Phosphorylation of the 47 KDa protein in gamma-thrombin-stimulated human platelets does not activate phospholipase A2: Evidence against lipocortin

Vol. 141, No. 2, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 459-465

December 15, 1986

PHOSPHORYLATION OF THE 47 KDa PROTEIN IN GAMMA-THROMBINSTIMULATED HUMAN PLATELETS DOES NOT ACTIVATE PHOSPHOLIPASE A2: EVIDENCE AGAINSTLIPOCORTIN Michael F. Crouch and Eduardo G. Lapetina Department of MolecularBiology, Burroughs WellcomeCo., 3030 Cornwallis Road, Research Triangle Park, North Carolina 27709 Received October 28, 1986

SUMMARY. Intact human platelets were stimulated w i t h alpha or gamma thrombin in the presence and absence of epinephrine and the ability of these agonists to stimulate aggregation, arachidonic acid release and protein phosphorylation was measured. Epinephrine alone had no effect on any of these events. Both alpha and gamma thrombin induced platelet aggregation which was potentiated in each case by epinephrine. Similarly,both thrombin species were able to induce the phosphorylation of platelet 20 KDa and 47KDa proteins. The g a m m a thrombin-induced phosphorylation was slightly enhanced by epinephrine. In contrast, only alpha thrombin was capable of inducing significant arachidonic acid release and the small release induced by gamma thrombin was reduced by epinephrine. These results show that the agonist-induced phosphorylationof the 47KDa protein by protein kinase C does not impart the ability to activate phospholipaseA2 in human platelets, and questions the s u g g e s t i o n t h a t t h e 4 7 K D a p r o t e i n is l i p o c o r t i n .

© 1986 Academic Press, Inc.

Arachidonic acid of human platelets is preferentially esterified at the twoposition of membrane phospholipids ( 1 ) . Upon stimulation of these cells with an appropriate agonist, such as thrombin, arachidonicacid is released (2) which can then be converted through cyclo-oxygenaseand lipoxygenasepathways to products which can further modulate platelet function (3,4). The enzymes responsible for arachidonate release from phospholipidsare thought

to

be phospholipases

A2

acting

on

phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and phosphatidic acid (5,6,7). Control of phospholipaseA2 activity has been considered over the past decade to reside in the intracellular

Ca2+ concentration, such that agonists which initially

raised the Ca2+ level inside the cell then, as a consequence, released arachidonic acid from membrane substrates.

Since inositol phospholipid hydrolysis is thought to be

intimately involved in the mechanism of Ca2+ mobilization in platelets, phospholipaseC

459

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and phospholipase A 2 activities are generally thought to be sequential events in the receptor-activated platelet (3,8,9). Much more recently, however, it has been suggested (1O) that release of esterified arachidonic acid from rabbit platelet phospholipids is under the control of an intracellular protein, termed 'lipocortin'. Touquiand coworkers (10) presented evidence that lipocortin is a substrate for protein kinase C and in the unphosphorylated state exerts a tonic inhibitory action on phospholipase A2.

However, the binding of a

stimulatory agonist to its receptor is able to phosphorylate the 47KDa protein, and this was proposed to reduce its phospholipase A2-inhibitory activity, thereby allowing arachidonic acid release to occur. This mechanism of control of phospholipase A2 activity would explain the results of some more recent experiments which have shown an independence of phospholipase A2 and the intraeellular Ca2+ concentration (11). However, they are at odds with the observation that

phorbol esters,

although potent

stimulators

of 47KDa protein

phosphorylation, are unable to release esterified arachidonic acid from human platelets (11,12), even in the presence of ionomyein(11). We have examined further the question of whether 47KDa protein phosphorylation induced by agonist-receptor interaction is a possible control mechanism of phospholipase A2 activity. Our results do not support this suggestion.

