scholarly journals Concomitant activation of Giprotein-coupled receptor and protein kinase C or phospholipase C is required for platelet aggregation

FEBS Letters ◽  
1999 ◽  
Vol 460 (1) ◽  
pp. 37-40 ◽  
Author(s):  
Fabio M Pulcinelli ◽  
Maria Teresa Ciampa ◽  
Mara Favilla ◽  
Pasquale Pignatelli ◽  
Silvia Riondino ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Platelet Aggregation ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Kinase C

scholarly journals Platelet aggregation induced by α2-adrenoceptor and protein kinase C activation. A novel synergism

1989 ◽  
Vol 263 (2) ◽  
pp. 377-385 ◽  
Author(s):  
W Siess ◽  
E G Lapetina
Keyword(s):  
Protein Kinase C ◽  
Platelet Aggregation ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Platelet Rich Plasma ◽  
Shape Change ◽  
Protein Activation ◽  
Kinase C ◽  
Gi Protein ◽  
Protein Kinase C Activation

Adrenaline or UK 14304 (a specific alpha 2-adrenoceptor agonist) and phorbol ester (phorbol 12,13-dibutyrate; PdBu) or bioactive diacylglycerols (sn-1,2-dioctanoylglycerol; DiC8) synergistically induced platelet aggregation and ATP secretion. The effect on aggregation was more pronounced than the effect on secretion, and it was observed in aspirinized, platelet-rich plasma or suspensions of washed aspirinized platelets containing ADP scavengers. No prior shape change was found. In the presence of adrenaline, DiC8 induced reversible aggregation and PdBu evoked irreversible aggregation that correlated with the different kinetics of DiC8- and PdBu-induced protein kinase C activation. Adrenaline and UK 14304 did not induce or enhance phosphorylation induced by DiC8 or PdBu of myosin light chain (20 kDa), the substrate of protein kinase C (47 kDa), or a 38 kDa protein. Immunoprecipitation studies using a Gcommon alpha antiserum or a Gi alpha antiserum showed that Gi alpha is not phosphorylated after exposure of platelets to PdBu or PdBu plus adrenaline. Adrenaline, PdBu or adrenaline plus PdBu did not cause stimulation of phospholipase C as reflected in production of [32P]phosphatidic acid. Adrenaline caused a small increase of Ca2+ in the platelet cytosol of platelets loaded with Indo-1; this effect was also observed in the absence of extracellular Ca2+. However, under conditions of maximal aggregation induced by adrenaline plus PdBu, no increase of cytosolic Ca2+ was observed. Platelet aggregation induced by PdBu plus adrenaline was not inhibited by a high intracellular concentration of the calcium chelator Quin-2. These experiments indicate that alpha 2-adrenoceptor agonists, known to interact with Gi, and protein kinase C activators synergistically induced platelet aggregation through a novel mechanism. The synergism occurs distally to Gi protein activation and protein kinase C-dependent protein phosphorylation and does not involve phospholipase C activation or Ca2+ mobilization.

Effect of the Polyamine-Spermine on Agonist-Induced Human Platelet Activation - Specific Inhibition of "Aggregation-Independent" Events Induced by Thrombin, but not by Collagen, Thromboxane Mimetic, Phorbol Ester or Calcium lonophore

1987 ◽  
Vol 57 (02) ◽  
pp. 191-195 ◽  
Author(s):  
Sunil Joseph ◽  
Sushila Krishnamurthi ◽  
Vijay V Kakkar
Keyword(s):  
Protein Kinase C ◽  
Platelet Aggregation ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Specific Inhibitor ◽  
Threshold Concentration ◽  
Specific Inhibition ◽  
Independent Events ◽  
Kinase C ◽  
Platelet Receptor

