Epinephrine and the Ca2+ionophore A23187 synergistically induce platelet aggregation without protein kinase C activation

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

Biochemical Journal ◽

10.1042/bj2630377 ◽

1989 ◽

Vol 263 (2) ◽

pp. 377-385 ◽

Cited By ~ 34

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.

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Calcium mobilization and glycoprotein IIb-IIIa complex ligands in epinephrine-stimulated platelets

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1991.260.5.h1619 ◽

1991 ◽

Vol 260 (5) ◽

pp. H1619-H1624

Author(s):

J. A. Ware ◽

M. T. Decenzo ◽

M. Smith ◽

M. Saitoh

Keyword(s):

Protein Kinase C ◽

Platelet Aggregation ◽

Protein Kinase ◽

Synthetic Peptide ◽

Platelet Surface ◽

Epinephrine Administration ◽

Kinase C ◽

Fibrinogen Binding ◽

Peptide Analogues ◽

Protein Kinase C Activation

In the presence of extracellular Ca2+, epinephrine induces a rise in cytoplasmic Ca2+ ([Ca2+]i) that is associated with fibrinogen binding to the platelet surface, platelet aggregation, and enhancement of the thrombin-stimulated [Ca2+]i rise and protein phosphorylation. Whether the [Ca2+]i rise induced by epinephrine results from Ca2+ entry associated with fibrinogen binding to its receptor on the platelet surface, the glycoprotein (gp) IIb-IIIa complex, is unknown. To determine the importance of the occupancy of the gp IIb-IIIa receptor on platelet function after epinephrine administration, we studied the effects of two monoclonal antibodies (M-148 and 7E3) and two synthetic peptide analogues to fibrinogen (synthetic tetrapeptides Arg-Gly-Asp-Ser (RGDS) and dodecapeptide His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val [gamma-(400-411)]), all of which bind to gp IIb-IIIa and inhibit fibrinogen binding and platelet aggregation on the epinephrine-induced rise in [Ca2+]i and enhancement of thrombin's phosphorylation of the 47-kDa substrate of protein kinase C (p47). None of the gp IIb-IIIa ligands significantly enhanced or inhibited the epinephrine-induced [Ca2+]i rise or its augmentation of p47 phosphorylation after thrombin administration; however, the synergistic [Ca2+]i rise that follows addition of both epinephrine and thrombin was reduced by both antibodies and both peptides. Thus ligand binding of gp IIb-IIIa does not influence the epinephrine-induced [Ca2+]i rise or its promotion of protein kinase C activation by thrombin; these events can be dissociated from the synergistic [Ca2+]i rise.

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Mechanism of thrombin-induced rise in platelet fructose 2,6-bisphosphate content. Studies using phorbol myristate acetate, dioctanoylglycerol and ionophore A23187

Biochemical Journal ◽

10.1042/bj2440547 ◽

1987 ◽

Vol 244 (3) ◽

pp. 547-551 ◽

Cited By ~ 9

Author(s):

V Vasta ◽

P Bruni ◽

M Farnararo

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Phorbol Myristate Acetate ◽

Ionophore A23187 ◽

Myristate Acetate ◽

Kinase C ◽

Protein Kinase C Activation

The mechanism by which thrombin increases platelet fructose 2,6-bisphosphate content was investigated. The action of thrombin was mimicked by phorbol 12 myristate 13-acetate and 1,2-dioctanoylglycerol. Ca2+ with A23187 potentiated the action of both these compounds. The action of thrombin required mobilization of intracellular and extracellular Ca2+ and was not decreased by indomethacin. This study suggests that protein kinase C activation and Ca2+ mobilization are both involved in the activation of glycolysis by thrombin.

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Ionophore A23187-induced protein-tyrosine phosphorylation of human platelets: Possible synergism between Ca2+ mobilization and protein kinase C activation

Biochemical and Biophysical Research Communications ◽

10.1016/0006-291x(91)91506-8 ◽

1991 ◽

Vol 174 (2) ◽

pp. 922-927 ◽

Cited By ~ 30

Author(s):

Hiroshi Takayama ◽

Toru Nakamura ◽

Shigeru Yanagi ◽

Takanobu Taniguchi ◽

Shun-ichi Nakamura ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Tyrosine Phosphorylation ◽

Human Platelets ◽

Ionophore A23187 ◽

Protein Tyrosine Phosphorylation ◽

Protein Tyrosine ◽

Kinase C ◽

Protein Kinase C Activation

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Platelet phospholipase D is activated by protein kinase C via an integrin αIIbβ3-independent mechanism

Biochemical Journal ◽

10.1042/bj3100623 ◽

1995 ◽

Vol 310 (2) ◽

pp. 623-628 ◽

Cited By ~ 32

Author(s):

E A Martinson ◽

S Scheible ◽

A Greinacher ◽

P Presek

Keyword(s):

Protein Kinase C ◽

Platelet Aggregation ◽

Protein Kinase ◽

Phospholipase D ◽

Major Protein ◽

Ionophore A23187 ◽

Type I ◽

Specific Product ◽

Kinase C ◽

Fibrinogen Binding

Blood platelets contain phospholipase D (PLD) that is rapidly activated following platelet stimulation. It is currently unclear, however, where PLD fits into the signalling cascade that leads to aggregation and secretion. Therefore we investigated the mechanism of activation of PLD in human platelets, using the formation of the PLD-specific product phosphatidylethanol as a measure of PLD activity. PLD was activated by a number of platelet agonists that also cause the activation of protein kinase C, including thrombin, collagen, the Ca2+ ionophore A23187 and the thromboxane A2-mimetic U46619. Phorbol 12-myristate 13-acetate (PMA), a direct activator of protein kinase C, also increased PLD activity. A selective inhibitor of protein kinase C, Ro-31-8220, totally blocked the stimulation of PLD by thrombin or PMA under conditions in which it also inhibited phosphorylation of pleckstrin, the major protein kinase C substrate in platelets. Ro-31-8220 additionally inhibited A23187-stimulated PLD activity, indicating that Ca2+ activation of PLD also occurs via a protein kinase C-dependent pathway. In the presence of the fibrinogen antagonist peptide RGDS, which inhibits fibrinogen binding to integrin alpha IIb beta 3 and allows little or no aggregation to occur, thrombin- and PMA-stimulated PLD activity was still observed, indicating that PLD activation is not simply a consequence of platelet aggregation. Furthermore, these agonists were able to stimulate PLD in platelets from a Glanzmann's thrombasthenia type I patient lacking the integrin alpha IIb beta 3 complex, which indicates that activation of PLD is also independent of the recruitment of integrin alpha IIb beta 3. Taken together, our results show that PLD is activated by a pathway involving protein kinase C, and suggest that PLD might be involved in signal transduction events occurring upstream of integrin alpha IIb beta 3 activation and fibrinogen binding, which are prerequisites for full platelet aggregation.

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