Inhibition by sodium nitroprusside or PGE1 of tyrosine phosphorylation induced in platelets by thrombin or ADP.

Inhibition by sodium nitroprusside or PGE, of tyrosine phosphorylation induced in platelets by thrombin or ADP ATSUSHI

ODA, BRIAN

J. DRUKER,

MARIANNE

SMITH,

AND

EDWIN

W. SALZMAN

Department of Surgery, Beth Israel Hospital, Boston 02215; and Division of Cellular Molecular Biology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115 Oda, Atsushi, Brian J. Druker, Marianne Smith, and Edwin W. Salzman. Inhibition by sodium nitroprusside or

PGE, of tyrosine phosphorylation induced in platelets by thrombin or ADP. Am. J. Physiol. 262 (Cell Physiol. 31): C701C707, 1992.-Upon platelet activation, numerousproteins are known to be tyrosine phosphorylated.To investigate the mechanismsof the regulation of tyrosine phosphorylation and its physiological significance, the effects on tyrosine phosphorylation of agents that elevate the platelet level of the cyclic nucleotidesCAMP and cGMP were examinedin aspirin-treated gel-filtered platelets by Western blotting with a specific antiphosphotyrosineantibody. The effects of theseagentson other aspectsof platelet activation, i.e., aggregation, secretion, and elevation of the concentration of cytosolic ionized calcium ([Ca’+]i), were alsoexamined in parallel experiments. Tyrosine phosphorylation in platelets activated by cu-thrombin (1 nM) was inhibited by prostaglandin (PG) E, (2 PM) or by sodium nitroprusside (100 PM). Elevation of [Ca’+]i, aggregation,and serotonin secretion was also strongly inhibited. On the other hand, a higher concentration of cw-thrombin(10 nM) induced tyrosine phosphorylation of the sameproteins, elevation of [Ca*+]i,platelet aggregation,and serotonin secretion, irrespective of pretreatment of platelets by either PGE, or sodium nitroprusside. Inhibition by sodium nitroprusside of tyrosine phosphorylation induced by cu-thrombin (1 nM) was accompanied by an increasedconcentration of cGMP. &BrcGMP (2 mM) also inhibited tyrosine phosphorylation and aggregation, although lessthan sodiumnitroprusside.ADP (20 PM) induced platelet shapechange and tyrosine phosphorylation of only a few proteins; these effects were also inhibited by either PGE, or sodium nitroprusside. Thus tyrosine phosphorylation in platelets can be inhibited by elevation of either CAMP or cGMP, an effect that is overcome by a high concentration of thrombin, resulting in granule secretion and aggregation.Some of the proteins that are tyrosine phosphorylated may be important in the regulation of platelet functions. calcium; guaninenucleotide-binding protein; pp6Osrc

cGMP-dependent kinases share some substrates in platelets (28, 30-32), suggesting the existence of pathways common to CAMP and cGMP in the regulation of platelet functions. However, Pumiglia et al. (23) recently reported that cGMP-elevating agents like sodium nitroprusside or 8bromoguanosine 3’,5’-cyclic monophosphate (8BrcGMP) inhibited thrombin-induced platelet aggregation but not tyrosine phosphorylation, whereas CAMPelevating agents like prostaglandin (PG) I2 or dibutyryl 3’,5’-cyclic monophosphate (DBcAMP) adenosine strongly inhibited both platelet aggregation and tyrosine phosphorylation (23). These issues led us to address the possible inhibition of tyrosine phosphorylation by agents that elevate cGMP. We found that tyrosine phosphorylation induced by cu-thrombin (1 nM) or by ADP (20 PM) was inhibited by sodium nitroprusside, which was previously shown to elevate cGMP but not CAMP within platelets (19, 32) and that [ Ca2+]i elevation, granule secretion, and aggregation were also inhibited. MATERIALS AND METHODS