SummarySpermine, a naturally occurring polyamine, has previously been described as an inhibitor of purified phospholipase C and protein kinase C in cell-free systems. The present study examines the effect of spermine on platelet aggregation, dense-granule secretion and thromboxane (Tx) B2 synthesis induced by a variety of agonists, which cause the activation of one or both enzymes to different extents. These studies revealed that, while spermine (10 mM) inhibited platelet aggregation in response to all the agonists examined, [14C]-5-hydroxytryptamine (5HT) release and TxB2 synthesis induced by thrombin (0.2 U/ml) and collagen (10-40 μg/ml) alone, were inhibited by spermine, the percentage inhibition being >90% for both responses with thrombin, 30% for 5HT release and 80% for TxB2 synthesis with collagen. The inhibition of collagen-induced [14C]-5HT secretion by spermine was due entirely to the inhibition of aggregation-dependent TxA2 synthesis as addition of a sub-threshold concentration of U46619, which induced no secretion on its own, totally restored collagen-induced [14C]-5HT secretion to the levels seen in the absence of spermine. Moreover, collagen-induced TxB2 formation in unstirred platelets, which occurred independently of aggregation was not significantly affected by spermine (10 mM). However, the inhibition of maximal thrombin-induced [14C]-5HT secretion and TxB2 synthesis, which are both aggregation-independent phenomena, could be attributed to the inhibition of thrombin-induced diacylglycerol formation and intracellular calcium mobilization, which were both inhibited by 80% in the presence of spermine. These results suggest that spermine may be a specific inhibitor of thrombin-induced platelet responses rather than a generally effective inhibitor of platelet phospholipase C and protein kinase C or the activation of these enzymes in response to agpnists other than thrombin, at least when added exogenously to intact platelets. This thrombin-specific inhibition may be due to an interference of the binding of thrombin to its platelet receptor and/or interference at a step following thrombin binding, which is involved in the subsequent coupling processes.

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 The phospholipase C/protein kinase C pathway is involved in cathepsin G-induced human platelet activation: comparison with thrombin

1996 ◽  
Vol 313 (2) ◽  
pp. 401-408 ◽  
Author(s):  
Mustapha SI-TAHAR ◽  
Patricia RENESTO ◽  
Hervé FALET ◽  
Francine RENDU ◽  
Michel CHIGNARD
Keyword(s):  
Protein Kinase C ◽  
Platelet Aggregation ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Platelet Activation ◽  
Inositol Phosphates ◽  
Cathepsin G ◽  
Dependent Manner ◽  
Functional Responses ◽  
Kinase C

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 μM). Time-course studies (0-180 s) comparing equivalent concentrations of cathepsin G (3 μM) 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.

SYNERGISM OF Gi-DISSOCIATION AND PROTEIN KINASE C STIMULATION IN PLATELET ACTIVATION

1987 ◽  
Author(s):  
W Siess ◽  
E G Lapetina
Keyword(s):  
Protein Kinase C ◽  
Platelet Aggregation ◽  
Protein Kinase ◽  
Phosphatidic Acid ◽  
Protein Phosphorylation ◽  
Phospholipase C ◽  
Phorbol Esters ◽  
Platelet Rich Plasma ◽  
Kinase C ◽  
Α2 Adrenoceptor

Epinephrine or UK 14304 (a specific (α2-adrenoceptor agonist) synergizes with phorbol esters (phorbol 12,13-dibutyrate, PdBu) or bioactive diacylglycerols (sn-1,2-dioctanoylglycerol, DiC8) to induce aggregation and ATP-secretion of platelets. The effect on aggregation is more pronounced than on secretion, and it is observed in aspirinized platelet-rich plasma or suspensions of washed platelets containing ADP-scavengers. No prior shape change is found. In the presence of epinephrine, DiCg induces reversible aggregation and PdBu evokes irreversible aggregation that correlates with the effects, on protein phosphorylation. Epinephrine and UK 14304 neither induce nor enhance the phosphorylation of myosin light chain (20kDa), the substrate of protein kinase C (47kDa), or a 38kDa protein evoked by DiCg) or PdBu. Epinephrine does not cause, stimulation of phospholipase C as reflected by the production of inositol mono-, bis- and tris-phosphate or phosphatidic acid. Even under conditions of maximal aggregation induced by epinephrine plus PdBu, formation of 32p-phOSphatidic acid is not observed. The synergistic action of epinephrine and PdBu does not depend on extracellular Ca2+. Primary aggregation induced by epinephrine, but not platelet aggregation induced by PdBu plus epinephrine, is inhibited by high intracellular concentrations of the calcium chelator quin2. Prostacyclin prevents platelet aggregation but does not affect protein phosphorylation induced by PdBu plus epinephrine.The experiments indicate that α2-adrenoceptor agonists may induce primary aggregation by a mechanism involving release of membrane-bound Ca2+. The synergism with protein kinase C is, however, caused by a mechanism that occurs distally to protein phosphorylation and is not related to Phospholipase C activation and Ca2+-fluxes across the Dlasma membrane or in the cvtosol. Evidence is presented suoportina the view that this mechanism miqht be related to the dissociation of Gi caused by α2-adrenoceptor activation.

