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 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.

Protein kinase C and cyclic AMP modulate thrombin-induced platelet-activating factor synthesis in human endothelial cells

1991 ◽  
Vol 1093 (1) ◽  
pp. 55-64 ◽  
Author(s):  
Regine Heller ◽  
Federico Bussolino ◽  
Dario Ghigo ◽  
Giovanni Garbarino ◽  
Henning Schröder ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Platelet Activating Factor ◽  
Human Endothelial Cells ◽  
Kinase C

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.

Activation of human platelets by protein kinase C antibody: role for surface phosphorylation in homeostasis

1996 ◽  
Vol 271 (5) ◽  
pp. H2134-H2144 ◽  
Author(s):  
A. Babinska ◽  
Y. H. Ehrlich ◽  
E. Kornecki
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Surface Protein ◽  
Human Platelets ◽  
Surface Proteins ◽  
Protein Kinase Activity ◽  
Platelet Surface ◽  
Inhibitory Peptides ◽  
Kinase C ◽  
Fibrinogen Binding

A monoclonal antibody that inhibits protein kinase C (PKC) activity, as well as PKC pseudosubstrate inhibitory peptides, was found to cause aggregation of human platelets followed by granular secretion. Binding of this antibody to the platelet surface was demonstrated directly by flow cytometry and immunofluorescence microscopy. Assays of ecto-protein kinase activity revealed that this antibody inhibits the phosphorylation of five proteins on the platelet surface. The platelet aggregation induced by extracellular PKC inhibitors could be blocked by the addition of the membrane-impermeable phosphatase inhibitor, microcystin. Thus the inhibition of surface protein phosphorylation together with continuous dephosphorylation, namely, a decrease in the phosphorylation state of surface proteins, causes the activation of platelets. The aggregation caused by decreased surface phosphorylation appears to be initiated by the exposure of active fibrinogen-binding sites on the platelet surface, as demonstrated by the formation of fibrinogen-dependent microaggregates, as the first step in this process. We conclude that the phosphorylation of surface proteins by a platelet ecto-protein kinase C protects platelets from spontaneous aggregation and thus can play an important role in homeostatic mechanisms that maintain circulating platelets in a resting state.

scholarly journals 4,4′-Di-isothiocyanatostilbene-2,2′-disulphonic acid (‘DIDS’) activates protein kinase C and Na+/H+ exchange in human platelets via α2A-adrenergic receptors

1993 ◽  
Vol 293 (2) ◽  
pp. 523-530 ◽  
Author(s):  
R Nieuwland ◽  
G Van Willigen ◽  
J W Akkerman
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Binding Sites ◽  
Adrenergic Receptors ◽  
Adrenergic Receptor ◽  
Early Event ◽  
Phosphoinositide Metabolism ◽  
Kinase C ◽  
Fibrinogen Binding ◽  
Disulphonic Acid

Most agonists stimulate platelets by inducing Ca2+ mobilization, Ca2+ influx and protein kinase C (PKC) activation leading to Na+/H+ exchange, exposure of fibrinogen-binding sites and aggregation. In contrast, previous studies showed that adrenaline induces exposure of fibrinogen-binding sites and aggregation without appreciable changes in cystolic Ca2+ content or PKC activity. In the present study we investigated platelet responses mediated via alpha 2A-adrenergic receptors, using 4,4′-di-isothiocyanatostilbene-2,2′-disulphonic acid (DIDS), which is known to bind to this type of receptor. Addition of DIDS (2-20 microM) induced (i) a rise in cytosolic pH of 0.23 +/- 0.05 pH unit (n = 5) as detected by BCECF fluorescence, due to activation of the Na+/H+ exchanger, (ii) a 3.5-4-fold increase in the phosphorylation of the 47 kDa protein, a major substrate of PKC, (iii) exposure of 81,072 +/- 7293 (n = 3) binding sites for 125I-fibrinogen per platelet, and (iv) irreversible aggregation. These responses occurred without changes in cytosolic [Ca2+], secretion of dense-granule contents and enhanced phosphoinositide metabolism, and were not affected by inhibition of thromboxane A2 generation (30 microM indomethacin). The alpha 2A-adrenergic-receptor antagonists oxymetazoline (500 microM) and yohimbine (1 mM) completely abolished DIDS-induced responses. Inhibition of PKC (1 microM staurosporine) prevented phosphorylation of the 47 kDa protein, the increase in Na+/H+ exchange and exposure of fibrinogen-binding sites. Thus our present data suggest that activation of PKC is an early event in DIDS-induced platelet activation via the alpha 2A-adrenergic receptor, which precedes any of the other known signal-transducing sequences.

