Diadenosine triphosphate (Ap3A) mediates human platelet aggregation by liberation of ADP

Vol. 118, No. 3, 1984

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

February 14, 1984

Pages 704-709

DIADENOSINE

TRIPHOSPHATE

(Ap3A)

MEDIATES

BY LIBERATION Jirrgen Fahrstrasse

17,

LDthje

lnstitut

fDr

HUMAN

PLATELET

AGGREGATION

OF ADP

and

Adaling

Ogilvie

Physiologische

Chemie,

D-852

Erlangen

GFR

Received December 30, 1983

Human platelets store considerable amounts of diadenosine 5’,S1r-P1,P3-triphosphate, which is released together with the homologue diadenosine tetraphosphate (Ap,,A) upon thrombin-induced aggregation (LUthje,J. & Ogilvie,A. (1983) Biochem. Biophys. Res. Conmrun. 115, 253-260). We now report that, when added to platelet-rich plasma at lo-20pM, diadenosine triphosphate gradually induces aggregation. The addition of diadenosine tetraphosphate antagonizes this effect by rapidly disaggregating the platelets. When another physiological but structurally unrelated stimulus, i.e. PAF (Platelet activating factor) is introduced into the system, diadenosine triphosphate drastically enhances and prolongs the aggregatory effect of PAF. Again, ApbA is antagonistic in this system. The mechanism of Ap3A-stimulation can be explained by the slow and continuous liberation of ADP from Ap A by the action of a hydrolyzing enzyme which is present in human plasma. Ou? studies suggest that Ap A may be physiologically important in providing a relatively longlived s F rmulus that can modulate platelet aggregation.

Diadenosine otic

triphosphate

cells

Based

using

on

nistic a potential might

a specific

in-vitro

role

(Ap3A)

data

it

the

signal

molecule

(2,8).

Both

platelet

act

of

as

is Ap3A,

only

marginally

itself

release

of of

PAF,

Abbreviations: 0006-291X/84

Here

aggregating

on

seems

platelet

in

of

of

high

of

platelet

were

3

A when

can

an

antago-

which

is

(3-6)

replication

and

which for

Ap,,A

has

to

of

influence in

Ap3A

state

induces

dinucleotides

aggregation

factor;PRP,

704

inactive

thrombin

interest.

incubated

$1.50

to

the

been

found

induced

by

to ADP

aggregation

(10,ll).

ADP-induced

aggregation

platelet-rich

be counteracted by

activating

0 1984 by Academic Press, Inc. of reproduction in any form reserved.

play

a metabolically

these

that

induced Ap

(1,2).

(Ap,+A)

stimulus able

we demonstrate which

might

DNA

exposure

physiological authors

potency

from

Ap,+A

function

aggregation ADP

as

released

important

eultary-

methodology

Ap3A

of

in

(7).

well

inhibitor

these (9).

mechanism

a slow

an

identified

tetraphosphate

“alarmone”

as

aggregation

however,

that

initiation

possible

competitive

probably

has

cular

an

Ap3A

The

suggested

the

been

chromatographic

diadenosine

are

platelet

With

as

recently

and

been

for

dinucleotides

a potent

which

Copyright All rights

store

aggregation.

process

has

homologue

act

platelets

very

enzymatic

toward

pleiotropically Human

has

plasma by

Ap,+A.

has

been

clarified

in

human

plasma.

platelet-rich

Ap3A

The

mole-

in

showing

plasma

(8)

Vol.

118,

No.

BIOCHEMICAL

3, 1984

AND

MATERIALS Reagents. [3H]Ap A was from Amersham and platelet act 1 vating factor (PAF) (calf intestine) was from Boehringer,

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

& METHODS (labeling were from Mannheim.

service). Sigma.

Ap A, ADP ph a sphatase

ApqA, Alkalrne

Preparation of platelet-rich plasma (PRP) and aggregation studies. Fresh blood was collected from volunteers into acid citrate/dextrose anticoagulant and centrifuged at room temperature for 15 minutes at 120 x g to yield PRP. Aggregation of platelets was followed photometrically in a Shimadzu photometer equipped with a mechanical stirring device and with a chart recorder. Standardization of the measurement as well as testing of platelet sensitivity was performed with ADP (1pM) as initiator of platelet aggregation (10,ll). Repurification of Ap3A and ApqAL The dinucleotides (10 nmol) were applied onto an HPLC system and chromatographed as described (1). The fractions pooled and desalted with a Sephadex G-10 column containing Ap A (Ap A) were (60 x 2.2 cm) 3 elute if with water. The fractions containing the nucleotides were concentrated by evaporation. in human p lasma. Measurement of 3 A-hydrolysis 13~]~p A (3.5pM; 22.7 Ci/mmol) was incubated wit 2 PRP at 37OC. Aliquots were withdra into x n and pipetted ice-cold trichloroacetic acid (10% final cont.). After centr’ifugation (12.OOOxg; 5 Min.; 4OC) the supernatants were neutral ized as described (12). Two microliters were spotted together with nucleotide markers on poly(ethyleneimine)-cellulose thin layers. The plates were first developped in water and, after drying, in 0.5 M LiCI. The spots visible under ultraviolet I ight were marked with a penci 1 and cut out. The thin layer pieces were overlayed with scintillation cocktail (toluene containing 0.5% 2,5-diphenyloxazole; 0.03% 1,4-bis-(4-methyl-5-phenyl-oxazol-2-yl)benzene) and counted in a Packard liquid scintillation counter. Incubation of[3H]Ap3A with water instead of PRP was run as a control. RESULTS Ap A induces

