Inhibition by antioxidants of agonist evoked cytosolic Ca++ increase, ATP secretion and aggregation of aspirinated human platelets

Vol. 139, No. 2, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 509-514

September 16, 1986

++

IN~{IBITION BY ANTIOXIDANTS OF AGONIST EVOKED CYTOSOLIC Ca INCREASE, ATP SECRETION AND AGGREGATION OF ASPIRINATED HUMAN PLATELETS o

Adolfo

Alexandre

, M.

Gabriella

Institute C.N.R.

Doni

o

, Emilia

of Biological

Padoin

University

of

Human

of Padova,

Renzo

Deana

Chemistry,

Unit for the Study of Mitochondrial °Institute

and

Physiology,

Physiology,

35131Padova,

Italy

Received July 7, 1986 SUMMARY: The synthetic antioxidants butylated hydroxytoluene (BHT), nordihydroguaiaretic acid and the one-electron donor l,l'-dimethylferrocene, ++ inhibit cytosolic Ca increase, shape change, aggregation and ATP secretion in aspirinated washed human platelets stimulated by thrombin, vasopressin and platelet-activating factor. The antioxidants also inhibit cy++ tosolic Ca increase originating from intracellular stores in the presence of EGTA. The effect of phorbol ester (TPA), which promotes platelet ++ aggregation and secretion without raising the cytosolic Ca , is also antioxidant-sensitive. Since agonist activation of aspirinated platelets does not involve cyclooxygenase or lipoxygenase metabolites, it is suggested that other yet unknown free radical-dependent pathways are involved in the mechanism of platelet activation, both in the protein kinase ++ C-independent events leading to the cytosolic Ca increase, and in those, largely protein kinase C-dependent, leading to aggregation and ATP secretion. @ 1986 A c a d e m i c Press, Inc.

The

interaction

such as thrombin, is followed

by

platelets

with

platelet-activating

by a rapid

i s accompanied

of

physiological

agents

(PAF), vasopressin, ADP, etc. 2+ increase of the cytosolic Ca concentration, which

functional

factor

responses,

namely shape change, aggregation 2+ in cytosolic Ca is largely due to an extra-

and secretion (i). The rise 2+ cellular Ca influx and, to a smaller extent,

*To whom correspondence

activating

to a release from the dense

should be addressed.

ABBREVIATIONS: BHT, 2,3-tert-butyl-4-methoxyphenol; BHA, 2,6-di-tert-butyl- 4-methylphenol; NDGA, nordihydreguaiaretic acid; BW 755C, 3-amino-lm- (trifluorometil)-fenil -2-pyrazoline; THR, thrombin; TPA, 12-O-tetradecanoyl phorbol acetate; PAF, platelet activating factor; EGTA, ethylene glycol bis (~ -aminoethylester),N,N,N',N'-tetraacetic acid. 0006-291 X / 8 6 $1.50

509

Copyright © 1986 by Academic Press, lnc. All rights of reproduction in any form reserved.

Vol. 139, No. 2, 1986

tubular

system,

cariocytes recently on

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

which

(2,

3).

clarified

platelets

by

is derived

The in

events

detail

activating

polyphosphoinositides

lic

Ca

and

(4, 5).

generating

from intracellular

2+

leading

to platelet

the

reticulum of mega-

activation

have

It has been shown that agonists

a phosphodiesterase

inositol 1,4,5-triphosphate, release

from the endoplasmic

second

activators

which

specifically

messengers

been act

splits

diacylglycerol

and

of protein kinase C and of calcium

stores respectively

(5-8). The rise of eytoso-

the activation of protein kinase C jointly cooperate

to the

induction of platelet aggregation and the release of the secretory granules.

Platelet

even

below-resting

protein

kinase

Conversely,

a

aggregation

C

and

exocytosis

are induced also at resting, or 2+ of the cytosolic Ca , provided that

concentrations is maximally

high

increase

activated of

(for

cytosolie

example by phorbol ester). 2+ Ca is a sufficient stimulus

also without activation of protein kinase C (i, 9). It has been reported E

and

the

one-electron

(I0,

ii) that some antioxidants

acceptor

nitroblue

tetrazolium

such as vitamin inhibit

platelet

aggregation and exocytosis.

