Br. J. Pharmacol. (1992), 105, 627-631
(D Macmillan Press Ltd, 1992
Okadaic acid inhibits activation of phospholipase C in human platelets by mimicking the actions of protein kinases A and C 'Trevor R. Walker & Steve P. Watson Department of Pharmacology, Mansfield Road, Oxford OXI 3QT 1 The effect of okadaic acid, a potent inhibitor of protein phosphatases 1 and 2A (PP1 and PP2A), on human platelets has been investigated. 2 Okadaic acid exerts a general increase in phosphorylation of platelet proteins but did not induce aggregation or secretion of 5-hydroxytryptamine (5-HT). Okadaic acid, however, did inhibit thrombininduced functional responses. 3 Maximally effective concentrations of prostacyclin, to elevate adenosine 3':5'-cyclic monophosphate (cyclic AMP), or phorbol dibutyrate, to activate protein kinase C, inhibited the formation of inositol phosphates by thrombin by approximately 60%. When used in combination, prostacyclin and phorbol dibutyrate reduced the levels of inositol phosphates induced by thrombin to 11%. 4 Okadaic acid (1 pM) decreased thrombin-induced formation of inositol phosphates by approximately 55% and increased the inhibitory action of prostacyclin or phorbol dibutyrate. Okadaic acid had no further effect when prostacyclin and phorbol dibutyrate were used in combination. 5 These results suggest that protein kinases A and C act to inhibit phospholipase C by distinct mechanisms and that their action is reversed by PP1 and/or PP2A. Keywords: Human platelets; okadaic acid; protein phosphatases; adenylyl cyclase; protein kinase C
Introduction In platelets, the major stimulatory second messengers are inositol 1,4,5-trisphosphate (IP3) and diacylgylcerol (DG) produced by hydrolysis of phosphatidylinositol 4,5-bisphosphate (Seiss, 1989). IP3 releases Ca2+ from intracellular stores and DG activates protein kinase C. In addition to other actions, Ca2 + activates calmodulin-dependent kinases which phosphorylate several platelet proteins including myosin light chains (20 kDa). Protein kinase C also phosphorylates a number of substrates in platelets, the predominant of which has a molecular mass of 47 kDa but no known function (Tyers et al., 1988). Ca2+ and protein kinase C interact synergistically to induce secretion; however, little is known about the mechanism of this process or proteins involved (Seiss & Lapetina, 1988). In the present study, we have used the selective phosphatase inhibitor okadaic acid, a major toxic component in diarrhetic shellfish poisoning and a potent tumour promoter (Cohen, 1990), to probe the role of protein phosphatases 1 and 2A (PP1 and PP2A) in the activation of human platelets. Karaki et al. (1989) have reported that okadaic acid inhibits activation of rabbit platelets by thrombin, possibly by potentiation of adenosine 3':5'-cyclic monophosphate (cyclic AMP)-mediated events. In the present study, we show that okadaic acid inhibits activation of human platelets by thrombin through potentiation of protein kinase A- and protein kinase Cmediated inhibition of phosphoinositide hydrolysis.
Methods Platelets were obtained from two sources. Blood was either drawn on the day of the experiment from aspirin-free volunteers with sterile 20mm sodium citrate as anticoagulant and platelet-rich plasma obtained by centrifugation at 200g for 20 min, or platelet concentrates were obtained from the Blood Transfusion Service, John Radcliffe Hospital, and used within 20h of donation. Platelets were isolated from platelet-rich plasma or the platelet concentrate by centrifugation at 1000g for 10min in the presence of prostacyclin to prevent aggre1
Author for correspondence.
gation. Platelets were resuspended in 1 ml of modified Tyrodes-HEPES (composition in mM: NaCi 138, NaH2PO4 0.36, KCl 2.9, NaHCO3 12, HEPES 20, glucose 5, EGTA 1, MgCl2 1; pH 7.3) and labelled with [3H]-inositol (50OpCi ml- ' for 3 h), [32P]-orthophosphate (2 mCi ml'- for 1 h) or [3H]-5hydroxytryptamine ([3H]-5-HT, 5 1iCi ml-1 for 1 h). Platelets were then centrifuged in the presence of prostacyclin at 1000 g for 10 min and resuspended at a concentration of 28 x 108ml-1 in the above buffer, containing indomethacin (10puM) to inhibit cyclo-oxygenase. EGTA was omitted in studies of aggregation; LiCl (10mM), which inhibits conversion of inositol monophosphate to free inositol, was added to [3H]-inositol-labelled platelets. Platelets were left at least 30 min before experimentation. Platelet suspensions (0.49ml) were prewarmed at 37°C for 5 min before addition of compounds as appropriate. Experiments were stopped by transfer to 940pl of chloroform/ methanol/HCl (50:100:1) for analyses of inositol phosphates, to 0.5 ml of 6% (v/v) glutaraldehyde in phosphate buffer (pH 7.3) for analyses of 5-HT secretion, or to Laemmli buffer for analyses of protein phosphorylation. Inositol phosphates, protein phosphorylation and 5-HT secretion were measured as described previously (Nunn & Watson, 1987). To prepare membrane and cytosol fractions, platelets were resuspended in 3 vol of lysis buffer (10 mm Tris, 1 mm EDTA, 0.1% (v/v) ,i-mercaptoethanol, 1 mm EGTA, pH 7.4) frozen rapidly in liquid nitrogen and thawed at 37°C; this process was repeated and the final suspension cooled to 4°C. The suspension was then sonicated on ice for 4 x 15 s before centrifugation at 1000 g for 10 min. The supernatant was removed and stored at 4°C; the pellet was resuspended in 3 vol of lysis buffer and the lysis procedure repeated. When lysis of all platelets had been achieved, after which no pellet formed when the suspension was centrifuged at 1000 g for 10min. the pooled supernatants were centrifuged at 100,000g at 4°C for 1 h. The supernatant represents the cytosol fraction while the pellet, resuspended in the original volume of lysis buffer, the membrane fraction. Protein phosphatase assays were performed on both membrane and cytosol fractions as described by Cohen et al. (1989) with 32P-labelled phosphorylase a and casein as substrates. Okadaic acid and labelled substrates for phosphatase assays were kindly given by Professor P. Cohen, Dundee. [3H]-inositol (15.4 Ci mmol - 1), [32P]-orthophosphate (8500-
628
T.R. WALKER & S.P. WATSON
9120 Ci mmol -1) and [3H]-5-HT (21.0 Ci mmol - 1) were from N.E.N., Du Pont (U.K.) Ltd., Stevenage. Thrombin and phorbol dibutyrate were from Sigma Chemical Company Ltd., Poole. Prostacyclin was kindly donated by Wellcome Laboratories (Beckenham, Kent, U.K.) All other reagents were of Analytical grade.
a Time (min) I
3
1
10
20
.4250
.490 .476
Results
-a50
Protein phosphatase assays
., ,
The activities of PP1 and PP2A were assayed in both particulate and cytosolic fractions of human platelets by use of specific radiolabelled substrates. The use of a small heat-stable protein termed inhibitor-2 allowed selective inhibition of PP1 activity whilst okadaic acid (1 pM) inhibited both PP1 and PP2A activity; PP2C is unaffected by okadaic acid and has a requirement for Mg2+ (10mM). The distribution of PP1 in platelets was similar in both particulate and cytosol fractions (Table 1). The specific activities of PP2A and PP2C were found to be higher in the cytosolic fraction than the particulate fraction (Table 1), which is in contrast to rabbit skeletal muscle where almost all PP2A and PP2C activity is found in the cytosolic fraction (Ingebritsen et al., 1983).