Materials. cu-Thrombin was a kind gift of Drs. John W. Fenton, Jr. (New York Department of Health, New York, NY) and John M. Maraganore (Biogen, Boston and Cambridge, MA). [‘*C]serotonin was from New England Nuclear (Boston, MA). Fura- acetoxymethyl ester (AM) was from Molecular Probes(Eugene,OR). PGE, wasfrom Biomol (Plymouth Meeting, PA). Aspirin, apyrase(type VIII), ADP, dimethyl sulfoxide (DMSO), N-2-hydroxyethylpiperazine-N’-2-ethanesulfonicacid (HEPES), sodium dodecyl sulfate (SDS), 2-mercaptoethanol, sodiumorthovanadate,tris(hydroxymethyl)aminomethane(Tris), hirudin, and sodiumnitroprusside were from Sigma (St. Louis, MO). Sepharose2B was from Pharmacia (Uppsala, Sweden). Nitrocellulose membrane (pore size, 0.2 pm) was from FOLLOWING ACTIVATION by thrombin or ADP, platelets & Schuell (Keene, NH). Gelatin and SDS-polyacrylundergo shape changes, secretion, and aggregation (28). Schleicher amide gel electrophoresis (PAGE) molecular standards were These changes are accompanied by activation of phos- from Bio-Rad (Richmond, CA) or from Amersham (Arlington pholipase C, phospholipase AZ, protein kinase C, and Heights, IL). Alkaline phosphatase (ALP)-conjugated antielevation of the cytosolic concentration of ionized cal- mouseimmunoglobulin (Ig) G, nitro blue tetrazolium (NBT), cium ([Ca”+]i) (21, 28). and 5-bromo-4-chloro-3-indoyl phosphate (BCIP) were from In addition, numerous proteins become phosphorylPromega (Madison, WI). Antiphosphotyrosine murine monoated on tyrosine residues (3-5, 7, 8, 10, 20, 23, 25) clonal antibody (4GlO) has beenpreviously described(13, 14). Platelet preparation. Human blood was drawn by venipuncfollowing thrombin or ADP stimulation. The physiologture into 1:lO volume of 3.8% (wt/vol) trisodium citrate and ical significance of tyrosine phosphorylation is unknown, but the unusually high levels of tyrosine kinases in gently mixed. Platelet-rich plasma (PRP) was prepared by centrifuging the whole blood at 200g for 20 min and aspirating platelets (9, 11) suggest the possibility of an important PRP to no lessthan 2 cm abovebuffy coat. PRP wasincubated physiological role. with aspirin (2 mM) for 30 min at room temperature. PGE, (1 Activation of phospholipase C, elevation of [ Ca2+]i, PM) was added from a stock of absoluteethanol (1 mM). The activation of protein kinase C, aggregation, and secretion PRP wasspun at 800g to form a soft platelet pellet. The pellet can be inhibited by either adenosine 3’,5’-cyclic monowas resuspendedin 1 ml of a modified HEPES-Tyrode buffer phosphate (CAMP) or guanosine 3’,5’-cyclic monophos[(in mM) 129 NaCl, 8.9 NaHC03, 0.8 KH2P04, 0.8 MgCl,, 5.6 phate (cGMP) (2, 6, 16, 17, 19, 22, 26-32). CAMP- and dextrose, and 10 HEPES, pH 7.41also containing apyrase(2 0363-6143/92 $2.00 Copyright 0 1992 the AmericanPhysiological Society c701 Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (134.129.182.074) on November 22, 2018. Copyright © 1992 the American Physiological Society. All rights reserved.

