Vol. 171, No. 2, 1990 September 14, 1990

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ELEVATION OF CAMP, BUT NOT cGMP, INHIBITS THROMBIN-STIMULATED TYROSINE PHOSPHORYLATION IN HUMAN PLATELETS

Kevin M. Pumiglia’*, Chi-Kuang Huang* and Maurice B. Feinsteid Departments of ‘Pharmacology andsPathology University of CT Health Center, Farmington,CT 06030 Received

July

30,

1990

Summary: Platelets have abundant tyrosine kinase activities, and activation of platelets results in the increased tyrosine phosphorylation of numerous protein substrates. The stimulation of tyrosine phosphorylation elicited by thrombin can be completely inhibited by preincubation with 1Omn prostacyclin (PGI,), bM PGD,, or 1mM N2,2’-0-dibutyryl-CAMP. In contrast,incubation of platelets with agents that increase cGMP (sodium nitroprusside or with 1mM 8-Bromo-cGMP) was without effect. The inhibition by prostacyclin was dose dependent, with an IC,, of approximately 3nM, corresponding to the dose range necessary to inhibit other platelet activation processes. These results demonstrate a novel pathway by which agents which raise CAMP may inhibit platelet signal transduction and differential mechanism of action between compounds which raise CAMP and those which elevate cGMP. 0 1990Academic Press,Inc.

In response to thrombin platelets rapidly undergo shape change, adhesion, secretion, aggregation and production of thromboxane A,. These cellular changes are triggered by the activation of phospholipases C and A,; the result being an increase in intracellular

calcium

concentrations, the activation of protein kinase C and the release of arachidonic acid from phospholipids (1) , Recently it has been demostrated that platelets contain high levels of the tyrosine kinases pp60c”,

pp59*,

and pp56*

relative to other cell types (2,3). It is now

well established that exposure of platelets to agonists such as thrombin, AVF”,

or the thromboxane

analog U46619l,

all result

collagen, PAF,

in an increase in platelet

phosphotyrosine (45). While the role of tyrosine phosphorylation

in signal transduction

in

* To whom correspondence should be addressed. CAMP: adenosine 3’5’qclic monophosphate; cGMP: guanosine 3’5’~cyclic monophosphate; EDRF: endothelium derived relaxing factor; Bt,-CAMP: ; 8-Br-cGMP: 8Bromo-cGMP; PIP,: phosphatidylinositol4,5,-bisphosphate; PAF: platelet activating factor; AVP: arginine vasopressin; EGTA: [ethylenebis(oxyethylene-nitrilo)] tetraacetic acid; HEPES: N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid; PIPES: piperazine-NJ?-b&(2ethanesulfonic acid. Abbreviations:

0006-291x/90 Copyright AN rights

$1.50

0 1990 by Academic Press, of reproduction in any form

Inc. reserved.

738

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the platelet remains largely unknown, it has been correlated with secretion, aggregation, phosphoinositide

hydrolysis and the activation of protein kinase C (5,6,7,8,).

It is well appreciated that products of the vascular endothelium,

namely PGI, and nitric

oxide (EDRF), can inhibit platelet function (9,10,11,) by elevating the levels of intracellular CAMP and cGMP repectively.

Elevation of cyclic nucleotide levels in human platelets has

been reported to inhibit PIP, hydrolysis, the rise in intracellular calcium, and the subsequent activation of protein kinase C (11,12,13). Sodium nitroprusside (SNP), a potent nitrodilator has been found to stimulate guanylate cyclase and inhibit platelet aggregation, in a fashion consistent with its use as an “exogenous” EDRF (14). As it has been suggested that tyrosine phosphorylation

may play an important role in platelet signal transduction, this study was

undertaken to determine if compounds which raise the intracellular concentration

of cyclic

nucleotides could affect tyrosine phosphorylation stimulated by thrombin. Materials