V
--
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.447 -431
.424 .20 OA (Im) 00.10.3 1 0 0.1 0.3 1 0 0.10.31 00.10.31
b
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Phosphorylation studies
ev
10
Okadaic acid (1 pM) induced a general increase in phosphorylation of proteins over 60min (Figures 1 and 2) similar to that observed in other cell types e.g. rat hepatocytes and adipocytes (Haystead et al., 1989). The rate of phosphorylation varied greatly between proteins (Figure 2) while, in marked contrast, a protein of mol. wt. 24kDa was unaffected by okadaic acid (Figure 1). The threshold dose of okadaic acid was 0.3 JM (Figure la).
0.
I1 RM
0
-
Action of okadaic acid on secretion and aggregation Okadaic acid (1 pM) did not induce aggregation or secretion of 5-HT from platelets despite the marked changes in protein phosphorylation which occur over 60 min. Preincubation with okadaic acid (1 M) for 10min, however, inhibited thrombininduced aggregation and secretion (Figures 3 and 4). This was particularly marked for low concentrations of thrombin, for example, okadaic acid reduced the secretion of 5-HT by 0.1 unit ml-1 thrombin from 38% to 4% (Figure 4).
Formation of inositol phosphates Okadaic acid exerted a dose- and time-dependent inhibition of thrombin-induced formation of total [3H]-inositol phosphates (Figure 5). Pre-incubation with 1 gM okadaic acid for 10 min inhibited thrombin-induced formation of inositol phosphates by approximately 56%. Moreover, significant inhibition was observed at the earliest time point monitored (5 s)
Increasing distance from origin Figure 1 (a) Effect of okadaic acid on protein phosphorylation. Platelets were prelabelled with 32P Pi and incubated with okadaic acid (1 pM) for varying times. Separation of proteins was achieved by gradient polyacrylamide gel electrophoresis (7.5%-15%), and autoradiography of the resulting gel indicates the extent of phosphorylation. Incubation time in the presence of okadaic acid (OA) is indicated at the top of the figure and the concentration indicated at the bottom. Relative molecular weights (in kDa) of distinct proteins are shown to the right of the figure. (b) Densitometric scan of basal protein phosphorylation (lower) and protein phosphorylation in the presence of okadaic acid (OA, 1 pM), pre-incubated for 10min (upper). Increased phosphorylation is expressed as an increase in optical density. Molecular weights of distinct proteins are indicated in kDa at the top of the figure.
following exposure to thrombin (Figure 5) making it likely that this effect contributes to the inhibition of thrombininduced aggregation and secretion by okadaic acid. Staurosporine, the non-selective kinase inhibitor (Tamaoki et al.,
Table 1 Protein phosphatase activity in human platelet cytosol and membrane fractions
PP1 PP2A PP2C
Cytosol
Particulate (mu mg-' protein)
(mu mg 1 protein)
Rabbit skeletal muscle (mu mg-' protein)
0.27 + 0.05 0.22 + 0.04 0.03 + 0.005
0.19 + 0.005 0.35 + 0.01 0.06 + 0.01
1.13 + 0.09 0.35 + 0.04 0.022 + 0.002
Activity is expressed as mean mu mg- I protein + s.e.mean from 6 experiments (one unit of activity is that amount of enzyme which catalyses the dephosphorylation of 1 pmol of substrate in 1 min). The activities of protein phosphatases 1 and 2A (PP1 and PP2A) were assayed with [32P]-phosphorylase a, PP1 can be selectively blocked with inhibitor-i whilst PP1 and PP2A can be blocked by okadaic acid (1 gM); assays were carried out as described in Methods. Casein is a substrate for both PP2A and PP2C therefore the assay for PP2C contained not only [32P]-casein but also okadaic acid (5yM) to block any PP2A activity and Mg2+ (10mM) as PP2C has a requirement for this divalent cation. Specific activity of phosphatases in rabbit skeletal muscle was reported by Ingebritsen et al. (1983), in this case expressed as mu mg- 1 protein.
OKADAIC ACID INHIBITS PLATELET ACTIVATION
629
a
8000'
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[Okadaic acid] (>.M) bI
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Time (min) Figure 2 Effect of okadaic acid on the rate of protein phosphorylation. Platelets were prepared as described for Figure la, and incubated with okadaic acid (1pM) for 10min. Results are expressed as c.p.m. (-) 25OkDa; (A) 5OkDa; (0) 76kDa; (0) 9OkDa. These symbols correspond to proteins indicated in Figure la.
120 100 80-
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Figure 3 Effect of okadaic acid on thrombin-induced aggregation. A typical aggregation trace induced by a range of thrombin concentrations (0.06 units ml - '-l unit ml -) (a) was repeated in the presence of okadaic acid (1pM, 10 min pre-incubation) (b). This is representative of three other similar experiments. 80
60-
40) -0
5
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115
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Figure 5 Effect of okadaic acid on thrombin-induced formation of inositol phosphates. (a) Dose-response curve for inhibition by okadaic acid. Platelets were prelabelled with [3H]-inositol and pre-incubated with okadaic acid (0.1-1 m, as indicated) for 10min. Platelets were then stimulated with a maximal dose of thrombin (1 unit ml-') for 5 min. Experiments were performed in triplicate and results are from 3 experiments and expressed as % of control inositol phosphate formation with s.e.mean shown by vertical bars. (b) Platelets were prelabelled with [3H]-inositol and pre-incubated with okadaic acid (1 gM) for 10min as indicated: (A) presence of okadaic acid; (El) absence of okadaic acid. Platelets were then stimulated with a maximal dose of thrombin (1 unit ml- 1) for varying times. Experiments were performed in triplicate and results are expressed as % inositol phosphate formation with s.e.mean shown by vertical bars; this is a representative experiment with similar results to two others. (c) Time-dependent effect of okadaic acid on thrombin-induced inositol phosphate formation. Platelets were prelabelled with [3H]-inositol and pre-incubated with okadaic acid (1 gM). Pre-incubation times with okadaic acid were as follows: lOs, 30s, min, 3 min, 10min and 20min. Platelets were then stimulated with a maximal dose of thrombin (1 unit ml-) for 5 min. The time scale relates to the total incubation time in the presence of okadaic acid. Experiments were performed in triplicate and results are expressed as % inositol phosphate formation with s.e.mean shown by vertical bars; this is a representative experiment with similar results to two others.