These observations were generally discussed in

terms

cyclooxygenase,

of

inhibition

of thromboxane

A

of

the

(ii). More recently,

hydrogen

2 peroxide

platelet

aggregation,

increase and

the

it was shown that small amounts of

efficacy

conversely

leading to impaired synthesis

of

that

several agonists in

some

in promoting

conditions

exogenous

catalase prevents aggregation (12).

the

The present paper shows that a variety of antioxidant agents inhibit 2+ increase of cytosolic Ca , aggregation and secretion of aspirinated

platelets

activated

vasopressin.

by

physiological

Platelet aggregation

stimuli

such

as

thrombin,

PAF

and

and secretion induced by tumor promoting

phorbol ester (TPA) is also inhibited in the presence of antioxidants. MATERIALS AND METHODS Materials: Quin 2 acetoxymethylester and ionomycin were purchased from Calbiochem, butylated hydroxytoluene (BHT), butylhydroxyanisol (BHA) and l,l'-dimethylferrocene from Aldrich, nordihydroguaiaretic acid, vasopressin and platelet activating factor from Sigma. Thrombin (Topostasin) was obtained from Roche, BW 755C from Wellcome and ATP-reagent kit from LKB. All other reagents were of analytical grade. Methods: Fresh blood was drawn from healthy volunteers, who had denied taking drugs for the previous i0 days, and immediately mixed with onesixth volume of acid citrate-dextrose anticoagulant (0.i M sodium citrate,

510

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 139, No, 2, 1986

7 mM citric acid,

0.14 M dextrose, pH 6.5). Platelet-rich plasma, prepared

by 5 min centrifugation at 700 x g, was incubated for 40 min at 37°C with 20 ~M quin 2 acetoxymethyl ester. The platelets were pelleted by centrifugation at 500 x g at room temperature for 20 min. The cells were gently resuspended in prewarmed medium consisting of 145 mM NaCI, 5 mM KCI, i mM MgSO , i0 mM HEPES, i0 mM glucose, pH 7.4 and incubated for 20 min at 37°~ with i00 ~M aspirin. The platelet count was adjusted to about i x i0 cells/ml and the suspension left at room temperature. 5 min before the measurements, aliquots of the suspension were equilibrated at 37°C and the external calcium was adjusted by addition of CaCI or EGTA. 2+ 2 Changes in the cytosolic free Ca concentration were measured following the fluorescence of the indicator quin 2 as described by Tsien et al. (13), with a Perkin-Elmer LS3 spectrofluorimeter in a cuvette termostatted at 37°C and magnetically Hallam et al. (3). Simultaneous

stirred.

measurement

of

Calibrations were performed according to ATP

secretion

was

carried

out

at

37°C

with a LKB Luminometer by adding i0 ~i of luciferin/luciferase reagent to 240 ~I of quin 2 loaded aspirinated platelet suspension. Each trace was calibrated by addition of a standard ATP solution. Aggregation was evaluated in parallel at 37°C with an Elvi (Logos) aggregometer. All types of experiments were performed with at least five different preparations. RESULTS AND DISCUSSION All the experiments described in this report were performed with aspirinated

platelets.

prevents from

the

This

production

arachidonic

acid.

treatment, of In

the

sin

and PAF

evidence

are

powerful

these

to promote platelet activation,

which

inactivates activating

conditions,

that platelet activation

agent

cyclooxygenase, thromboxane

such as thrombin,

These properties

A

2 acid fails

while arachidonie

other stimuli

still efficient.

the

vasopres-

are generally

taken as

does not necessarily involve metabolites

of the cyclooxygenase and lipoxygenase pathways. In the experiments incubated presence the

with

i

mM

of Fig.

external

of various antioxidants

one-electron

donor

i quin 2-1oaded aspirinated platelets were 2+ Ca and activated with thrombin in the (the phenol BHT, the orto-quinol NDGA and

l,l'-dimethylferrocene

plus

ascorbate).