and Methods

Platelet Pretxzrution: Human platelet concentrates were obtained from the American Red Cross, Farmington CT., within 24 hours of collection. Contaminating blood cells were removed by centrifugation at 700xg for 45 seconds, prior to treatment with 200 CIM aspirin for 20 minutes to inactivate cyclooxygenase. Platelets were then pelleted by centrifugation at 700xg for 10 minutes followed by resuspension and washing in wash medium (145 mM NaCl, 5mM KCl, 5.5mM Dextrose, 0.2mM EGTA, 1OmM PIPES pH 6.5). Cells were pelleted as indicated above and resuspended in modified Tyrodes solution (145 mM NaCl, 5 mM KCl, 5.5 mM dextrose, 0.04 mM CaCl, 1mM MgCl, l0 mM HEPES, pH 7.4) at a final concentration of lX109 as determined by counting in a haemocytometer with phase contrast microscopy. $ Platelets (lo@ 1) in modified Tyrode’s buffer were incubated at 23°C. Reaction were quenched by adding an equal volume of stopping buffer consisting of 3% SDS, 5% P-mercaptoethanol, 10% glycerol, 60mM Tris-HCl (pH 6.8), and 1OmM EDTA followed by immediate heating in a boiling water bath for 3 min. Samples were then subjected to standard SDS-PAGE on 7.5% polyacrylamide gels, then electrotransfered (2 hours at 400 mAmps) onto nitrocellulose (Schleicher and Schuell) or Immobilon-P (Millipore) essentially as described by Towbin et al. (15), in a transfer buffer of 150mM glycine, 20mM Trizma base, 20% MeOH. Protein transfers were evaluated by staining with Ponceau Red, 0.075% in 5% acetic acid before immunoblotting. Platelet phosphotyrosine was analyzed by Western blot utilizing a polyclonal anti-phosphotyrosine antibody raised in rabbit as originally described by Ek and Heldin (16), which previously was well characterized as specific for phosphotyrosine (17,18). Nitrocellulose membranes were blocked with 5% BSA in a buffer of 155mM NaCl and 3OmM Tris-HCl (pH 7.4) before incubation with anti-phosphotyrosine antibodies (1:lOOO in 5% BSA). Excess antibody was washed away with 150mM NaCl and 9mM HEPES (pH 7.2) prior to detection of bound antibody with [rz?]-labeled protein A (bCi/rnl in 5% BSA). Blots were developed on Kodak X-Omat films at -70°C for 2-4 days. Nitrocellulose blots were overlayed with developed autoradiograms and the bands of interest excised and added to 5mls of Polyfluor scintillant cocktail (Packard) then counted in a Minaxi 4000 series scintillation counter (Packard) with a window setting of O-80 keV to detect bound [?I-Protein A. Protein bands 739

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at 65kd and 79kd were routinely assayed, as their high signal made for accurate excision, and their response to thrombin corresponded to that of other substrates, Matetiah: Bovine serum albumin fraction V, Tris-HCl, prostacyclin, prostaglandin D,, Bt,CAMP, S-br-cGMP, sodium nitoprusside and human thrombin (2000 Units/mg) were purchased from Sigma. Electrophoresis reagents were purchased from Bio-Rad, and HEPES was obtained from Calbiochem, all other reagents were of reagent grade and were obtained from J.T. Baker Biochemical. Protein A (9.8 pCi/pg) was purchased from New England Nuclear. Results

The anti-phosphotyrosine

antibody detected more than 20 tyrosine phosphorylated

substrates by Western blot analysis. Thrombin produced a rise in the phosphotyrosine in a number of substrates, most notably proteins with molecular weights of 128,118, 100,79,65 and 39 kDa, but also in a number of other bands of lesser prominence (Fig. 1) The bands detected correspond closely to those reported by Ferrell and Martin (4). As demonstrated in figures 1 and 2, incubation with 1OOnm PGI, for 2 minutes completely inhibited the increase in phosphotyrosine induced by thrombin. cyclase through a different

PGD, (GM), which activates adenylate

receptor than PGI, also completely inhibited the thrombin

stimulated tyrosine phosphorylation

(data not shown). In addition, treatment with 1mM Bb-

KDA

128 -

79 -

65 60 -

FIGURE 1. Stimulation of tyrosine phosphorylation and its inhibition by PGI, and Bt,Platelets were exposed to PGI, (1OOnM) or SNP CAMP, but not SNP or 8-Br-cGMP.