scholarly journals Mastoparan promotes exocytosis and increases intracellular cyclic AMP in human platelets. Evidence for the existence of a Ge-like mechanism of secretion

1992 ◽  
Vol 281 (2) ◽  
pp. 465-472 ◽  
Author(s):  
C P D Wheeler-Jones ◽  
T Saermark ◽  
V V Kakkar ◽  
K S Authi
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
G Protein ◽  
Human Platelets ◽  
Dependent Manner ◽  
Kinase C ◽  
Reversible Electroporation ◽  
Dose Dependent ◽  
Stimulation Of

Recent studies have shown that mastoparan, an amphiphilic peptide derived from wasp venom, accelerates guanine nucleotide exchange and GTPase activity of purified GTP-binding proteins. In the present study we have examined the functional consequences of exposure of intact human platelets to mastoparan. Mastoparan promoted rapid (less than or equal to 1 min) dose-dependent increases in 5-hydroxy[14C]tryptamine and beta-thromboglobulin release from dense-granule and alpha-granule populations respectively. The exocytotic response did not result from a lytic effect of mastoparan and occurred in the complete absence of platelet shape change and aggregation. Liberation of [3H]arachidonate and increases in cytosolic [Ca2+] (detected with fura 2) were not observed in platelets stimulated with mastoparan. Similarly, in platelets preloaded with [3H]inositol during reversible electroporation, mastoparan did not cause the accumulation of [3H]inositol phosphates. Mastoparan-induced secretion was unaffected by preincubation with either the protein kinase C inhibitor staurosporine (10 nM-10 microM) or prostacyclin (PGI2; 100 ng/ml) and was not accompanied by phosphorylation of the 45 kDa protein kinase C substrate or the 20 kDa protein normally associated with platelet activation. The G-protein inhibitor guanosine 5′-[beta-thio]diphosphate (GDP[S]; 1 mM) attenuated the secretion induced by mastoparan in both intact and saponin-permeabilized platelets. Encapsulation of GDP[S] during reversible permeabilization inhibited mastoparan-induced secretion, providing evidence for an intracellular action of GDP[S]. In all these studies thrombin (0.05-0.2 unit/ml) elicited characteristic responses, and thrombin-induced secretion was inhibited by staurosporine, PGI2 and GDP[S]. Mastoparan also increased intra-platelet cyclic AMP in a dose-dependent manner. Mastoparan and PGI2 increased 32P incorporation into a protein of approx. 24 kDa, whereas phosphorylation of a 50 kDa substrate was only seen in PGI2-stimulated platelets. These results indicate that mastoparan promotes secretion by a mechanism which does not involve stimulation of phospholipase C and suggest that the secretory event may result either from a direct fusogenic action of mastoparan and/or from stimulation of the putative exocytosis-linked G-protein, Ge.

scholarly journals Regulation of glycoprotein IIB/IIIA exposure on platelets stimulated with alpha-thrombin

Blood ◽  
1992 ◽  
Vol 79 (1) ◽  
pp. 82-90 ◽  
Author(s):  
G van Willigen ◽  
JW Akkerman
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Binding Sites ◽  
Platelet Activating Factor ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Adenosine Diphosphate ◽  
Kinase C ◽  
Protein Kinase C Inhibitor

Previous studies have shown that binding sites for fibrinogen on platelets stimulated with platelet-activating factor (PAF), adenosine diphosphate or epinephrine rapidly close in the absence of fibrinogen. In the present study we investigated whether alpha-thrombin induced similar changes in the glycoprotein (GP) IIB/IIIA-complex. Whereas 80% of binding sites exposed by PAF closed within 30 minutes (22 degrees C), alpha-thrombin (0.1 U/mL) triggered long-lasting exposure of binding sites for [125I]-fibrinogen and [125I]-fibronectin. Even removal of alpha-thrombin with an excess of hirudin failed to close the binding sites. Similar to PAF, alpha-thrombin-exposed sites rapidly closed after addition of the protein kinase C inhibitor staurosporine (1 mumol/L) or dibutyryl cyclic adenosine monophosphate (250 mumol/L). In contrast, prostacyclin (PGI2, 10 ng/mL), which induced rapid closure of binding sites in platelets stimulated with PAF, failed to close the sites in alpha-thrombin-treated platelets. Removal of alpha-thrombin from the platelets restored the PGI2-sensitivity. These data indicate that a short interaction between alpha-thrombin and platelets triggers long-lasting exposure of GPIIB/IIIA. Furthermore, as long as alpha- thrombin remains bound to the platelets, agonists that activate the PGI2-receptor are unable to close GPIIB/IIIA.

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

1988 ◽  
Vol 249 (2) ◽  
pp. 487-493 ◽  
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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