All

the

antioxidants tested strongly inhibit the agonist-induced increase of cyto2+ solic Ca , as well as the shape change, aggregation and ATP secretion. The

antioxidants

ionomycin-promoted

per

se

did

cytosolic

Ca

not 2+

affect increase

the

rate

(not

shown).

curve of the BHT inhibition of platelet activation 50%

inhibition

of

the

thrombin-promoted

secretion is obtained at 20-80 ~M BHT,

cytosolic

and

extent The

of

dose-effect

is reported in Fig. 2. 2+ Ca increase and ATP

while a similar effect on aggrega-

tion is observed at slightly higher concentrations of the antioxidant.

511

the

Vol. 139, No. 2, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A

8

100

C

CONTROL A

CONTROL

I NM-

ATOP

500nM-

.,,.,.;,,~,NOGA FC ~BHT

f

°,T, I/

50

/ F C ~NDGA 8HT

20OHM,~ ..U

100 nM- ' ~ ' '

(D

THR

CONTROL

I

i

50

THR

L

100 150 [BHT] (pM)

(i)

Fi$. i. Antioxidant inhibition of platelet functions. Quin 2-1oaded aspirinated platelets were incubated with 1 mM CaCI and stimulated with 0.15 2 U/ml thrombin (THR). When indicated 50 ~ M BHT, 65 ~ M nordihydroguaiaretic acid (NDGA) and 65 ~ M dimethylferrocene (FC) plus 2 mM ascorbate were added 3 min before THR. A) quin 2 fluorescence, B) ATP secretion. The traces are corrected for some quenching effect of the antioxidants. C) platelet aggregation.

~

. Dose-response effect of BHT on thrombin induced: cytosolie free increase (A), platelet aggregation ( 0 ) and ATP secretion (I). Experimental conditions as in Fi E . i.

Fig.

3

shows

thrombin-induced of

extraeellular

also

under

these

that,

rise

in

of

agreement

with

cytosolic

free

However

the

calcium.

conditions,

where

previous

calcium BHT

the

reports

is very

inhibitory

intracellular

(2,

3),

the

low in the absence effect

stores

is

are

evident the

sole

is also observed

with

2+ source

of the cytosolic

The

antioxidant

vasopressin, solic

Ca

2+

PAF

inhibition

or ADP

increase

on ATP secretion

Ca

are

as

of platelet

agonist.

reported

and a g g r e g a t i o n

function

Experiments

in Fig.

4.

on the inhibition

Similar

with v a s o p r e s s i n

results

and PAF

are

of cytoobtained

(not shown).

200 nM~M~

CONTROL

150 nM-

~%%k~ 80 p~.1 BHT

~ EGTA BHT ~00 nM- "

~

~

1~ 160 tJM BHT

I

~min-':

THR 2+

Fig. 3. Rise of cytosolic Ca in quin 2-1oaded platelets in the absence of external calcium: inhibitory effect of BHT. ExDer~mental conditions as in Fig. 1 except that 1 mM EGTA was substituted for Ca

512

f 200

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 139, No. 2, 1986

~a2÷]i

A

B

C

5oooM-., CONTRO"7'CO"TRO' CO.R ' O' BHT

VP

Pmlin~

PAF

ADP 2+

Fig. _:4. BHT inhibition of agonist promoted Ca increase. When indicated i ~ M vasopressin (VP), 75 nM PAF and I O ~ M ADP were added, while 5 0 ~ M BHT was added 3 min before the agonist.

The kinase

tumor

C.

It

promoters has

been

phorbol

esters

reported

that

are powerful

they

induce

activators

platelet

of protein

aggregation

and

2+ ATP

secretion

ties

in the

are taken

vated,

as evidence

aggregation

sting

absence

cytosolic

and

Ca

2+

fect

of

antioxidants

also

in

view

of

on

phorbol

report

aggregation

ATP

evoked

have

(results

not shown).

As

already

er p r o m o t e d the se

been

by

pathways.