C702

TYROSINE

PHOSPHORYLATION

AND

U/ml) and hirudin (0.1 U/ml). To make gel-filtered platelets, the platelet suspension was then layered onto a Sepharose 2B gel column (9 ml bed volume) preequilibrated with a modified HEPES-Tyrode buffer containing 1 mM CaCl,. Platelet aggregation. Gel-filtered platelets were adjusted to a concentration of 3 x lO’/ml in a modified HEPES-Tyrode buffer containing 1 mM CaCl,. Aggregation and shape change were monitored at 37°C at a continuous stirring rate of 1,000 rpm by a lumiaggregometer (Chronolog, Harvertown, PA). Serotonin secretion. To measure serotonin release, PRP (15 ml) was incubated with aspirin (2 mM) and 2 &i [‘*C]serotonin. After 30 min at room temperature, PRP was gel-filtered as described above. At indicated times, secretion was stopped by the addition of an equal volume of ice-cold 2% paraformaldehyde containing 200 mM ethylene glycol-bis(P-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA). After centrifugation (8,000 g, 5 min), an aliquot of the supernatant was taken for scintillation counting. Secreted serotonin was expressed as a percentage of the total platelet-associated [‘*C]serotonin. &orimetric measurement of [ca2+], by fura-2. [Ca”+], was measured as described previously (33) by a SPEX spectrofluorimeter (Fluolog-2, Edison, NJ). Fluorescence was monitored with continuous stirring of platelet suspensions (3 X 10’ cells/ ml). Gel electrophoresis and Western blotting to detect tyrosine phosphorylation. Platelet stimulation was terminated by the addition of an equal volume of two times concentrated Laemmli’s sample buffer (15) with 2-mercaptoethanol, 10 mM EGTA, and 1 mM sodium orthovanadate. After boiling at 95°C for 5 min, one-dimensional SDS electrophoresis was carried out with 10% polyacrylamide gel as described (15). Separated proteins were electrophoretically transferred from the gel onto nitrocellulose membrane in a buffer containing Tris (25 mM), glycine (192 mM), and 20% methanol at 0.2 A for 6 h at room temperature. To block residual protein binding sites, nitrocellose was incubated in Tris-buffered saline (TBS) [(in mM) 10 Tris, 150 NaCl, pH 8.01 containing 2% gelatin. The blots were washed with TBS with 0.05% Tween 20 (TBST) and incubated overnight with antiphosphotyrosine monoclonal murine antibody (13, 14) at a final concentration of 1.5 pg/ ml in TBST. The primary antibody was removed and the blots were washed four times in TBST. To detect antibody reactions, the blots were incubated with ALP-conjugated second antibody diluted 1:2,000 in TBST, washed four times in TBST, and placed in a buffer [(in mM) 100 Tris, 100 NaCl, 5 MgCl,, pH 9.51. Enzymatic color development was stopped by rinsing the filters in distilled water. The portion of nitrocellulose that contained the molecular standards was cut before blocking and stained with Amido Black. cGMP measurement by radioimmunoassay (RIA). Platelet aliquots were treated with trichloroacetic acid (TCA, final concentration 10%). After centrifugation (3,000 g) to remove protein, TCA was extracted by water-saturated ethyl ether (5x). The samples were evaporated to dryness under Nz. After reconstitution, RIA was conducted with a commercially available kit (New England Nuclear, Boston, MA). RESULTS

We investigated changes in protein tyrosine phosphorylation in platelets that were detected by Western blotting with a specific antiphosphotyrosine antibody. Aspirin-pretreated gel-filtered platelets [3 x 10’ cells/ ml, in a modified HEPES-Tyrode buffer containing CaCls (1 mM)] were stimulated by cu-thrombin (10 or 1 nM) with continuous stirring, and, at indicated times, aliquots of platelets that contained 2 x lo7 cells were

CYCLIC

NUCLEOTIDES

IN

PLATELETS

collected, and the pattern of tyrosine phosphorylation was analyzed (Fig. 1, A and B). The molecular mass of the most prominent band (60 kDa) corresponded to that

of pp6Osrc, which is known to be tyrosine phosphorylated under resting conditions (3-5, 7, 8, 20, 23, 25). Three proteins (34, 36, and 70 kDa) increased in the extent of their phosphorylation within 15 s but then became dephosphorylated. Proteins of relative molecular mass 50, 62,80,105,130,

and 145 kDa also rapidly

increased their

extent of tyrosine phosphorylation, but the level of phosphorylation was sustained. Three other proteins (40,100, and 115 kDa) were slowly phosphorylated over the time course of the experiments. There were also numerous other minor proteins that were tyrosine phosphorylated. As expected, treatment of platelets with a-thrombin (10 or 1 nM) resulted in aggregation (Fig. 2A), secretion (Fig. 2B), and elevation of [Ca’+]i (Fig. 3, A and B). We next investigated the effects of agents that elevate

A

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Fig. 1. Changes of tyrosine phosphorylation in platelets stimulated by cu-thrombin. a-Thrombin [lo nM (A) or 1 nM (B)] was added to a platelet suspension at time 0. Aliquots of platelet suspension were taken at 15 s, 1 min, and 2 min after addition of thrombin. Proteins were analyzed by 10% SDS-PAGE and subsequent Western blotting with a specific antiphosphotyrosine antibody. The positions of molecular markers are shown on the right. Lane 1 (for both A and B): tine 0 with thrombin alone; lanes 24: 15 s, 1 min, and 2 min after addition of thrombin alone, respectively; lane 5: 1 min after addition of sodium nitroprusside (100 1M); lanes 6-8: 15 s, 1 min, and 2 min after addition of thrombin to sodium nitroprusside-pretreated platelet suspensions, respectively; lane 9: 1 min after addition of PGEl (2 PM); lanes 10-12: 15 s, 1 min, and 2 min after addition of thrombin to PGE1-pretreated platelet suspensions, respectively. Representative of 8 different experiments with blood from different donors. PGEl (2 PM) or sodium nitroprusside (100 PM) was added 1 min before addition of thrombin.

Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (134.129.182.074) on November 22, 2018. Copyright © 1992 the American Physiological Society. All rights reserved.

TYROSINE

PHOSPHORYLATION Thrombin PGEl SNP

10

AND CYCLIC

nM

+ Thrombin + Thrombin

NUCLEOTIDES

alone

/“”

1 0 nM 10 nM

c703

IN PLATELETS

Thrombin

1 nM

80bromo cGMP + Thrombin 1 nM

PGEl + Thrombin SNP + Thrombin

t

Thrombin

10

t

nM

1 min

1 nM 1 nM

1 nM 1 min

Thrombin Thrombin Thrombin Thrombin Thrombin Thrombin

Time

Thrombin

alone

10 nM 10 nM +SNP 10 nM + El 1 nM 1 nM + SNP 1nM + El

Fig. 2. Aggregation or secretion induced by cu-thrombin. cu-Thrombin [ 10 nM (A) or 1 nM (B)] was added at time indicated by arrows. PGE, (2 PM) or sodium nitroprusside (SNP; 100 PM) was added 1 min before addition of thrombin. 8-BrcGMP was added 15 min before thrombin in A. Representative of 10 independent experiments with blood from different donors. C: [14C]serotonin secretion induced by a!-thrombin. Abscissa represents time after addition of a!-thrombin. PGEl or sodium nitroprusside was added as described in Fig. 1. Mean of 3 independent experiments is shown.

(min)

intracellular levels of CAMP or cGMP. Pretreatment of platelets with either sodium nitroprusside (100 PM) or PGE1 (2 PM) had virtually no effect on tyrosine phosphorylation by ar-thrombin (10 nM) (Fig. lA). The inhibition of aggregation (Fig. 2A) or secretion (Fig. 2C) by these agents was small. Calcium elevation was inhibited but was still higher than that induced by cu-thrombin (1 nM) alone (Fig. 3, A and B). However, the effect of CYthrombin (1 nM) on tyrosine phosphorylation was strongly inhibited by either sodium nitroprusside (100 PM) or PGE, (2 PM) (Fig. 1B). In some experiments, slow phosphorylation (5) of a 130.kDa protein occurred with ar-thrombin (1 nM), after treatment with sodium nitroprusside (100 PM), but not PGEl (2 PM). In parallel experiments, aggregation (Fig. 2B) and secretion (Fig. 2C) were abolished. Calcium elevation (Fig. 3B) was inhibited. Because the inhibitory effect of sodium nitroprusside was surprising in view of the previous report of Pumiglia et al. (23), we examined the dose response of sodium nitroprusside. Tyrosine phosphorylation induced by thrombin (1 nM, 1 min) was not inhibited by sodium nitroprusside (10 nM). However, from 100 nM to 100 PM sodium nitroprusside, tyrosine phosphorylation of several proteins was inhibited dose dependently (Fig.

4A). This observation is in agreement with the dose range of sodium nitroprusside that elevates cGMP (Fig. 4B). It is also consistent with the inhibitory effects on dense granule secretion (Fig. 4C). Sodium nitroprusside (10 and 100 PM) had a similar inhibitory effect on tyrosine phosphorylation and secretion. 8-BrcGMP inhibited tyrosine phosphorylation of 115 kDa protein induced by cu-thrombin (1 nM), but the effect was weak and only detectable in the early stages (15 s-l min) (Fig. 5). Inhibition of phosphorylation of 50- and 145-kDa protein was detected throughout the time course of the experiment (Fig. 5). 8-BrcGMP (2 mM) delayed aggregation by cu-thrombin (1 nM) (Fig. 2B) but had no effect on platelet aggregation induced by cu-thrombin (10 nM) (data not shown), and dense granule secretion was not inhibited 30 s, 1 min, or 2 min after stimulation (data not shown). As expected, 8-BrcGMP (2 mM, 15 min) had no effect on tyrosine phosphorylation induced by cu-thrombin (10 nM) (data not shown). ADP (20 PM) induced tyrosine phosphorylation in a few proteins (40, 62, and 130 kDa) (Fig. 6). The phosphorylation was inhibited by either sodium nitroprusside (100 PM) or PGEl (2 PM). Similarly, platelet shape change (Fig. 7A ) or elevation of [ Ca2+]i (Fig. 7B) induced by