Regulation of oxytocin secretion by the ovine corpus luteum: effect of activators of protein kinase C

1990 ◽  
Vol 124 (2) ◽  
pp. 225-232 ◽  
Author(s):  
J. J. Hirst ◽  
G. E. Rice ◽  
G. Jenkin ◽  
G. D. Thorburn
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Corpus Luteum ◽  
Phospholipase C ◽  
Tissue Slices ◽  
Kinase C ◽  
Luteal Tissue ◽  
Dose Dependent ◽  
Stimulation Of

ABSTRACT The effect of protein kinase C activation and dibutyryl cyclic AMP on oxytocin secretion by ovine luteal tissue slices was investigated. Several putative regulators of luteal oxytocin secretion were also examined. Oxytocin was secreted by luteal tissue slices at a basal rate of 234·4 ± 32·8 pmol/g per h (n = 24) during 60-min incubations.Activators of protein kinase C: phorbol 12,13-dibutyrate (n = 8), phorbol 12-myristate,13-acetate (n = 4) and 1,2-didecanoylglycerol (n = 5), caused a dose-dependent stimulation of oxytocin secretion in the presence of a calcium ionophore (A23187; 0·2 μmol/l). Phospholipase C (PLC; 50–250 units/l) also caused a dose-dependent stimulation of oxytocin secretion by luteal slices. Phospholipase C-stimulated oxytocin secretion was potentiated by the addition of an inhibitor of diacylglycerol kinase (R59 022; n = 4). These data suggest that the activation of protein kinase C has a role in the stimulation of luteal oxytocin secretion. The results are also consistent with the involvement of protein kinase C in PLC-stimulated oxytocin secretion. The cyclic AMP second messenger system does not appear to be involved in the control of oxytocin secretion by the corpus luteum. Journal of Endocrinology (1990) 124, 225–232

scholarly journals Prostacyclin inhibits platelet aggregation induced by phorbol ester or Ca2+ ionophore at steps distal to activation of protein kinase C and Ca2+-dependent protein kinases

1989 ◽  
Vol 258 (1) ◽  
pp. 57-65 ◽  
Author(s):  
W Siess ◽  
E G Lapetina
Keyword(s):  
Protein Kinase C ◽  
Platelet Aggregation ◽  
Protein Kinase ◽  
Protein Kinases ◽  
Shape Change ◽  
Ionophore A23187 ◽  
Maximal Effect ◽  
Kinase C ◽  
Dependent Protein ◽  
High Concentrations

Suspensions of aspirin-treated, 32P-prelabelled, washed platelets containing ADP scavengers in the buffer were activated with either phorbol 12,13-dibutyrate (PdBu) or the Ca2+ ionophore A23187. High concentrations of PdBu (greater than or equal to 50 nM) induced platelet aggregation and the protein kinase C (PKC)-dependent phosphorylation of proteins with molecular masses of 20 (myosin light chain), 38 and 47 kDa. No increase in cytosolic Ca2+ was observed. Preincubation of platelets with prostacyclin (PGI2) stimulated the phosphorylation of a 50 kDa protein [EC50 (concn. giving half-maximal effect) 0.6 ng of PGI2/ml] and completely abolished platelet aggregation [ID50 (concn. giving 50% inhibition) 0.5 ng of PGI2/ml] induced by PdBu, but had no effect on phosphorylation of the 20, 38 and 47 kDa proteins elicited by PdBu. The Ca2+ ionophore A23187 induced shape change, aggregation, mobilization of Ca2+, rapid phosphorylation of the 20 and 47 kDa proteins and the formation of phosphatidic acid. Preincubation of platelets with PGI2 (500 ng/ml) inhibited platelet aggregation, but not shape change, Ca2+ mobilization or the phosphorylation of the 20 and 47 kDa proteins induced by Ca2+ ionophore A23187. The results indicate that PGI2, through activation of cyclic AMP-dependent kinases, inhibits platelet aggregation at steps distal to protein phosphorylation evoked by protein kinase C and Ca2+-dependent protein kinases.

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