20-
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Thrombin (units ml-') Figure 4 Effect of okadaic acid on thrombin-induced 5hydroxytryptamine (5-HT) secretion. Platelets were prelabelled with [3H]-5-HT and stimulated with thrombin concentrations for min, in the presence (U) or absence (El) of okadaic acid (I P, 10min preincubation). Results are from 3 experiments and are shown as mean % 5-HT release with s.e.mean shown by vertical bars.
1986), inhibited the action of okadaic acid on thrombininduced formation of inositol phosphates (Table 2). This is consistent with the mechanism of action of okadaic acid being through potentiation of phosphorylation rather than acting as an antagonist at the thrombin receptor. Pretreatment of platelets with maximally effective concentrations of prostacyclin or phorbol dibutyrate reduced thrombin-induced formation of inositol phosphates to approximately 40% of control values in each case. In the presence of 1,UM okadaic acid, these responses were reduced to
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T.R. WALKER & S.P. WATSON
Table 2 Inhibition of thrombin-induced formation of total inositol phosphates d.p.m. Thrombin Thr + PGI2 Thr + PDBu Thr + OA Thr + PGI2 + OA Thr + PDBu + OA Thr + PGI2 + PDBu Thr + PGI2 + PDBu + OA Thr + staurosporine Thr + staurosporine + OA
1067 + 23 454 + 62 434 + 46 484 + 49 216 + 36 221 + 36 121 + 19 94 + 29 1116 + 48 1078 + 96
(%)
100 42.5 + 40.7 + 45.4 + 20.2 + 22.6 + 11.3 + 8.8 + 104.6 +
5.8 4.3 4.6 3.4 3.1
1.8 2.7 4.5 101 + 9.0
Results are expressed as d.p.m. or % + s.e.mean of maximal formation of inositol phosphates, induced by thrombin during 5 min stimulation (n > 4). Compounds are as follows: thrombin (Thr, 1 unitml-1), prostacyclin (PGI2) 2.5,pgml-', phorbol dibutyrate (PDBu) 300nM, okadaic acid (OA) 1,M staurosporine (3pM). Platelets were pre-incubated in the presence of okadaic acid for 10min prior to stimulation by
thrombin.
approximately 20% of controls (Table 2). The inhibitory actions of prostacyclin and phorbol dibutyrate were partially additive, reducing the formation of inositol phosphates to 110% of control values (Table 2). This was not significantly altered in the presence of 1 UM okadaic acid (P > 0.05; Table 2). These results suggest that prostacyclin and phorbol dibutyrate inhibit the activation of phospholipase C (PLC) by thrombin through predominantly different mechanisms and that okadaic acid may be able to mimic both of these actions.
Discussion Effect of okadaic acid
on
protein phosphorylation
Okadaic acid produces an increase in phosphorylation of a large number of platelet proteins which may reflect a net increase in activity of a single kinase or, more likely, a family
of protein kinases. The net increase in kinase activity could be brought about in two ways. The kinases may be intrinsically active and their action terminated by PP1 and/or PP2A or they may be activated by increased phosphorylation which is under regulation from PP1 and/or PP2A. Recently eleven novel serine/threonine protein kinases were identified in platelets, ten of which show increased activity following challenge with thrombin, possibly as a result of autophosphorylation (Ferrell & Martin, 1989). The general increase in phosphorylation may also reflect an alteration in the balance between kinases and phosphatases leading to increased activity of several kinases, the cascade being regulated at one or more points by PP1 and/or 2A. Action of okadaic acid
on
phospholipase C
It has been reported previously that okadaic acid mimics the inhibitory action of cyclic AMP-dependent protein kinase on vascular smooth muscle (Karaki et al., 1989). However okadaic acid did not alter the levels of cyclic AMP suggesting that it does not activate adenylyl cyclase or inhibit cyclic AMP phosphodiesterase. Karaki et al. (1989) proposed that inhibition of protein phosphatases by okadaic acid resulted in an increase in the effects caused by cyclic AMP-dependent protein kinase (protein kinase A). Prostacyclin mediates its inhibitory action on platelets through elevation of cyclic AMP and activation of protein kinase A (Seiss & Lapetina, 1989). One action of cyclic AMP is the inhibition of phosphoinositide hydrolysis following
receptor activation (Watson et al., 1984). Phorbol esters also inhibit formation of inositol phosphates by cell surface receptors and it has been postulated that this is a result of a negative feedback regulation by protein kinase C (Watson & Lapetina, 1985). The observation that the effect of phorbol dibutyrate is partly additive with that of prostacyclin suggests that they produce their effects by separate mechanisms. A number of classes of phospholipase C (PLC) have been identified in a variety of tissues, a, 6, y and 6 (Rhee et al., 1989); cDNA sequencing and immunoreactivity analyses suggest that subtypes of these classes exist (Wahl & Carpenter, 1991). Activation of protein kinase C has been reported to phosphorylate selectively the ai isoform of PLC in a number of cell lines as well as in bovine brain (Ryu et al., 1990). In contrast, protein kinase A selectively phosphorylates the y isoform of PLC in C6Bu1 cells (Kim et al., 1989). Phosphorylation of these isoforms of PLC has no apparent effect on their catalytic activity but may interfere with G protein interactions and therefore attenuate phosphoinositide hydrolysis in response to receptor activation (Kim et al., 1989; Ryu et al., 1990). Characterization of PLC enzymes in platelets has identified a number of subtypes, both cytosolic and membrane-bound (Baldassare et al., 1989; Banno et al., 1986; 1988; 1990). The relationship between platelet isozymes of PLC and those identified in other cell types has still to be established. Prostacyclin and phorbol ester may work at least, in part, by distinct mechanisms, possibly through selective phosphorylation of specific PLC isozymes. Alternatively cyclic AMP and protein kinase C may increase GTPase activity of the a subunit of the putative G protein Gp (Knight & Scrutton, 1987; Krishnamurthi et al., 1989). It has also been demonstrated that agents which raise cyclic AMP levels decrease the ability of thrombin to bind to its receptor, this correlates closely with the inhibition of thrombin-induced functional responses (Lerea et al., 1987). The observation that okadaic acid potentiates the effect of protein kinase A and protein kinase C but has no effect when both pathways are maximally activated suggests that the protein phosphatases PP1 and/or PP2A act to limit the actions of protein kinase A and protein kinase C on receptor-regulation of PLC.
Role of protein phosphatases in platelet activation The role of kinases in stimulus-secretion coupling has been extensively investigated (Hanks et al., 1988; Kikkawa et al., 1989; Taylor et al., 1990). More recently, attention has also focussed on the possible control of secretion by protein phosphatases. It has been reported that a platelet protein of 18 kDa molecular mass is dephosphorylated when platelets are stimulated with thrombin (Imaoka et al., 1983). It is known that dephosphorylation in some cell types is a critical event for cellular responses. For example, in Paramecium, rapid and marked dephosphorylation of a 65 kDa protein correlates temporally with synchronous exocytosis: both events peak within 1-3 s and are complete by 5 s (Zeiseniss & Plattner, 1985). The possibility that activation of a phosphatase may be a requirement in the initial stages of stimulus-secretion coupling has been suggested (Zeiseniss & Plattner, 1985). In platelets, little is known of the importance of this effect. It has been proposed that platelet protein phosphatases may be involved in down-regulation of platelet function (Sakon et al., 1990). However, we believe that the role of protein phosphatases is more complex, made evident by the ability of okadaic acid to attenuate functional responses in human platelets. Okadaic acid and other specific protein phosphatase inhibitors will allow investigation of the precise roles which PP1 and PP2A play in signal transduction. We are grateful to Professor P. Cohen for the gift of okadaic acid and advice with the phosphatase assays. T.R.W. is in receipt of a B.H.F. studentship and S.P.W. is a Royal Society University Research Fellow.