TYROSINE

c704

PHOSPHORYLATION

AND CYCLIC

NUCLEOTIDES

IN PLATELETS

Thrombin alone

2.5 Thrombin

E + El + SNP

t

Thrombin

10

t

nM

Thrombin

1 min

1 nM

1 min

Fig. 3. Cytosolic ionized calcium ([Ca”]J in thrombin-stimulated platelets loaded with fura-2. cy-Thrombin [lo nM (A) or 1 nM (B)] was added at time indicated by arrows. Abscissa represents time. Ordinate shows ratios of emission (505 nm) of fura- (alternately stimulated by 340 and 380 nm), which is a measure of [Ca*+]i in platelets in suspension. PGE, or sodium nitroprusside (SNP) was added as described in Fig. 1. Representative of 3 independent experiments.

kDa 200

97 69 46

ii oz

t Thrombin stimulated (1 nM, lmin)

Basal

’

Log Molar

concentration

Log Molar SNP concentration Fig. 4. Dose response of sodium nitroprusside (SNP) on tyrosine phosphorylation, cGMP accumulation, and inhibition of dense granule secretion, A: platelets were stimulated by &hrombin (1 nM) for 1 min. After lysis, proteins were analyzed by Western blotting with antiphosphotyrosine antibody. Dose-dependent inhibition of tryrosine phosphorylation by SNP was not limited to a particular protein. B: SNP dose dependently elevated cGMP levels in platelets. SNP elevated cGMP significantly (by paired t test; P < 0.05) from 100 nM in a dose-dependent manner. Results were from 3 experiments with different batches of platelets. C: [i4C]serotonin secretion induced by cY-thrombin (1 nM, 2 min) was inhibited by SNP dose dependently. Secretion measured with the addition of vehicle (distilled water) is 100%. Mean of 2 independent experiments is shown. SNP

ADP (20 PM) was inhibited by sodium nitroprusside (100 PM) or PGEi (2 PM). Under these conditions, ADP did not induce secretion (28). DISCUSSION

The present study confirms and extends previous reports of tyrosine phosphorylation during platelet activation (3-5, 7, 8, 20, 23, 25). The apparent molecular mass of tyrosine phosphorylated proteins induced by thrombin is largely in agreement with those reported by others. Subtle differences may be due to the use of different antibodies and differences in platelet preparation (7, 8). Inhibition of tyrosine phosphorylation by

Concentration

PGEi, presumably through elevation of CAMP concentration, is also consistent with previous reports (5,823). However, we found that cGMP-elevating agents sodium nitroprusside and 8-BrcGMP also inhibited tyrosine phosphorylation in platelets, suggesting a physiological role for cGMP in the regulation of tyrosine phosphorylation. The inhibitory effects were observed in the dose range of sodium nitroprusside that induces cGMP elevation. Furthermore, 8-BrcGMP inhibited tyrosine phosphorylation induced by &hrombin (1 nM). The weakness of its inhibitory effect was probably due to the inefficient entrance of this water-soluble cGMP analogue into the platelets. In contrast, 10 or 100 PM sodium nitroprusside, which maximally inhibited tyrosine phos-


TYROSINE

PHOSPHORYLATION

AND CYCLIC

NUCLEOTIDES

IN PLATELETS

c705

kDa -

200

-

116 07

-

E E t I-

.

-

E CI ?