(IO&M) for 2 minutes or Bt,-CAMP (1mM) or 8-Br-cGMP (1mM) for 15 minutes before stimulation with lU/ml thrombin for 5 minutes. The figure shows an autoradiagram of proteins separated by SDS-PAGE, immunoblotted with anti-phosphotyrosine antibody and probed with [‘zI]-Protein-A as described in Methods. Similiar results were obtained in two other experiments. 740

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THR+ CAMP

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THR+ SNP

THR+ CCMP

FIGURE 2. Inhibition of thrombin stimulated tyrosine phosphorylation in pp65 by PGI, and Bt&iVIP. Platelets were stimulated with lU/ml thrombin alone or after pretreatment

with PGI, Bt,cAMP, SNP, or 8-Br-cGMP as described for Figure 1. pp65 was excisedfrom inununoblots and bound [‘?I-Protein-A counted as described in Methods. Basal c.p.m. was subtracted from all samples and the data are expressed as the % of the thrombin stimulated increase in c.p.m. This experiment was duplicated in two other platelet preparations.

CAMP resulted in nearly complete phosphotyrosine

inhibition

of the thrombin

(figures 1 and 2), indicating that the inhibition

in the intracellular

stimulated

increase in

is mediated by an increase

CAMP concentration.

SNP, a potent activator of platelet guanylate cyclase, and 8-Br cGMP, a cell permeable cGMP analog, have been reported to inhibit thrombin secretion, phosphoinositide SNP(lO@M)

stimulated platelet aggregation,

hydrolysis, calcium mobilization

and protein phosphorylation.

can completely inhibit the aggregation response to lU/ml

thrombin in non-

aspirinized platelets and elevate the intracellular cGMP concentration lo-fold (19). We also observed that lO@M SNP or 1mM 8-br-cGMP

strongly inhibited thrombin-stimulated

platelet aggregation (data not shown). However, in contrast to the agents which elevated CAMP, those that elevated cGMP (100 PM SNP or 1mM 8-Br cGMP) produced no significant inhibition

of the thrombin-stimulated

tyrosine phosphorylation

response (Figs. 1

and 2). Tyrosine phosphorylation

was increased by thrombin in a dose-dependent

a maximal increase in pp79 phosphotyrosine levels, attained at O.SU/rnl thrombin.

fashion, with

of 2.12 ( 2 0.12 SEM) times that of resting

Preincubation of platelets with lO@M SNP had very

little effect on the dose-reponse to thrombin (Fig. 3B). In contrast, preincubation with 1OnM PGI, resulted in over 90% inhibition

at concentrations up to lU/ml

The dose-response for inhibition of thrombin-induced

thrombin

tyrosine phosphorylation

examined. As demonstrated in figure 4, PGI, inhibited tyrosine phosphorylation 741

(Fig. 3A). by PGI, was produced

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-

0.8 0.4

0.6

U/ml

.r” .z

2.8

0.8

1.0

1.2

Thrombin

-

%

0.0

0.2

0.4

U/ml

0.6

1 .o

0.8

Thrombin

FIGURE 3. Dose-response for thrombin-induced tyrosine phosphorylation of pp79: inhibition by PGI, but not SNP. Platelets were incubated with various doses of thrombin for 5 minutes, either untreated (0) or after 2 minutes pretreatment with 1OnM PGI, (A, 0) or 10% M SNP ( B, 0) . Proteins were separated and irnmunoblotted as in Figure 1. The pp79 band was cut out and counted as described, and c.p.m. were normalized to the level in non-stimulated platelets. This data is typical of two other experiments.

by 1 U/ml thrombin with an IC,, of approximately 3nM, with complete inhibition PGI,.

