition of platelet aggregation and secretion was observed. This combination of inhibitors was also required to inhibit effectively the phosphorylation of myosin light chain and the 47 kDa substrate of protein kinase C. Intracellular Ca2+ flux monitored by the fluorescent dye fura-2 showed that this was almost completely mediated by the aspirin-sensitive TxA2 pathway. We suggest that the aspirin-insensitive pathway is primarily mediated by phospholipase C formation of diacylglycerol to activate protein kinase C. The inhibition by apyrase suggests a strong dependency on autocrine stimulation by secreted ADP to fully activate both phospholipase C and express fibrinogen-binding sites mediating platelet aggregation. This alternate pathway of phospholipase C activation by ALB-6 may be mediated by cytoplasmic alkalinization [monitored by SNARF-1 (5′(6′)-carboxy-10-bismethylamino-3-hydroxy-spiro-[7H- benzo[c]xanthine-1′,7(3H)-isobenzofuran]-3′-one) fluorescence of the dye]. Both activation pathways are dependent on intact antibodies, since F(ab′)2 fragments of SYB-1, a monoclonal antibody against the CD9 antigen with activation characteristics identical with those of ALB-6, do not elicit activation. Besides thrombin, collagen is another physiological agonist shown to induce aspirin-insensitive activation. Similarities to ALB-6 in collagen sensitivity to apyrase in combination with aspirin inhibitors were noted with respect to aggregation and secretion, as well as a complete block of Ca2+ flux by aspirin. However, it is unlikely that collagen activation is mediated by the CD9 antigen, since SYB-1 F(ab′)2 fragments had no effect on collagen activation and aspirin also completely blocked the alkalinization response to collagen, in contrast with ALB-6.

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

1987 ◽  
Vol 243 (3) ◽  
pp. 667-678 ◽  
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.

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

1991 ◽  
Vol 273 (1) ◽  
pp. 115-120 ◽  
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.

scholarly journals Desensitization and resensitization of human platelets to 5-hydroxytryptamine at the level of signal transduction

1995 ◽  
Vol 307 (3) ◽  
pp. 775-782 ◽  
Author(s):  
P Roevens ◽  
D de Chaffoy de Courcelles
Keyword(s):  
Signal Transduction ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Time Course ◽  
Late Phase ◽  
Human Platelets ◽  
Uptake System ◽  
Kinase C ◽  
5Ht2 Receptor

Desensitization of platelets to 5-hydroxytryptamine (5HT) (1 microM), during active removal of the agonist by the platelet 5HT-uptake system, was studied at the level of signal transduction. Desensitization to 5HT was dose-dependent and homologous. Without occupation of the 5HT2 receptor, neither an increase in cytosolic [Ca2+] (30 nM ionomycin), nor a separate or simultaneous activation of protein kinase C (by 10 microM 1-oleoyl-2-acetylglycerol), could induce desensitization to 5HT (1 microM). During the early phase of desensitization, the 5HT2 receptor was coupled to phospholipase C, whereas during the late phase of desensitization this coupling was disconnected. However, after disappearance of the agonist, the coupling in the resting platelet recovered quickly, and was nearly complete (82%) after 30 min. During this resensitization, the 5HT-inducibility of activation of phospholipase C, of the increase in cytosolic [Ca2+] and of stimulation of protein kinase C were restored in parallel. The time course for resensitization of the 5HT-induced increase in cytosolic [Ca2+] was independent of the presence of extracellular Ca2+. It is concluded that, after dissociation of 5HT from the platelet 5HT2-receptor, 5HT-induced responses rapidly resensitize. Because of its short duration and the parallelism in recovery between the different ‘down-stream phospholipase C’ intracellular transduction signals, it is considered that desensitization arises from a reversible change in the transduction mechanism at a step up to or including the activation of phospholipase C. Neither desensitization nor resensitization to 5HT is dependent on the presence of extracellular Ca2+.

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