MATERIALS AND METHODS Materials. El~inephrine was obtained from S i g m a Chemical Co., [5,6,8,9,11,12,14,15-°H]araehidonic acid (80-135 Ci/mmol) was from Amersham, and carrier-free (32p)orthophosphoric acid was from ICN. Preparation of human platelets. Platelets were isolated from blood of healthy human donors who had not taken medication for at least the previous 2 weeks. The blood was anticoagulated with 3.8% trisodium citrate (6 ml in 60 ml blood), centrifuged for 20 rain at 800 g and the platelets resuspended in the appropriate volume of buffer or platelet poor plasma, as described below. The buffer contained (mM): NaCI, 138; KCI 2.9; Hepes, 20; NaH2PO4, 3.3; MgCI2, 1.0, and were maintained at 37°C and adjusted to pH 7.4. Depending on the experiment, the buffer was modified to contain 1 mM EGTA or NaH2PO4 was omitted. In all experiments, platelets were treated with 1 mM aspirin and were resuspended in the presence of apyrase (0.6 ADP'ase units/ml). During all experiments, just prior to each centrifugation of platelet samples, and also during periods of radioactive labelling, prostaeyelin (10O ng/ml) was added to inhibit platelet activation. Measurement of arachidonic acid release. Platelets from 200 ml of blood were isolated and resuspended in 5 ml of platelet poor plasma and incubated for 90 min with

460

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50 laCi of [3H]arachidonic acid. After labelling, platelets were diluted to 30 ml with platelet poor plasma, centrifuged at 800 g for 15 min, and resuspended in I0 ml of buffer containing EGTA. This wash procedure was repeated and the platelets were finally resuspended in buffer without EGTA. Aliquots (final volume 0.5 ml) of platelets were placed into tubes and agonist or buffer added. The lipoxygenase/cyclo-oxygenase inhibitor, BW755C (I00 I~M), was also added 1 min prior to the incubation to inhibit any metabolism of arachidonic acid via these pathways. Incubations were continued for specified periods and reactions were terminated by addition of 1.8 ml CHCI3/CH3OH/HCI (100:200:2). The phases were separated by addition of CHCI3 and H20 (0.6 ml each) and the lower organic phase was collected from each sample and dried in a vacuum evaporator. Lipids were resuspended in 50 lal of CHCI3 and were spotted onto thin layer silica gel chromatography plates (Whatman, LK 6D), and labelled arachidonic acid separated using the upper phase of an ethylacetate/iso-octane/acetic acid/water (90:50:20:100) solvent system (13). Radioactivity was localized using autoradiography and the corresponding areas on the plates were scraped and counted in a scintillation counter. Measurement of protein phosphorylation. P l a t e l e t s from i00 ml of blood were resuspended in 5 ml of buffer containing EGTA and without added phosphate. (32p)p i (5 mCi) was added and the p l a t e l e t s were incubated for 90 rain. A f t e r this time, p l a t e l e t s were diluted with 30 ml of buffer and c e n t r i f u g e d for 10 rain at 800 g. P l a t e l e t s were resuspended in 10-20 ml of buffer without EGTA but containing unlabelled phosphate, and aliquots were used for m e a s u r e m e n t of a g o n i s t - s t i m u l a t e d p r o t e i n phosphorylation. Samples (final volume 0.5 ml) of platelets were added to tubes with buffer or agonist, and the reactions terminated after designated times by taking a 50 lal aliquot and adding it to 50 lal of electrophoresis sample buffer. The proteins were separated using S D S - P A G E (14), and changes in 32p_ phosphorylation state were visualized by autoradiography, and quantitated by cutting out the corresponding proteins from the gel and counting in a liquid scintillation counter. RESULTS Stimulation of human p l a t e l e t s with alpha thrombin (10 nM) induced a rapid aggregation with l i t t l e or no d e t e c t a b l e lag period (Fig 1). G a m m a thrombin (50 nM) also stimulated aggregation, but this was preceded by a lag period of 15-45 see (Fig 1). However, once aggregation had begun, the r a t e of aggregation was similar for both alpha and gamma thrombins (Fig 1). Epinephrine (100 laM) p o t e n t i a t e d the aggregation for both agonists, but this was more pronounced for gamma thrombin, with an obvious d e c r e a s e in the lag period prior to aggregation