This concentration

by 1OnM

range corresponds well to the range of concentrations of PGI,

necessary to inhibit calcium mobilization

and DAG production

in response to lU/ml

thrombin (11). Sodium nitroprusside, at concentrations up to 5O@M, was without effect on tyrosine phosphorylation

stimulated by lU/ml

thrombin.

It is interesting to note that

exposure of platelets to either PGIz or SNP had very little effect on the basal levels of phosphotyrosine. Discussion

It is not yet understood where in the signal transduction

cascade in platelets the

activation of tyrosine phosphorylation takes place. Recent findings in PAP stimulated rabbit platelets suggest that tyrosine phosphorylation 742

is stimulated

prior to the activation

of

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1

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!

100

1000

FIGURE 4. Dose-response for PGI, inhibition of thrombin-stimulated tyrosine phosphorylation of pp79. Platelets were pretreated with various dosesof PGI, for 2 minutes

prior to stimulation with lU/rnl thrombin for 5 minutes. Samples were processed and quantitated as indicated in Methods, using pp79 as an indicator of the tyrosine phosphorylation. Data is expressedas % of the thrombin-stimulated rise in phosphotyrosine. Similiar results were obtained in a duplicate experiment.

phospholipase C, and is neccesssary for the activation of that enzyme (7,8). However our evidence in human platelets argues against this hypothesis. We find that agents which act distal to phospholipase

C, such as the calcium ionophore

stimulate tyrosine phosphorylation

A23187 and phorbol

to a degree comparable to that of thrombin’.

esters,

In addition

we find that tyrphostins, (synthetic inhibitors of certain tyrosine kinases)(20), completely inhibit thrombin-stimulated by thrombin

tyrosine phosphorylation,

but have no effect on PIP, hydrolysis

in human platelets (Pumiglia and Feinstein, unpublished

indicate that enhanced tyrosine phosphorylation phospholipase C.

data). Our data

may be secondary to the activation of

Hence, CAMP may inhibit tyrosine phosphorylation

ability to block the activation of phospholipase

by its well known

C and the subsequent mobilization

of

calcium and activation of protein kinase C. Alternatively

CAMP may directly modulate the activity of platelet tyrosine kinases or

phosphatases. However we observed no significant inhibition tyrosine phosphorylation, phosphorylation.

in contrast tyrphostins

significantly

of the substantial resting decrease basal tyrosine

Ferrell and Martin (4) also reported that PGE, produced no changes in

tyrosine phosphorylation,

[32P]phosphotyrosine

content

or pp60”S’c phosphorylation

in

unstimufated platelets, but they did not extend their study to the effect of PGE, on agonist-

stimulated tyrosine phosphorylation

(4). In addition, we find that agents which raise CAMP

r Banga, H.S., Pumiglia, KM., Lau, L.F., Huang, C-K., Casenellie, J. and Feinstein, M.B. (1990),

Manuscript in preparation. 743

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stimulated by A23187 or PMA (Pumiglia

and

Feinstein, unpublished data). These data argue against a direct effect on the participating kinase(s) and/or phosphatase(s), though this has not yet been conclusively ruled out. Compounds which raise intracellular cGMP, such as SNP or 8-Br-cGMP have also been reported to inhibit

phosphoinositide

hydrolysis and calcium mobilization

in intact platelets

(12,13). Recently it has been reported that cGMP affects the phospholipase preferentially,

being in fact a relatively weak inhibitor of phospholipase

Earlier studies showing inhibition by cGMP of phosphoinositide

A, pathway

C activation (21).

hydrolysis utilized platelets

that were not aspirinized (12,13), thus the inhibition of phospholipase C by cGMP in those cases may have been indirect, predominately due to the block of thromboxane

4

production

by cGMP (21). In our study platelets were routinely aspirinized to eliminate stimulation of phospholipase

C by the thrombin-induced

generation

of thromboxane

A,. Under these

conditions, SNP and 8-Br-cGMP were not capable of inhibiting the direct effect of thrombin on tyrosine phosphorylation. In conclusion, this paper presents the first evidence that agonist-stimulated phosphorylation

tyrosine

in the platelet can be blocked by agents that elevate CAMP but not cGMP.