46

t ADP

20

ADP alone POE1 + ADP SNP + ADP

uM

1 min

B 12

3

4

56

70

Fig. 5. Tyrosine phosphorylation in platelets stimulated by cY-thrombin (1 nM) with or without %BrcGMP pretreatment (2 mM, 15 min). Platelet suspensions were incubated for 15 min before addition of (Ythrombin (1 nM). Control cells were pretreated with distilled water for 15 min (vehicle for 8-BrcGMP). Tyrosine phosphorylation was examined as described in Fig. 1 and MATERIALS AND METHODS. Lane 1: time 0 for control platelets; lanes 2-4: 15 s, 1 min, and 2 min after addition of thrombin to control cell suspension, respectively; lane 5: 15 min after 8-BrcGMP treatment; lanes 6-8: 15 s, 1 min, and 2 min after addition of thrombin to the cGMP-pretreated platelet suspension, respectively. Representative of 3 independent experiments. Positions of molecular markers are identified on right. Molecular mass of the proteins whose phosphorylation was inhibited is indicated by the arrows on left.

kDa 200

97

-

66

-

45

-

31

62

40

2

3

456

76

9

10

11

N e I=

t

ADP

20 uM

1 min

116

1

ADP alone + SNP + El

12

Fig. 6. ADP (20 NM)-induced changes in tyrosine phosphorylation. Sodium nitroprusside (100 FM) or PGEl (2 PM) was added as described in Fig. 1. Lane 1: time 0 with ADP alone; lanes 2-4: 15 s, 1 min, and 2 min after addition of ADP alone, respectively; lane 5: 1 min after addition of sodium nitroprusside (100 PM); lanes 6-8: 15 s, 1 min, and 2 min after addition of ADP to sodium nitroprusside-pretreated platelet suspensions, respectively; lane 9: 1 min after addition of PGEi (2 pM); lanes 10-12: 15 s, 1 min, and 2 min after addition of ADP to PGEipretreated platelet suspensions, respectively. Representative of 4 different experiments with blood from different donors. Molecular mass of the proteins that increased or decreased their phosphorylation is indicated by arrows on left.

phorylation by 1 nM thrombin, induced an 8- or 22-fold increase from the basal value of cGMP, respectively, within 1 min. Over this concentration range, sodium nitroprusside does not elevate CAMP (19, 32). These results are in contrast to the report of Pumiglia et al. (23), who did not observe an inhibitory effect of sodium nitroprusside or &BrcGMP on tyrosine phosphorylation in thrombin-stimulated platelets. However, most of their experiments were conducted at a concentration of 1 U/ml (-10 nM) thrombin. As we have shown, the inhibitory effects of the stated concentration of sodium nitroprusside or &BrcGMP on tyrosine phos-

Fig. 7. Effects of PGEi (2 PM) or sodium nitroprusside (SNP; 100 PM) on shape change and elevation of [Ca’+], induced by ADP. A: ADP (20 PM) was added at time indicated by arrows. Downward deflection in light transmittance in the aggregometer represents “shape change” of platelets. Representative of 5 different experiments. B: [Ca”+h in ADPstimulated platelets loaded with fura- was measured as described in Fig. 3. Arrow indicates time of addition of ADP (20 PM). Representative of 3 independent experiments.

phorylation and platelet function were only observed with cu-thrombin (1 nM) and could be overcome by an excess of a-thrombin (10 nM, 1 U/ml). Deana et al. (2) also reported that aggregation induced by a low dose but not by a high dose of thrombin was inhibited by sodium nitroprusside or 8-BrcGMP. Because most platelet agonists, including thrombin, epinephrine, collagen, and ADP were found to elevate cGMP in platelets, it was at one time proposed that cGMP mediates platelet activation (1). However, subsequent studies showed that cGMP-elevating agents [e.g., sodium nitroprusside, nitric oxide (NO), or %BrcGMP], added to platelets before agonists such as thrombin, inhibit calcium elevation, phospholipase C activation, protein kinase C activation, aggregation, and secretion in a fashion similar to the effect of elevation of CAMP (2, 6, 16, 17, 19, 22, 26-32). Recently, Radomski et al. (24) described the presence of an NO- and cGMP-generating system in platelets, acting as an intrinsic negative-feedback pathway, analogous to that in endothelial cells. Our study is consistent with these reports and indicates that tyrosine phosphorylation is another common target of CAMP and cGMP. The inhibitory effect of CAMP may appear to be greater than that of cGMP, depending on the relative concentration of cyclic nucleotides (23).