(Fig 1). The d e g r e e of p o t e n t i a t i o n by epinephrine

was somewhat variable between platelet preparations. Alpha thrombin (10 nM) induced the phosphorylation of two main proteins in h u m a n platelet.% 20 K D a and 47 KDa, which are substrates for myosin light chain kinase and protein kinase C, respectively. Fig 2 shows the degree of phosphorylation of the 47 K D a protein after a 60 sec incubation with alpha thrombin. G a m m a

thrombin (50 nM)-induced

phosphorylation was preceded by a lag time of about 30 see (not shown) as described previously (15), but after 60 sec the degree of phosphorylation was only slightly less that 461

Vol. 141, No. 2, 1 9 8 6

BIOCHEMICAL AND BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S

i

A.

B.

j ~ , ~-thrombin

C,

0

10 nM

i

3'o

6o

sec

Z

O

09 z < oE II"1-

y-thrombin

.-I

z LU

t

t Fig. 1.

~" r-thrombin " ¢

Alpha and gamma thrombin-induced aggregation of human platelets. Allquots (0.5 ml) of washed human platelets were placed in aggregometer tubes and either alpha (10 nM) or gamma (50 nM) thrombin added. The aggregation was recorded as an increase in light transmission through the platelet suspension. The three sets of tracings represent (A) aggregation induced by alpha and gamma thrombin alone, and aggregation stimulated by alpha thrombin (B) and gamma thrombin (C) in the absence and presence of epinephrine (Epin; ioo pM).

t h a t i n d u c e d by a l p h a t h r o m b i n (Fig 2). In t h e p r e s e n c e of e p i n e p h r i n e (100 }aM) t h e g a m m a - t h r o m b i n r e s p o n s e was s l i g h t l y p o t e n t i a t e d , a n d was i n d i s t i n g u i s h a b l e f r o m t h a t

600

d

o rr iZ o o

5OO 400

300 D

~

HH

2oo

o.

100 Z

0

c,-T

y-T

y. T +

Epin.

Epin.

Fig. 2.

[32p]47 KDa Protein phosphorylation of human platelets stimulated by alpha and gamma thrombin. Washed human platelets were labelled with (32p)Pi for 90 rain, washed, and samples (0.5 ml) were then incubated for 60 see in the presence of alpha (a-T; I0 nM) or gamma (x-T; 50 nM) thrombin with or without epinephrine (Epin; 100 pM). Changes in protein phosphorylation were measured by separating the proteins using SDS-PAGE, visualizing labelled bands by autoradiography and eounting appropriate bands in a scintillation eounter. Results are the mean + SE of 4-7 samples from two separate experiments. 462

O Z

Vol. 141, No. 2, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

._1

or~

700

FZ 0 0

600

z~ o 500 II w rn ...1 I..u

400 300

Z

oa -tO

200

.< n.-

100 0

Fig. 3.

of

alpha

o,-T

y -T

y-T qEpin.

Epin.

Effects of alpha and gamma thrombin o n arachidonic acid liberation from human platelets. Platelets were suspended in 5 ml of platelet poor plasma and incubated with [3H]arachidonic acid (50 IJCi) for 90 rain. Platelets were then washed and samples (0.5 ml) were placed in tubes with either alpha (a-T; 10 nM) or gamma (y-T; 50 nM) thrombin in the absence or presence of epinephrine (Epin; 100 laM). The incubation was continued for 60 sec and the amount of released arachidonic acid was masured as described in Materials and Methods. Results are the mean _+ SE of 4 samples from two separate experiments.

thrombin

(Fig 2).

Epinephrine

alone

produced

no

effect

on

protein

phosphorylation (Fig 2). Alpha thrombin (10 nM) was also a potent stimulus of arachidonic acid release from human platelets. This response was maximal after 30 see (not shown), after which there was a slight decline, indicating re-esteriflcation of the fatty acid into membrane phospholipids.