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References BALDASSARE, J.J., HENDERSON, P.A. & FISHER, G.J. (1989). Isolation and characterization of one soluble and two membrane-associated forms of phosphoinositide-specific phospholipase C from human platelets. Biochemistry, 28, 6010-6016. BANNO, Y., NAKASHIMA, S. & NOZAWA, Y. (1986). Partial purification of phosphoinositide phospholipase C from human platelet cytosol; characterization of its three forms. Biochem. Biophys. Res. Commun., 136, 713-721. BANNO, Y., YADA, Y. & NOZAWA, Y. (1988). Purification and characterization of membrane-bound phospholipase C specific for phosphoinositides from human platelets. J. Biol. Chem., 263, 11459-11465. BANNO, Y., YU, A., NAKASHIMA, T., HOMMA, Y., TAKENAWA, T. & NOZAWA, Y. (1990). Purification and characterization of a cytosolic phosphoinositide-phospholipase C (gamma 2-type) from human platelets. Biochem. Biophys. Res. Commun., 167, 396-401. COHEN, P. (1990). Okadaic acid: a new probe for the study of cellular regulation. Trends Biochem. Sci., 15, 98-102. COHEN, P., ALEMANY, S., HEMMINGS, B.A., RESINK, T.J., STRAL-
FORS, P. & LIM TUNG, H.Y. (1989). Protein phosphatase-1 and protein phosphatase-2A from rabbit skeletal muscle. Methods Enzymol., 159, 390-508. FERRELL Jr., J.E. & MARTIN, G.S. (1989). Thrombin stimulates the activities of multiple previously unidentified protein kinases in platelets. J. Biol. Chem., 264, 20723-20729. HANKS, S.K., QUINN, A.M. & HUNTER, T. (1988). The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science, 241, 42-52. HAYSTEAD, T.A.J., SIM, A.T.R., CARLING, D., HONNOR, R.C., TSUKI-
TANI, T., COHEN, P. & HARDIE, D.G. (1989). Effects of the tumour promoter okadaic acid on intracellular protein phosphorylation and metabolism. Nature, 337, 78-81. IMAOKA, T., LYNHAM, JA. & HASLAM, R.J. (1983). Purification and characterization of the 47,000-dalton protein phosphorylated during degranulation of human platelets. J. Biol. Chem., 258, 11404-11414. INGEBRITSEN, T.S., STEWART, A.A. & COHEN, P. (1983). The protein phosphatases involved in cellular regulation. 6. Measurement of type-1 and type-2 protein phosphatases in extracts of mammalian tissues; an assessment of their physiological roles. Eur. J. Biochem., 132, 297-307. KARAKI, H., MITSUI, M., NAGASE, H., OZAKI, H. SHIBATA, S. & UEMURA, D. (1989). Inhibitory action of a toxic okadaic acid, iso-
lated from the black sponge on smooth muscle and platelets. Br. J. Pharmacol., 98, 590-596. KIKKAWA, U., KISHIMOTO, A. & NISHIZUKA, Y. (1989). The protein kinase C family: heterogeneity and its implications. Annu. Rev. Biochem., 58, 31-44. KIM, U.-H., KIM, J.W. & RHEE, S.G. (1989). Phosphorylation of phospholipase C-gamma by cAMP-dependent protein kinase. J. Biol. Chem., 264, 20167-20170. KNIGHT, D.E. & SCRUTTON, M.C. (1987). Secretion of 5hydroxytryptamine from electropermeabilised human platelets. Effects of GTP and cyclic 3',5'-AMP. FEBS Lett., 223, 47-52. KRISHNAMURTHI, S., WHEELER-JONES, C.P.D. & KAKKAR, V.V.
(1989). Effect of phorbol ester treatment on receptor-mediated versus G-protein-activator-mediated responses in platelets. Evidence for a two-site action of phorbol ester at the level of Gprotein function. Biochem. J., 262, 77-81. LEREA, K.M., GLOMSET, J.A. & KREBS, E.G. (1987). Agents that elevate cAMP levels in platelets decrease thrombin binding. J. Biol. Chem., 262, 282-288. NUNN, D.L. & WATSON, S.P. (1987). A diacylglycerol kinase inhibitor, R59022, potentiates secretion by and aggregation of thrombinstimulated human platelets. Biochem. J., 243, 809-813. RHEE, S.G., SUH, P.-G., RYU, S.-H. & LEE, S.Y. (1989). Studies of inositol phospholipid-specific phospholipase C. Science, 244, 546-550. RYU, S.-H., KIM, U.-H., WAHL, M.I., BROWN, A.B., CARPENTER, G.,
HUANG, K.-P. & RHEE, S.G. (1990). Feedback regulation of phospholipase C-beta by protein kinase C. J. Biol. Chem., 265, 1794117945. SAKON, M., KAMBAYASHI, J., KAJIWARA, Y., UEMURA, Y., SHIBA, E.,
KAWASAKI, T. & MORI, T. (1990). Platelet protein phosphatases and their endogenous substrates. Biochem. Int., 22, 149-161. SIESS, W. (1989). Molecular mechanisms of platelet activation. Physiol. Rev., 69, 58-178. SEISS, W. & LAPETINA, E.G. (1988). Ca2l mobilization primes protein kinase C in human platelets. Ca2+ and phorbol esters stimulate platelet aggregation and secretion synergistically through protein kinase C. Biochem. J., 255, 309-318. SEISS, W. & LAPETINA, E.G. (1989). Prostacyclin inhibits platelet aggregation induced by phorbol ester or Ca"+ ionophore at steps distal to activation of protein kinase C and Ca2"-dependent protein kinases. Biochem. J., 258, 57-65. TAMAOKI, T., NOMOTO, H., TAKAHASHI, I., KATO, Y., MORIMOTO, M. & TOMITA, F. (1986). Staurosporine, a potent inhibitor of phospholipid/Ca2" dependent protein kinase. Biochem. Biophys. Res. Commun., 135, 397-402. TAYLOR, S.S., BUECHLER, J.A. & YONEMOTO, W. (1990). cAMPdependent protein kinase: framework for a diverse family of regulatory enzymes. Annu. Rev. Biochem., 59, 971-1005. TYERS, M., RACHUBINSKI, R.A., STEWART, M.I., VARRIOCHIO, A.M.,
SHORR, R.G.L., HASLAM, R.J. & HARLEY, C.B. (1988). Molecular cloning and expression of the major protein kinase C substrate of platelets. Nature, 333, 470-473. WAHL, M. & CARPENTER, G. (1991). Selective phospholipase C activation. BioEssays, 13, 107-113. WATSON, S.P. & LAPETINA, E.G. (1985). 1,2-Diacylglycerol and phorbol ester inhibit agonist-induced formation of inositol phosphates in human platelets: possible implications for negative feedback regulation of inositol phospholipid hydrolysis. Proc. Nati. Acad. Sci. U.S.A., 82, 2623-2626. WATSON, S.P., McCONNELL, R.T. & LAPETINA, E.G. (1984). The rapid formation of inositol phosphates in human platelets by thrombin is inhibited by prostacyclin. J. Biol. Chem., 259, 13199-13203. ZIESENISS, E. & PLATTNER, H. (1985). Synchronous exocytosis in Paramecium cells involves very rapid (less than or equal to 1 s), reversible dephosphorylation of a 65-kD phosphoprotein in exocytosis-competent strains. J. Cell Biol., 101, 2028-2035.