aggregation

-3

diadenosine pared

triphosphate

platelet-rich

Addition

of

increasing

of

Ap3A

at

human

(Ap3A) from

20 IJM

induced,

aggregation

of

the

platelets. on

plasma

& DISCUSSION

iluman

healthy

platelets

To

investigate

platelets, donors

after

for

a lag (Figure

we

of

used

In

effect

of

freshly

bioassay several

1).

the

(10, minutes,

contrast,

pre13). a slowly aggregation

Transmission

Aggregation

human platelets induced by Ap A. Platelet-rich was freshly prepared from the blood of he41 thy donors. Aggregation of platelets was followed photometrically. a) Diadenosine triphosphate (Ap3A) was added to give a concentration of 20,~M. Diadenosine tetraphosphate (Ap,,A) was then added at 100 pfl as indicated by the second arrow. b) Aggregatlon induced by ADP (0.5,uM). of

705

Vol. 118, No. 3, 1984 induced and

by

showed

stimulus with

an

and

identical

tetraphosphate the

of

aggregation

(9).

aggregating

effects

aggregation

was

A13A

enhances

We also the

as

minutes

(Figure

enhancement

concentration

2 also This

demonstrates is

0.5

be Ap3A

of

of

Ap3A

a

consistent

added

at

our

lipid

etc.

(14-16). by

9 /uM

alone

rapidly

that

ADP

with

a competitive

again

on Figure 2: Effects of Ap3A and Ap,+A let activating factor PAF. b) Aggregation by P~~~~one.‘*$‘&f”,~~ added at a cont. of 50,uM. AP,,A w as then of 50,uM.

from

before

10 and

overrode

at

the the

leuco-

after

several

a dramatic at

Ap4A

the when

by

conditions -8 x 10 M

3.8

caused

Ap4A,

the.

same

spontaneous added

later

platelets. disaggregating

mechanism

aggregation

for

effect

Ap4A-inhibition

induced

.“f)A~P~A(~~;)a~~~~~~7dor~e:~~:’ Lastly,AOP3was pipetted

706

(PAF).

and

inhibited

dissociated

30puM.

induced

various

added

Ap3A

2).

of

factor

homologue

Ap3A (Figure

observed

stimulation

reversion

PAF, the

extends ADP-induced

platelets

spontaneous

plitelet

The

under PAF

Furthermore, PAF

result

we always

aggregation

other

diadenosine

aggregation

released in

any

inhibit

activating

platelet

of

dinucleotide,

This

Ap4A.

the

nmol

that

between

platelet on

as

remove

the

system, by

whereas

effect.

(SO PM),

To

competitively

in

ADP 10

potency.

role

followed

with

with

ADP.

concentrations

aggregation no

of

counteracted

effect effect factor,

When

traces

can

mM

at

a regulatory

platelet

observed

Fig.

could

aggregation

showed

Ap4A.

to

Ap4A

inflammation,

2).

disaggregation high

play

of

concentration

A up

aggregating

thrombosis, immediate

3

that

with

the

may

caused

at

maximal

activating

which

such

which

the

platelet

Ap

Ap3A

chrcmatographing

disaggregating

al.

ihdiately

started

Ap A sample, we repurified 3 Figure 1 also demonstrates

strong

et

Using

investigated

cytes

had

Harrison

no

RESEARCH COMMUNICATIONS

~)IM) of

by

showed

our

results.

(Ap4A)

finding

excluded

which from

than

A contamination

definitely

system,

component

obtained

(Tess

reversion. was

HPLC

aggregating

AND BIOPH%ICAL

low concentrations

at

spontaneous

possible Ap3A

ADP

BIOCHEMICAL

by

at

of of

plate-

a cont.

vol.

118,

No.