As endothelium-derived

PGI,

of tyrosine phosphorylation

is the most potent platelet antagonist known, its inhibition

may be an important and novel mechanism for regulating certain

platelet functions. Further study of the mechanism of action of CAMP will be important to understanding the mechanism of regulation of tyrosine phosphorylation

in platelets, and its

role in signal transduction. This work was supported by NIH grants HL18937 (MBF) and AI-20943 CKH is the recipient of an American Heart Association Established Investigator

Acknowledgments

(CKH). Award.

References

1. Kroll, M.H., and Shafer, A.I. (1989) Blood 74, 1181-1195. 2. Golden, A. and Brugge, J.S. (1989) Proc. Natl. Acad. Sci. USA 86, 901-905. 3. Horak, I.D., Cocoran, M.L., Thompson, PA, Wahl, L.M., and Bolen, J.D. (1990) Oncogene 5,597-602. 4. Ferrell, J.E. and Martin, G.S. (1988) Mol. and Cell Bio. 8, 3603-3608. 5. Nakamura, S-I. and Yamamura, H. (1989) J. Biol. Chem. 264, 7089-7091. 6. Lerea, K.M., Tonks, N.K, Krebs, E.G., Fisher, E.H., and Glomset, J.A. (1989) Biochemistry 28, 9286-9292. 7. Dhar, A., Paul, AK, and Shukla, S.D. (1990) Molecular Pharmacology 37~519-525. 8. Salari, S., Duronio. V., Howard, S.L., Demos, M., Jones, K., Reany, A, Hudson, AT., and Pelech, S.L. (1990) FEBS L&t. 263, 104-108. 9. Moncada, S., Gryglewski, R., Bunting, S., and Vane J.R. (1976) Nature 263:663-665. 744

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10. Azuma, A., Ishikawa, M., and Sekizaki, S. (1986), 88, 411-415. 11. Zavoico, G.B., Halenda, S.P., Chester, D., and Feinstein, M.B. (1985) In Prostaglandins, Leukotrienes, and Lipoxins (Bailey ed.) pp 345-356 Plenum Publishing Corp., 12. Nakashima, S., Tohmatsu, T., Hattori, H., Okanao, Y., and Nowzawa, Y. (1986) Biochem. Biophys. Res. Connn. 135, 1099-1104. 13. Takai, Y., Kaibuchi, K., Matsubara, T. and Nishizuka, Y. (1981) Biochem. Biophys. Res. Comm. 101, 61-67. 14. Gerzer, R., Karrenbrock, B., Seiss, W., and Heim, J-M. (1988) Thromb. Res. 52, 11-21. 15. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76, 4350-4354. 16. Ek, B., and Heldin, C-H. (1984) J. Biol. Chem. 259, 11145-11152. 17. Gomez-Cambronero, J., Yamazaki, M., Metwally, F., Molski, T.F.P., Bonak, V.A., Huang, C-K, Becker, E.L., and Sha’afi, R.I. (1989) Proc. Natl. Acad. Sci. USA 86, 3569-3573. 18. Huang, C-K, Iaramee, G.R. and Casnelhe, J. (1988) Biochem. Biophys. Res. Comm. 151, 794-801. 19. Harbrugge, M., Friedrich, C., Eigenthaler, M., Schanzenbacher, P., and Walter, U. (1990) J. Biol. Chem. 265, 3088-3093. 20. Yaish, P., Gazit, A., Gilon,C., and Levitski,k (1988) Science 242, 933-935. 21. Sane, D.C., Bielawska, A, Greenburg, C.S., and Hunan, Y.A. (1989) Biochem. Biophys. Res. Comm. 165, 708-714.

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