(Received September 6, 1991 Revised November 5, 1991 Accepted November 8, 1991)
(D Macmillan Press Ltd, 1992
Okadaic acid inhibits activation of phospholipase C in human platelets by mimicking the actions of protein kinases A and C 'Trevor R. Walker & Steve P. Watson Department of Pharmacology, Mansfield Road, Oxford OXI 3QT 1 The effect of okadaic acid, a potent inhibitor of protein phosphatases 1 and 2A (PP1 and PP2A), on human platelets has been investigated. 2 Okadaic acid exerts a general increase in phosphorylation of platelet proteins but did not induce aggregation or secretion of 5-hydroxytryptamine (5-HT). Okadaic acid, however, did inhibit thrombininduced functional responses. 3 Maximally effective concentrations of prostacyclin, to elevate adenosine 3':5'-cyclic monophosphate (cyclic AMP), or phorbol dibutyrate, to activate protein kinase C, inhibited the formation of inositol phosphates by thrombin by approximately 60%. When used in combination, prostacyclin and phorbol dibutyrate reduced the levels of inositol phosphates induced by thrombin to 11%. 4 Okadaic acid (1 pM) decreased thrombin-induced formation of inositol phosphates by approximately 55% and increased the inhibitory action of prostacyclin or phorbol dibutyrate. Okadaic acid had no further effect when prostacyclin and phorbol dibutyrate were used in combination. 5 These results suggest that protein kinases A and C act to inhibit phospholipase C by distinct mechanisms and that their action is reversed by PP1 and/or PP2A. Keywords: Human platelets; okadaic acid; protein phosphatases; adenylyl cyclase; protein kinase C
Introduction In platelets, the major stimulatory second messengers are inositol 1,4,5-trisphosphate (IP3) and diacylgylcerol (DG) produced by hydrolysis of phosphatidylinositol 4,5-bisphosphate (Seiss, 1989). IP3 releases Ca2+ from intracellular stores and DG activates protein kinase C. In addition to other actions, Ca2 + activates calmodulin-dependent kinases which phosphorylate several platelet proteins including myosin light chains (20 kDa). Protein kinase C also phosphorylates a number of substrates in platelets, the predominant of which has a molecular mass of 47 kDa but no known function (Tyers et al., 1988). Ca2+ and protein kinase C interact synergistically to induce secretion; however, little is known about the mechanism of this process or proteins involved (Seiss & Lapetina, 1988). In the present study, we have used the selective phosphatase inhibitor okadaic acid, a major toxic component in diarrhetic shellfish poisoning and a potent tumour promoter (Cohen, 1990), to probe the role of protein phosphatases 1 and 2A (PP1 and PP2A) in the activation of human platelets. Karaki et al. (1989) have reported that okadaic acid inhibits activation of rabbit platelets by thrombin, possibly by potentiation of adenosine 3':5'-cyclic monophosphate (cyclic AMP)-mediated events. In the present study, we show that okadaic acid inhibits activation of human platelets by thrombin through potentiation of protein kinase A- and protein kinase Cmediated inhibition of phosphoinositide hydrolysis.
Methods Platelets were obtained from two sources. Blood was either drawn on the day of the experiment from aspirin-free volunteers with sterile 20mm sodium citrate as anticoagulant and platelet-rich plasma obtained by centrifugation at 200g for 20 min, or platelet concentrates were obtained from the Blood Transfusion Service, John Radcliffe Hospital, and used within 20h of donation. Platelets were isolated from platelet-rich plasma or the platelet concentrate by centrifugation at 1000g for 10min in the presence of prostacyclin to prevent aggre1
Author for correspondence.
gation. Platelets were resuspended in 1 ml of modified Tyrodes-HEPES (composition in mM: NaCi 138, NaH2PO4 0.36, KCl 2.9, NaHCO3 12, HEPES 20, glucose 5, EGTA 1, MgCl2 1; pH 7.3) and labelled with [3H]-inositol (50OpCi ml- ' for 3 h), [32P]-orthophosphate (2 mCi ml'- for 1 h) or [3H]-5hydroxytryptamine ([3H]-5-HT, 5 1iCi ml-1 for 1 h). Platelets were then centrifuged in the presence of prostacyclin at 1000 g for 10 min and resuspended at a concentration of 28 x 108ml-1 in the above buffer, containing indomethacin (10puM) to inhibit cyclo-oxygenase. EGTA was omitted in studies of aggregation; LiCl (10mM), which inhibits conversion of inositol monophosphate to free inositol, was added to [3H]-inositol-labelled platelets. Platelets were left at least 30 min before experimentation. Platelet suspensions (0.49ml) were prewarmed at 37°C for 5 min before addition of compounds as appropriate. Experiments were stopped by transfer to 940pl of chloroform/ methanol/HCl (50:100:1) for analyses of inositol phosphates, to 0.5 ml of 6% (v/v) glutaraldehyde in phosphate buffer (pH 7.3) for analyses of 5-HT secretion, or to Laemmli buffer for analyses of protein phosphorylation. Inositol phosphates, protein phosphorylation and 5-HT secretion were measured as described previously (Nunn & Watson, 1987). To prepare membrane and cytosol fractions, platelets were resuspended in 3 vol of lysis buffer (10 mm Tris, 1 mm EDTA, 0.1% (v/v) ,i-mercaptoethanol, 1 mm EGTA, pH 7.4) frozen rapidly in liquid nitrogen and thawed at 37°C; this process was repeated and the final suspension cooled to 4°C. The suspension was then sonicated on ice for 4 x 15 s before centrifugation at 1000 g for 10 min. The supernatant was removed and stored at 4°C; the pellet was resuspended in 3 vol of lysis buffer and the lysis procedure repeated. When lysis of all platelets had been achieved, after which no pellet formed when the suspension was centrifuged at 1000 g for 10min. the pooled supernatants were centrifuged at 100,000g at 4°C for 1 h. The supernatant represents the cytosol fraction while the pellet, resuspended in the original volume of lysis buffer, the membrane fraction. Protein phosphatase assays were performed on both membrane and cytosol fractions as described by Cohen et al. (1989) with 32P-labelled phosphorylase a and casein as substrates. Okadaic acid and labelled substrates for phosphatase assays were kindly given by Professor P. Cohen, Dundee. [3H]-inositol (15.4 Ci mmol - 1), [32P]-orthophosphate (8500-
628
T.R. WALKER & S.P. WATSON
9120 Ci mmol -1) and [3H]-5-HT (21.0 Ci mmol - 1) were from N.E.N., Du Pont (U.K.) Ltd., Stevenage. Thrombin and phorbol dibutyrate were from Sigma Chemical Company Ltd., Poole. Prostacyclin was kindly donated by Wellcome Laboratories (Beckenham, Kent, U.K.) All other reagents were of Analytical grade.