The arachidonic acid release after a 60 see incubation with alpha

thrombin was near maximal (Fig 3), while gamma thrombin (50 nM) was virtually ineffective at inducing araehidonate release (Fig 3), and in the presence of epinephrine (100 laM) the release was even less (Fig 3).

Epinephrine alone did not release any

e s t e r i f i e d a r a c h i d o n i c a c i d (Fig 3).

DISCUSSION It is well known that following receptor activation by thrombin that there is a rapid activation of phospholipase C and this is followed by an increase in the activity of p h o s p h o l i p a s e A 2 (3,8,9,15).

It

was a s s u m e d f o r m a n y y e a r s t h a t t h e s e t w o e v e n t s w e r e

a s s o c i a t e d , and e v e n t s w h i c h w e r e a c o n s e q u e n c e o f p h o s p h o l i p a s e C a c t i v a t i o n w e r e 463

Vol. 141, No. 2, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

responsible for the release of araehidonie acid from membrane phospholipids. r e c e n t years, the

most popular candidate for the release of

Over

araehidonic acid was an

increase in the intraeellular Ca 2+ concentration, which has been shown to be due, at least in part,

to the generation of inositol trisphosphate

(IP 3) from

phosphoinositide

hydrolysis (17,18,19). Indeed, IP 3 has been shown to be able to release arachidonic acid from permeabilized platelets (20,21). recently shown that

However, Pollock

both ionomycin and

arachidonie acid from human platelets,

and

coworkers

(11)

have

collagen (10 lag/ml) were able to

release

but collagen accomplished this

without

elevating the intracellular Ca 2+ c o n c e n t r a t i o n to levels which were threshhold for the ionomycin-induced effect. Their conclusion was that the receptor-induced release of arachidonie

acid

from membrane phospholipids was a result of

than an elevation in the A

cytosolic

a mechanism

other

Ca 2+ concentration.

possible mechanism for the r e c e p t o r - m e d i a t e d arachidonate release has just

been proposed by Touqui et al (10) as discussed in the Introduction. We have examined their suggestion

that liberation

of

arachidonic

acid

is controlled

phosphorylation s t a t e of the 47 KDa protein which they recognized as

by

the

lipocortin,

by

comparing the effects of alpha and gamma thrombin on araehidonic acid release and protein

phosphorylation using intact human platelets. Alpha thrombin possesses all of

the p l a t e l e t - s t i m u l a t i n g qualities of native thrombin, whereas gamma thrombin is unable to

release

significant quantities

of araehidonie acid,

but will induce other platelet

responses such as the release of intracellular Ca 2+ stores and aggregation (15). Alpha thrombin induced a considerable phosphorylation of the 47KDa protein, and gamma thrombin was almost as effective. In combination

with

epinephrine, g a m m a

thrombin-induced phosphorylation was the same as that found for alpha thrombin. contrast,

although

release,

gamma thrombin

absence of

alpha

epinephrine.

thrombin was

was a potent

virtually ineffective,

of arachidonic

both

in

acid

the presence and

Under these conditions, epinephrine clearly potentiated the

gamma thrombin-induced aggregation. Thus, there ability of an agonist

stimulus

In

is a clear dissociation between the

to phosphorylate the 47KDa protein

acid. From these results, we can conclude either that (1)

and

release arachidonic

the 47KDa protein does not

possess lipocortin activity in the human platelet (as was suggested by Touqui et al (I0) 464

Vol. 141, No. 2, 1986

for

the rabbit platelet)

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and

so this does not represent

a ubiquitous modulator of

phospholipase A 2 activity in platelets or (2) the 47 KDa protein is lipocortin in human platelets, but there is another more important control mechanism of arachidonic acid liberation in these ceils which is independent of protein kinase C and Ca 2+ mobilization.

ACKNOWLEDGEMENT We thank Dr. John Fenton for his generous gift of alpha and gamma thrombins.

REFERENCES i. 2. 3. 4. 5. 6. 7. 8. 9. I0. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

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