C706

TYROSINE

PHOSPHORYLATION

AND CYCLIC

Furthermore, the results of our study are consistent with the existence of a physiological role for tyrosine phosphorylation in the regulation of platelet function. ADP, which only induced shape change and, occasionally, weak primary aggregation in aspirin-treated platelets in the absence of added fibrinogen, led to tyrosine phosphorylation of only a few proteins, and its effects on tyrosine phosphorylation, shape change, and elevation of [ Ca2+]i were inhibited by sodium nitroprusside or PGE,. cu-Thrombin (10 nM)-induced tyrosine phosphorylation was virtually unaffected by sodium nitroprusside or PGE1, and impairment of aggregation and secretion by those inhibitors was minimal. Inhibition of tyrosine phosphorylation was well correlated with inhibition of dense granule secretion. Previously, Inazu et al. (12) observed that H202 plus vanadata induced tyrosine phosphorylation, presumably through inhibition of tyrosine phosphatases, and that the phosphorylation temporally preceded aggregation. In permeabilized platelets, vanadate and molybdate also induce tyrosine phosphorylation and granule secretion, probably through inhibition of tyrosine phosphatases (18). Our data are consistent with those observations and suggest that tyrosine phosphorylation accompanies several aspects of platelet function, such as granule secretion and aggregation. Recently, we examined individual thrombin-stimulated platelets loaded with indo-l, another calcium indicator, by flow cytometry and determined that only platelets that elevated their free calcium concentration secreted their granule contents, suggesting that granule secretion is calcium dependent (21). However, in parallel experiments we found that ADP also elevated cytosolic calcium but failed to induce granule secretion, indicating that factors in addition to calcium elevation were necessary (21). Such an additional factor may be increased tyrosine phosphorylation of specific cellular proteins. Platelet activation by aluminum fluoride, presumably through a direct effect on guanine nucleotide-binding proteins (G proteins) associated with phospholipase C (28), was previously shown to be inhibited by agents that increase CAMP or cGMP (2, 17). G proteins may be common early targets of both cyclic nucleotides. Ferrel and Martin (4) observed that either phorbol ester (a protein kinase C activator) alone or calcium ionophore (A23187) alone induced weak tyrosine phosphorylation in platelets, but the effect of the two agonists in combination was much enhanced. Tyrosine phosphorylation, at least in part, is likely to be the consequence of calcium elevation and protein kinase C activation, as a result of increased phospholipase C activity. This relation is supported by the present study, since calcium elevation was inhibited when tyrosine phosphorylation was inhibited. Furthermore, platelet aggregation was reported to be required for tyrosine phosphorylation of some proteins (5,8). Inhibition of aggregation may be another cause of inhibition of tyrosine phosphorylation by sodium nitroprusside or PGE1. Antiaggregatory effects of cGMP are generally accepted in platelets (2, 6, 16, 17, 19, 22, 2632), and the use of a high concentration of thrombin that largely overcame the antiaggregatory effects of sodium nitroprusside may be the reason why the previous report

NUCLEOTIDES

IN PLATELETS

did not describe any inhibition by sodium nitroprusside of thrombin-induced tyrosine phosphorylation (23). Such mechanisms and others that are affected by both CAMP and cGMP may be important in the regulation of tyrosine phosphorylation. It is possible that some proteins are tyrosine phosphorylated under direct regulation by G proteins in platelets. Such a mechanism was described in neutrophils very recently (9). The kind donation of ar-thrombin from Drs. John. M. Maraganore and John W. Fenton is gratefully acknowledged. This work was supported by National Heart, Lung, and Blood Institute Grants HL-38820 and HL-33014. Address for reprint requests: E. W. Salzman, Dept. of Surgery, Beth Israel Hospital, 330 Brookline Ave., Boston, MA 02215. Received 15 April 1991; accepted in final form 15 October 1991. REFERENCES T. M., S. N. Dixit, and A. H. Kang. Effect of cyclic 3’,5’guanosine monophosphate on human platelet function. J. Lab. Clin. Med. 88: 215-221,1976.