a Time (min) I
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1
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20
.4250
.490 .476
Results
-a50
Protein phosphatase assays
., ,
The activities of PP1 and PP2A were assayed in both particulate and cytosolic fractions of human platelets by use of specific radiolabelled substrates. The use of a small heat-stable protein termed inhibitor-2 allowed selective inhibition of PP1 activity whilst okadaic acid (1 pM) inhibited both PP1 and PP2A activity; PP2C is unaffected by okadaic acid and has a requirement for Mg2+ (10mM). The distribution of PP1 in platelets was similar in both particulate and cytosol fractions (Table 1). The specific activities of PP2A and PP2C were found to be higher in the cytosolic fraction than the particulate fraction (Table 1), which is in contrast to rabbit skeletal muscle where almost all PP2A and PP2C activity is found in the cytosolic fraction (Ingebritsen et al., 1983).
V
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.424 .20 OA (Im) 00.10.3 1 0 0.1 0.3 1 0 0.10.31 00.10.31
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Phosphorylation studies
ev
10
Okadaic acid (1 pM) induced a general increase in phosphorylation of proteins over 60min (Figures 1 and 2) similar to that observed in other cell types e.g. rat hepatocytes and adipocytes (Haystead et al., 1989). The rate of phosphorylation varied greatly between proteins (Figure 2) while, in marked contrast, a protein of mol. wt. 24kDa was unaffected by okadaic acid (Figure 1). The threshold dose of okadaic acid was 0.3 JM (Figure la).
0.
I1 RM
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-
Action of okadaic acid on secretion and aggregation Okadaic acid (1 pM) did not induce aggregation or secretion of 5-HT from platelets despite the marked changes in protein phosphorylation which occur over 60 min. Preincubation with okadaic acid (1 M) for 10min, however, inhibited thrombininduced aggregation and secretion (Figures 3 and 4). This was particularly marked for low concentrations of thrombin, for example, okadaic acid reduced the secretion of 5-HT by 0.1 unit ml-1 thrombin from 38% to 4% (Figure 4).
Formation of inositol phosphates Okadaic acid exerted a dose- and time-dependent inhibition of thrombin-induced formation of total [3H]-inositol phosphates (Figure 5). Pre-incubation with 1 gM okadaic acid for 10 min inhibited thrombin-induced formation of inositol phosphates by approximately 56%. Moreover, significant inhibition was observed at the earliest time point monitored (5 s)
Increasing distance from origin Figure 1 (a) Effect of okadaic acid on protein phosphorylation. Platelets were prelabelled with 32P Pi and incubated with okadaic acid (1 pM) for varying times. Separation of proteins was achieved by gradient polyacrylamide gel electrophoresis (7.5%-15%), and autoradiography of the resulting gel indicates the extent of phosphorylation. Incubation time in the presence of okadaic acid (OA) is indicated at the top of the figure and the concentration indicated at the bottom. Relative molecular weights (in kDa) of distinct proteins are shown to the right of the figure. (b) Densitometric scan of basal protein phosphorylation (lower) and protein phosphorylation in the presence of okadaic acid (OA, 1 pM), pre-incubated for 10min (upper). Increased phosphorylation is expressed as an increase in optical density. Molecular weights of distinct proteins are indicated in kDa at the top of the figure.
following exposure to thrombin (Figure 5) making it likely that this effect contributes to the inhibition of thrombininduced aggregation and secretion by okadaic acid. Staurosporine, the non-selective kinase inhibitor (Tamaoki et al.,
Table 1 Protein phosphatase activity in human platelet cytosol and membrane fractions
PP1 PP2A PP2C
Cytosol
Particulate (mu mg-' protein)
(mu mg 1 protein)
Rabbit skeletal muscle (mu mg-' protein)
0.27 + 0.05 0.22 + 0.04 0.03 + 0.005
0.19 + 0.005 0.35 + 0.01 0.06 + 0.01
1.13 + 0.09 0.35 + 0.04 0.022 + 0.002
Activity is expressed as mean mu mg- I protein + s.e.mean from 6 experiments (one unit of activity is that amount of enzyme which catalyses the dephosphorylation of 1 pmol of substrate in 1 min). The activities of protein phosphatases 1 and 2A (PP1 and PP2A) were assayed with [32P]-phosphorylase a, PP1 can be selectively blocked with inhibitor-i whilst PP1 and PP2A can be blocked by okadaic acid (1 gM); assays were carried out as described in Methods. Casein is a substrate for both PP2A and PP2C therefore the assay for PP2C contained not only [32P]-casein but also okadaic acid (5yM) to block any PP2A activity and Mg2+ (10mM) as PP2C has a requirement for this divalent cation. Specific activity of phosphatases in rabbit skeletal muscle was reported by Ingebritsen et al. (1983), in this case expressed as mu mg- 1 protein.
OKADAIC ACID INHIBITS PLATELET ACTIVATION
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Time (min) Figure 2 Effect of okadaic acid on the rate of protein phosphorylation. Platelets were prepared as described for Figure la, and incubated with okadaic acid (1pM) for 10min. Results are expressed as c.p.m. (-) 25OkDa; (A) 5OkDa; (0) 76kDa; (0) 9OkDa. These symbols correspond to proteins indicated in Figure la.
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Figure 3 Effect of okadaic acid on thrombin-induced aggregation. A typical aggregation trace induced by a range of thrombin concentrations (0.06 units ml - '-l unit ml -) (a) was repeated in the presence of okadaic acid (1pM, 10 min pre-incubation) (b). This is representative of three other similar experiments. 80
60-
40) -0
5
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Figure 5 Effect of okadaic acid on thrombin-induced formation of inositol phosphates. (a) Dose-response curve for inhibition by okadaic acid. Platelets were prelabelled with [3H]-inositol and pre-incubated with okadaic acid (0.1-1 m, as indicated) for 10min. Platelets were then stimulated with a maximal dose of thrombin (1 unit ml-') for 5 min. Experiments were performed in triplicate and results are from 3 experiments and expressed as % of control inositol phosphate formation with s.e.mean shown by vertical bars. (b) Platelets were prelabelled with [3H]-inositol and pre-incubated with okadaic acid (1 gM) for 10min as indicated: (A) presence of okadaic acid; (El) absence of okadaic acid. Platelets were then stimulated with a maximal dose of thrombin (1 unit ml- 1) for varying times. Experiments were performed in triplicate and results are expressed as % inositol phosphate formation with s.e.mean shown by vertical bars; this is a representative experiment with similar results to two others. (c) Time-dependent effect of okadaic acid on thrombin-induced inositol phosphate formation. Platelets were prelabelled with [3H]-inositol and pre-incubated with okadaic acid (1 gM). Pre-incubation times with okadaic acid were as follows: lOs, 30s, min, 3 min, 10min and 20min. Platelets were then stimulated with a maximal dose of thrombin (1 unit ml-) for 5 min. The time scale relates to the total incubation time in the presence of okadaic acid. Experiments were performed in triplicate and results are expressed as % inositol phosphate formation with s.e.mean shown by vertical bars; this is a representative experiment with similar results to two others.
