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

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.

Download Full-text

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

Biochemical Journal ◽

10.1042/bj2580057 ◽

1989 ◽

Vol 258 (1) ◽

pp. 57-65 ◽

Cited By ~ 32

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.

Download Full-text

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

FEBS Letters ◽

10.1016/0014-5793(89)80144-9 ◽

1989 ◽

Vol 243 (2) ◽

pp. 275-279 ◽

Cited By ~ 9

Author(s):

Christine Olbrich ◽

Wolfgang Siess

Keyword(s):

Protein Kinase C ◽

Platelet Aggregation ◽

Protein Kinase ◽

Ionophore A23187 ◽

Induce Platelet Aggregation ◽

Kinase C ◽

Protein Kinase C Activation

Download Full-text

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.

Download Full-text

Bradykinin and vasopressin activate phospholipase D in rat Leydig cells by a protein kinase C-dependent mechanism

Journal of Endocrinology ◽

10.1677/joe.0.1360119 ◽

1993 ◽

Vol 136 (1) ◽

pp. 119-126 ◽

Cited By ~ 10

Author(s):

A. M. Vinggaard ◽

H. S. Hansen

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Phospholipase D ◽

Leydig Cells ◽

Platelet Activating Factor ◽

Ionophore A23187 ◽

Dependent Manner ◽

Kinase C ◽

Dependent Mechanism

ABSTRACT In the present study we report that bradykinin stimulated phospholipase D activity in rat Leydig cells. Bradykinin added for 8 min stimulated choline formation in a dose-dependent manner and, in the presence of ethanol, bradykinin (100 nmol/l) stimulated transphosphatidylation by phospholipase D resulting in the formation of phosphatidylethanol. This stimulation was abolished after down-regulation of protein kinase C by long-term pretreatment for 22 h with phorbol 12-myristate 13-acetate (PMA). The stimulation of phospholipase D by the simultaneous addition for 8 min of maximum concentrations of PMA and vasopressin (AVP), PMA and bradykinin, or AVP and bradykinin produced no additive phosphatidylethanol or choline response, suggesting that AVP, bradykinin and PMA stimulated phospholipase D-catalysed phosphatidylcholine hydrolysis by a similar protein kinase C-dependent mechanism. Furthermore, LH (10 ng/ml), insulin (500 nmol/l), GH (100 ng/ml), interleukin-1β (5 U/ml) and platelet-activating factor (200 nmol/l) were found not to activate phospholipase D, whereas the Ca2+ ionophore A23187 (10 μmol/l) stimulated phosphatidylethanol formation, suggesting that Ca2+ might be a regulator of phospholipase D in Leydig cells. Journal of Endocrinology (1993) 136, 119–126

Download Full-text

The regulation of phospholipase D activity and its role in sn-1,2-diradylglycerol formation in bombesin- and phorbol 12-myristate 13-acetate-stimulated Swiss 3T3 cells

Biochemical Journal ◽

10.1042/bj2800431 ◽

1991 ◽

Vol 280 (2) ◽

pp. 431-438 ◽

Cited By ~ 43

Author(s):

S J Cook ◽

C P Briscoe ◽

M J O Wakelam

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Cyclic Amp ◽

Phospholipase D ◽

Inositol Phosphate ◽

Ionophore A23187 ◽

3T3 Cells ◽

Kinase C ◽

Protein Kinase C Inhibitor ◽

Swiss 3T3

Addition of the phorbol ester phorbol 12-myristate 13-acetate (PMA) to quiescent Swiss 3T3 cells resulted in a sustained increase in sn-1,2-diradylglycerol (DG) mass and [3H]DG in [3H]palmitate-labelled cells where phosphatidylcholine was the major labelled phospholipid. This occurred in the absence of inositol phosphate accumulation. In [3H]palmitate-labelled cells both bombesin and PMA stimulated the formation of phosphatidylbutanol ([3H]PtdBut) in the presence of 0.3% (v/v) butan-1-ol. The kinetics of [3H]PtdBut formation were consistent with phospholipase D (PLD) activation preceding sustained DG formation. The inclusion of butan-1-ol inhibited 70% of PMA-stimulated DG formation but only 30% of the bombesin response. The ability of bombesin and PMA to stimulate the accumulation of [3H]PtdBut was completely abolished in Swiss 3T3 cells which had been pre-treated with 400 nM-PMA for 48 h to down-regulate protein kinase C activity. PMA-stimulated [3H]PtdBut formation was inhibited by 90% by the protein kinase C inhibitor Ro-31-8220 (10 microM), but bombesin-stimulated PtdBut accumulation was inhibited by at most 50% by the same concentration of inhibitor. Cyclic AMP-elevating agents, i.e. forskolin, dibutyryl cyclic AMP and isobutylmethylxanthine, did not inhibit bombesin stimulation of PLD activity. Bombesin-stimulated PLD activity was inhibited by 50% by buffering of the extracellular Ca2+ concentration to 150 nM, but combination of this treatment with Ro-31-8220 addition was less than additive. Ionophore A23187 alone was able to stimulate PLD activity, but this response was inhibited 50% by Ro-31-8220. Thapsigargin was unable to stimulate PLD activity and had no modulatory effect upon bombesin-stimulated PLD activity at any agonist concentration. The results are discussed in terms of the role of PLD in DG generation and the regulation of PLD activity both by bombesin and by PMA.

Download Full-text