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Jr., and S. Martin. Tyrosine-specific protein phosphorylation is regulated by glycoprotein IIb-IIIa in platelets. Proc. Natl. Acad. Sci. USA 86: 2234-2238, 1989. Fitzpatrick, F. A., and R. R. Gorman. Regulatory role of cyclic adenosine 3’,5’ -monophosphate on the platelet cyclooxygenase and platelet function. B&him. Biophys. Acta 582: 44-58, 1979. Golden, A., and J. S. Brugge. Thrombin treatment induces rapid changes in tyrosine phosphorylation in platelets. Proc. Natl. Acad. Sci. USA 86: 901-905,1989. Golden, A., J. S. Brugge, and S. J. Shattil. Role of membrane glycoprotein IIb-IIIa in agonist-induced tyrosine phosphorylation. J. CeZZBioZ. 111: 3117-3127,199O. Grinstein, S., and W. Furuya. Tyrosine phosphorylation and oxygen consumption induced by G proteins in neutrophils. Am. J. Physiol. 260 (Cell Physiol. 29): C1019-C1027, 1991. Gutlind, J. S., P. M. Lacal, and K. C. Robbins. Thrombindependent association of phosphatidylinositol-3 kinase with p6Osrc and p59fyn in human platelets. Mol. CeZZ.BioZ. 8: 3603-3610, 1990.

I. D., M. L. Corcoran, P. A. Thompson, L. M. Wahl, 11. Horak, and J. B. Bolen. Expression of p60fyn in human platelets. Oncogene 5: 597-602,199O. 12. Inazu, T., T. Taniguchi, S. Yanagi, and H. Yamamura.

Protein-tyrosine phosphorylation and aggregation of intact human platelets by vanadate with Hz02. Biochem. Biophys. Res. Commun. 170: 259-263, 13. Kanakura, Y. Torimoto,

1990. Y., B. Druker, and J. D.

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transduction of the human granulocyte-macrophage colony-stimulating factor and interleukin-3 receptors involves tyrosine phosphorylation of a common set of cytoplasmic proteins. Blood 76: 706-715,199O.

14. Kanakura, Y., B. Druker, J. DiCarlo, S. A. Cannistra, J. D. Griffin. Phorbol 12-myristate 13-acetate inhibits

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granulocyte-macrophage colony stimulating factor-induced protein tyrosine phosphorylation in a human factor-dependent hematopoietic cell line. J. BioZ. Chem. 256: 490-495, 1991. 15. Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature Land. 227: 680-685, 1970. 16. Lanz,

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Thrombin-induced protein phosphorylation in human platelets.

Cardiovascular function during induced hypotension by fenoldopam or sodium nitroprusside in anesthetized dogs.

Thrombin-induced lysosomal exocytosis in human platelets is dependent on secondary activation by ADP and regulated by endothelial-derived substances.

In vitro inhibition by endothelins of thrombin-induced aggregation and Ca2+ mobilization in human platelets.

Platelet activation by diacylglycerol or ionomycin is inhibited by nitroprusside.

The signal transduction induced by thrombin in human platelets.

Elevation of cAMP, but not cGMP, inhibits thrombin-stimulated tyrosine phosphorylation in human platelets.

Effect of ADP on PGE1 formation in blood platelets from patients with depression, mania and schizophrenia.

β3 in thrombin-induced human platelets.

-)-alpha-methyl-p-tyrosine pretreatment or anterior diencephalic lesions.

Human platelet secretion and aggregation induced by calcium ionophores. Inhibition by PGE1 and dibutyryl cyclic AMP.

threonine and tyrosine kinases regulates ADP-induced thromboxane generation in platelets.

Letter: Inhibition of platelet function by nitroprusside.

Sodium nitroprusside-induced hypothermia in mice.

Inhibition of tyrosine phosphorylation potentiates substrate-induced neurite growth.

Inhibition of PAF-induced activation of human platelets by verapamil.

Protein-tyrosine phosphorylation and aggregation of intact human platelets by vanadate with H2O2.

Thrombin derivatives obtained by autolytic or limited tryptic cleavage.

Thrombin induced surface changes of human platelets.

Activation of platelets by endocannabinoids: distinct agonists or arachidonate reservoirs?

Thrombin, collagen and A23187 stimulated endogenous platelet arachidonate metabolism: differential inhibition by PGE1, local anesthetics and a serine-protease inhibitor.

Inhibitory effect of Bcl-xL gene on toxicity induced by sodium nitroprusside in SH-SY5Y cells.

Inhibition of PGE1 induced intestinal secretion by the synthetic enkephalin analogue FK 33-824.

Suspected sodium nitroprusside-induced cyanide intoxication.