20-
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Thrombin (units ml-') Figure 4 Effect of okadaic acid on thrombin-induced 5hydroxytryptamine (5-HT) secretion. Platelets were prelabelled with [3H]-5-HT and stimulated with thrombin concentrations for min, in the presence (U) or absence (El) of okadaic acid (I P, 10min preincubation). Results are from 3 experiments and are shown as mean % 5-HT release with s.e.mean shown by vertical bars.
1986), inhibited the action of okadaic acid on thrombininduced formation of inositol phosphates (Table 2). This is consistent with the mechanism of action of okadaic acid being through potentiation of phosphorylation rather than acting as an antagonist at the thrombin receptor. Pretreatment of platelets with maximally effective concentrations of prostacyclin or phorbol dibutyrate reduced thrombin-induced formation of inositol phosphates to approximately 40% of control values in each case. In the presence of 1,UM okadaic acid, these responses were reduced to
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T.R. WALKER & S.P. WATSON
Table 2 Inhibition of thrombin-induced formation of total inositol phosphates d.p.m. Thrombin Thr + PGI2 Thr + PDBu Thr + OA Thr + PGI2 + OA Thr + PDBu + OA Thr + PGI2 + PDBu Thr + PGI2 + PDBu + OA Thr + staurosporine Thr + staurosporine + OA
1067 + 23 454 + 62 434 + 46 484 + 49 216 + 36 221 + 36 121 + 19 94 + 29 1116 + 48 1078 + 96
(%)
100 42.5 + 40.7 + 45.4 + 20.2 + 22.6 + 11.3 + 8.8 + 104.6 +
5.8 4.3 4.6 3.4 3.1
1.8 2.7 4.5 101 + 9.0
Results are expressed as d.p.m. or % + s.e.mean of maximal formation of inositol phosphates, induced by thrombin during 5 min stimulation (n > 4). Compounds are as follows: thrombin (Thr, 1 unitml-1), prostacyclin (PGI2) 2.5,pgml-', phorbol dibutyrate (PDBu) 300nM, okadaic acid (OA) 1,M staurosporine (3pM). Platelets were pre-incubated in the presence of okadaic acid for 10min prior to stimulation by
thrombin.
approximately 20% of controls (Table 2). The inhibitory actions of prostacyclin and phorbol dibutyrate were partially additive, reducing the formation of inositol phosphates to 110% of control values (Table 2). This was not significantly altered in the presence of 1 UM okadaic acid (P > 0.05; Table 2). These results suggest that prostacyclin and phorbol dibutyrate inhibit the activation of phospholipase C (PLC) by thrombin through predominantly different mechanisms and that okadaic acid may be able to mimic both of these actions.
Discussion Effect of okadaic acid
on
protein phosphorylation
Okadaic acid produces an increase in phosphorylation of a large number of platelet proteins which may reflect a net increase in activity of a single kinase or, more likely, a family
of protein kinases. The net increase in kinase activity could be brought about in two ways. The kinases may be intrinsically active and their action terminated by PP1 and/or PP2A or they may be activated by increased phosphorylation which is under regulation from PP1 and/or PP2A. Recently eleven novel serine/threonine protein kinases were identified in platelets, ten of which show increased activity following challenge with thrombin, possibly as a result of autophosphorylation (Ferrell & Martin, 1989). The general increase in phosphorylation may also reflect an alteration in the balance between kinases and phosphatases leading to increased activity of several kinases, the cascade being regulated at one or more points by PP1 and/or 2A. Action of okadaic acid
on
phospholipase C
It has been reported previously that okadaic acid mimics the inhibitory action of cyclic AMP-dependent protein kinase on vascular smooth muscle (Karaki et al., 1989). However okadaic acid did not alter the levels of cyclic AMP suggesting that it does not activate adenylyl cyclase or inhibit cyclic AMP phosphodiesterase. Karaki et al. (1989) proposed that inhibition of protein phosphatases by okadaic acid resulted in an increase in the effects caused by cyclic AMP-dependent protein kinase (protein kinase A). Prostacyclin mediates its inhibitory action on platelets through elevation of cyclic AMP and activation of protein kinase A (Seiss & Lapetina, 1989). One action of cyclic AMP is the inhibition of phosphoinositide hydrolysis following
receptor activation (Watson et al., 1984). Phorbol esters also inhibit formation of inositol phosphates by cell surface receptors and it has been postulated that this is a result of a negative feedback regulation by protein kinase C (Watson & Lapetina, 1985). The observation that the effect of phorbol dibutyrate is partly additive with that of prostacyclin suggests that they produce their effects by separate mechanisms. A number of classes of phospholipase C (PLC) have been identified in a variety of tissues, a, 6, y and 6 (Rhee et al., 1989); cDNA sequencing and immunoreactivity analyses suggest that subtypes of these classes exist (Wahl & Carpenter, 1991). Activation of protein kinase C has been reported to phosphorylate selectively the ai isoform of PLC in a number of cell lines as well as in bovine brain (Ryu et al., 1990). In contrast, protein kinase A selectively phosphorylates the y isoform of PLC in C6Bu1 cells (Kim et al., 1989). Phosphorylation of these isoforms of PLC has no apparent effect on their catalytic activity but may interfere with G protein interactions and therefore attenuate phosphoinositide hydrolysis in response to receptor activation (Kim et al., 1989; Ryu et al., 1990). Characterization of PLC enzymes in platelets has identified a number of subtypes, both cytosolic and membrane-bound (Baldassare et al., 1989; Banno et al., 1986; 1988; 1990). The relationship between platelet isozymes of PLC and those identified in other cell types has still to be established. Prostacyclin and phorbol ester may work at least, in part, by distinct mechanisms, possibly through selective phosphorylation of specific PLC isozymes. Alternatively cyclic AMP and protein kinase C may increase GTPase activity of the a subunit of the putative G protein Gp (Knight & Scrutton, 1987; Krishnamurthi et al., 1989). It has also been demonstrated that agents which raise cyclic AMP levels decrease the ability of thrombin to bind to its receptor, this correlates closely with the inhibition of thrombin-induced functional responses (Lerea et al., 1987). The observation that okadaic acid potentiates the effect of protein kinase A and protein kinase C but has no effect when both pathways are maximally activated suggests that the protein phosphatases PP1 and/or PP2A act to limit the actions of protein kinase A and protein kinase C on receptor-regulation of PLC.