755C,

a

by

that

(12). TPA

obtained

A

the

of metabolites further

non-specific

ester

shown

BHA,

of

indication inhibitor

promoted

5,

and

of

the

the

ester,

the

the

phorbol

plus

T

ascorbate

platelets,

wheth-

does not require

or of the lipoxygenaineffectiveness

lipoxygenase,

on

the

of

---,,.

f

-5

CONTROL

TPA

Fig. 5. BHT inhibition of phorbol ester-promoted ATP secretion (A) and aggregation (B). 0.12 nM TPA was added at the arrow; when indicated 8 0 ~ M BHT was added S min before TPA.

513

BW

thrombin

CONTROL

/

and

Similar

B ATP

ef-

activation,

aggregation

ferrocene

the c y c l o o x y g e n a s e is

study

with

both

of aspirinated

this

to

acti-

below-re-

by antioxidants.

or by phorbol

of

or even

platelet

interferes

in Fig.

NDGA

C is m a x i m a l l y

important

affected

activation

stimuli

kinase

(9). These proper-

at resting

therefore

catalase

As

with

mentioned,

increase

also

are markedly

physiological

involvement

occur

It was

the

Ca

if the p r o t e i n

levels.

the

results

that,

exocytosis

ester-induced secretion

of cytosolic

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 139, No. 2, 1986

activation

of

inhibitory

action

this

report

aspirinated of

cannot

the be

platelets

(not

antioxidants ascribed

to

shown).

It

follows

on platelet functions their

scavenging

of

that

the

described free

in

radicals

involved in this type of reactions. It has been previously

reported that platelet functions are sensitive

to antioxidants

(ii),

cy

agonists

(12,

14)

suggest

that

other

of

platelet

that H 0 and superoxide ions potentiate the effica2 2

H 0 and 2 2 radicals are produced upon platelet activation (15). Our

results

dent reactions at

the

level

increased

are involved of

the

cytosolic

and

that

possibly

superoxide

as yet undefined free radical

in the processes

depen-

of platelet activation,

both

protein kinase C independent events leading to the 2+ Ca , and of those, largely protein kinase C-depen-

dent, leading to aggregation and ATP secretion. ACKNOWLEDGMENTS We wish to thank Miss Monica Vettore for excellent secretarial work and Mr. Gianluigi Gioachin for technical assistance. REFERENCES i. 2. 3.

Rink, T.J. and Hallam, T.J. (1984) Trends Biochem. Sci. 9, 215-219. Hallam, T.J., Thompson, N.T., Scrutton, M.C. and Rink, T.J. (1984) Biochem. J. 221, 897-901. Hallam, T.J., Sanehez, A. and Rink, T.J. (1984) Biochem. J. 218, 819-

4.

827. Berridge, M.J. and Irvine, R.F.

5. 6. 7.

Brass, L.F. and Joseph, S.K. (1985) J. Biol. Chem. 260, 15172-15177. Nishizuka, Y. (1984) Nature 304, 693-698. Israels, S.J., Robinson, P., Docherty, J.C. and Gerrard, J.M. (1985)

(1984) Nature 312, 315-320.

ii.

Thrombosis Res. 40, 499-509. Adunyah, 8.E. and Dean, W.L. (1985) Biochem. Biophys. Res. 1274-1280. Rink, T.J., Sanchez, A. and Mallam, T.J. (1983) Nature 305, Vargaftig, B.B., Tranier, Y. and Chignard, M. (1975) Eur. col. 33, 19-29. White, J.G., Rao, G.H.R. and Gerrard, J.M. (1977) Amer.

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88, 387-398. Del Principe,

8. 9. I0.

13. 14. 15.

Comm. 128, 317-319. J. PharmaJ.

Pathol.

D., Menichelli, A., De Matteis, W., Di Corpo, M.L., Di Giulio, S. and Finazzi-Agr$, A. (1985) FEBS Lett. 185, 142-146. Tsien, R.Y., Pozzan, T. and Rink, T.J. (1982) J. Cell. Biol. 94, 325334. Handin, R.I., Karabin, R. and Boxer, G.J. (1977) J. Clin. Invest. 69, 969-965. Marcus, A.J. (1972) Semin Hematol. 16, 188-195.

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