Role of protein phosphatases in platelet activation The role of kinases in stimulus-secretion coupling has been extensively investigated (Hanks et al., 1988; Kikkawa et al., 1989; Taylor et al., 1990). More recently, attention has also focussed on the possible control of secretion by protein phosphatases. It has been reported that a platelet protein of 18 kDa molecular mass is dephosphorylated when platelets are stimulated with thrombin (Imaoka et al., 1983). It is known that dephosphorylation in some cell types is a critical event for cellular responses. For example, in Paramecium, rapid and marked dephosphorylation of a 65 kDa protein correlates temporally with synchronous exocytosis: both events peak within 1-3 s and are complete by 5 s (Zeiseniss & Plattner, 1985). The possibility that activation of a phosphatase may be a requirement in the initial stages of stimulus-secretion coupling has been suggested (Zeiseniss & Plattner, 1985). In platelets, little is known of the importance of this effect. It has been proposed that platelet protein phosphatases may be involved in down-regulation of platelet function (Sakon et al., 1990). However, we believe that the role of protein phosphatases is more complex, made evident by the ability of okadaic acid to attenuate functional responses in human platelets. Okadaic acid and other specific protein phosphatase inhibitors will allow investigation of the precise roles which PP1 and PP2A play in signal transduction. We are grateful to Professor P. Cohen for the gift of okadaic acid and advice with the phosphatase assays. T.R.W. is in receipt of a B.H.F. studentship and S.P.W. is a Royal Society University Research Fellow.
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FORS, P. & LIM TUNG, H.Y. (1989). Protein phosphatase-1 and protein phosphatase-2A from rabbit skeletal muscle. Methods Enzymol., 159, 390-508. FERRELL Jr., J.E. & MARTIN, G.S. (1989). Thrombin stimulates the activities of multiple previously unidentified protein kinases in platelets. J. Biol. Chem., 264, 20723-20729. HANKS, S.K., QUINN, A.M. & HUNTER, T. (1988). The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science, 241, 42-52. HAYSTEAD, T.A.J., SIM, A.T.R., CARLING, D., HONNOR, R.C., TSUKI-
TANI, T., COHEN, P. & HARDIE, D.G. (1989). Effects of the tumour promoter okadaic acid on intracellular protein phosphorylation and metabolism. Nature, 337, 78-81. IMAOKA, T., LYNHAM, JA. & HASLAM, R.J. (1983). Purification and characterization of the 47,000-dalton protein phosphorylated during degranulation of human platelets. J. Biol. Chem., 258, 11404-11414. INGEBRITSEN, T.S., STEWART, A.A. & COHEN, P. (1983). The protein phosphatases involved in cellular regulation. 6. Measurement of type-1 and type-2 protein phosphatases in extracts of mammalian tissues; an assessment of their physiological roles. Eur. J. Biochem., 132, 297-307. KARAKI, H., MITSUI, M., NAGASE, H., OZAKI, H. SHIBATA, S. & UEMURA, D. (1989). Inhibitory action of a toxic okadaic acid, iso-
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(1989). Effect of phorbol ester treatment on receptor-mediated versus G-protein-activator-mediated responses in platelets. Evidence for a two-site action of phorbol ester at the level of Gprotein function. Biochem. J., 262, 77-81. LEREA, K.M., GLOMSET, J.A. & KREBS, E.G. (1987). Agents that elevate cAMP levels in platelets decrease thrombin binding. J. Biol. Chem., 262, 282-288. NUNN, D.L. & WATSON, S.P. (1987). A diacylglycerol kinase inhibitor, R59022, potentiates secretion by and aggregation of thrombinstimulated human platelets. Biochem. J., 243, 809-813. RHEE, S.G., SUH, P.-G., RYU, S.-H. & LEE, S.Y. (1989). Studies of inositol phospholipid-specific phospholipase C. Science, 244, 546-550. RYU, S.-H., KIM, U.-H., WAHL, M.I., BROWN, A.B., CARPENTER, G.,
HUANG, K.-P. & RHEE, S.G. (1990). Feedback regulation of phospholipase C-beta by protein kinase C. J. Biol. Chem., 265, 1794117945. SAKON, M., KAMBAYASHI, J., KAJIWARA, Y., UEMURA, Y., SHIBA, E.,
KAWASAKI, T. & MORI, T. (1990). Platelet protein phosphatases and their endogenous substrates. Biochem. Int., 22, 149-161. SIESS, W. (1989). Molecular mechanisms of platelet activation. Physiol. Rev., 69, 58-178. SEISS, W. & LAPETINA, E.G. (1988). Ca2l mobilization primes protein kinase C in human platelets. Ca2+ and phorbol esters stimulate platelet aggregation and secretion synergistically through protein kinase C. Biochem. J., 255, 309-318. SEISS, W. & LAPETINA, E.G. (1989). Prostacyclin inhibits platelet aggregation induced by phorbol ester or Ca"+ ionophore at steps distal to activation of protein kinase C and Ca2"-dependent protein kinases. Biochem. J., 258, 57-65. TAMAOKI, T., NOMOTO, H., TAKAHASHI, I., KATO, Y., MORIMOTO, M. & TOMITA, F. (1986). Staurosporine, a potent inhibitor of phospholipid/Ca2" dependent protein kinase. Biochem. Biophys. Res. Commun., 135, 397-402. TAYLOR, S.S., BUECHLER, J.A. & YONEMOTO, W. (1990). cAMPdependent protein kinase: framework for a diverse family of regulatory enzymes. Annu. Rev. Biochem., 59, 971-1005. TYERS, M., RACHUBINSKI, R.A., STEWART, M.I., VARRIOCHIO, A.M.,
SHORR, R.G.L., HASLAM, R.J. & HARLEY, C.B. (1988). Molecular cloning and expression of the major protein kinase C substrate of platelets. Nature, 333, 470-473. WAHL, M. & CARPENTER, G. (1991). Selective phospholipase C activation. BioEssays, 13, 107-113. WATSON, S.P. & LAPETINA, E.G. (1985). 1,2-Diacylglycerol and phorbol ester inhibit agonist-induced formation of inositol phosphates in human platelets: possible implications for negative feedback regulation of inositol phospholipid hydrolysis. Proc. Nati. Acad. Sci. U.S.A., 82, 2623-2626. WATSON, S.P., McCONNELL, R.T. & LAPETINA, E.G. (1984). The rapid formation of inositol phosphates in human platelets by thrombin is inhibited by prostacyclin. J. Biol. Chem., 259, 13199-13203. ZIESENISS, E. & PLATTNER, H. (1985). Synchronous exocytosis in Paramecium cells involves very rapid (less than or equal to 1 s), reversible dephosphorylation of a 65-kD phosphoprotein in exocytosis-competent strains. J. Cell Biol., 101, 2028-2035.
(Received September 6, 1991 Revised November 5, 1991 Accepted November 8, 1991)