BIOCHEMICAL

3, 15’84

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

f-5

6-

6

4

8

15 TIME

3:

Fiqure Ci/mmol)

Liberation was incubated

as described

ADP-induced tides

an

liberates

Ap3A

suggested

cule,

namely

with

platelet-poor

yielded

identical

plasma the

and

are

plasma.

diadenosine

was

then

further

triphosphatase whether

it

for

Ap3A

(17,181

prove

that

to every lar

3),

the

time These

those

obtained

to

Ap3A

aggregation course experiments from

phosphatase ADP

experiments

released involving

prevents from

the

dinucleo-

Moreover,

that

both

slowly

a rate

should

of

in

of

aggregation yielded

a single

not is

by

of

Ap3A

aggregation

aggregation

mediated

Ap A is responsible 3 use of alkaline

for

the

Ap3A by

have

experiments

which

is

less

in

than ADP

constant that

Ap3A. -

5%

to

me-

were

us

portions very

simi-

4). Additional

was at

a potent

prompted

(Fig.

aggregation

phosphatase

707

hydrolases

sufficient

curves of

the

phos-

of

A

whether

unspecific

considerations

ADP

in

known

Our

3

PRP)

are

ADP,

adding

application

an

of ADP 3 H’JAp

of

(18-20).

liberates

These

The

of[

yet

Specific

generate 1.

3).

activities is

Ap,+A

hydrolysis

Fig.

(Fig. formation

plasma

for

continuously

shown

molecomplexes.

Incubation

It

enzyme.

Ap3A

PRP

enzyme

by

a stimulatory

x g centrifugation

the

as

induced

platelet

simultaneous

the

well

of of in

platelets. in

aggregation

adenosine.

12,000

a specific

plasma

of

growing

the to

the

as

Assuming

minute.

Alkaline that

slow

simulate

is

platelet-rich

(Fig.

the

incubated

activity

or

minute

was

after

with

described

diate

the

in

suggesting

associated

stimulus.

that on.

generation

for

degraded

(obtained

course

slow

reflected

been

per

the

Ap3A

phodiesterase,

aggregation

show aggregat

time

responsible

results, not

in

The

that

be

plasma

platelet

22.7 treated

reversible.

Ap3A

were

clearly

on

tritium-labelled of

which

experiments

possibility

this,

AMP,

Our

human

might

human plasma. [3H]Ap A (3.5,uM; Aliquots were wit fi drawn and

in

37’C. & METHODS.

influence

in

the ADP,

degradation

and

Ap3A at

be completely

ADP

confirm

slow

to

from PRP

(9).

antagonistic

proved

Ap3A

ADP with

MATERIALS

aggregation

have

effects

To

under

of

30 (min)

obtained

a concentration

proof from that

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 118, No. 3, 1984

1

3

min

Figure 4: Aggregation induced by repeated additions of small amounts of or by a single application of Ap A. Aggregating nucleotides were added of PRP at the time marked by the3dashed line. a) PRP without any additions. b) Repeated additions of ADP. At the time indicated by the dashed line of ADP was first pipetted to give a concentration of O.Ol,uM. This addition was repeated every minute. c) Single addition of Ap A to give a final of 10,uM. d) Repeated additions of ADP as in exp.b 3but with ten-fold

amounts ADP

imnediately tant

to

induced act is

as

of

ADP (0.1

(1,uM).

The

destroyed the by the

A.

3

stimulatory

e)

Control

results

from

but such

after

addition

Ap

3A

of

(Fig.

phosphatase support

agent,

phosphatase-sensitive,

the

liberated

Heat-denatured The

aggregation

indicate

ADP

enzyme. Ap

,uM).

triangles

as

the as

5).

did

not

conclusion

a source

of

a single Ap4A

(0.1

1,ul cont. higher

addition

of

mM).

Ap A itself

3

is

prevent

aggregation

that an

ADP to 1 ml

Ap A itself

aggregating

3

resis-

does

product

ADP.

Ap3A

b

J/

Figure 5: Effect of alkaline phosphatase (S,ug/ml) on platelet a) Aggregation induced by Ap A (10,utl). induced by Ap3A. phatase was added shortly before Ap A. c) Haat-denatured induced tase was added before Ap A. d) Contro13aggregation In exp. a and c , Ap,,A w 1 s added at 0.1 mM.

708

aggregation b) Alkaline phosalkaline phosphaby ADP (1,uM).

not that

Vol.

118,

The also

No.

aggregatory When

Figs.

4 and

in

human

antagonistic by

liberating

mainly Further

reasons

not

yet

the

unusual

has

work in-vivo

should to

for

modulate

such

are

turn

platelet

human

applied,

always

varying

“intrinsic”

3

which

donors we

observed traces

reflected can

was

in

also

a

be

seen

sensitivities

A and

during

on

of can

of

an

enzymatic

Our

results

activity provide

polyphosphates signal these

effects

are

elicit

in

the

evidence which

nucleotides in-vitro

which

aggregation

3

intracellular that

both

Ap A mediates

diadenosine

confirm

Ap4A,

activation,

aggregation.

depends

as to

COMMUNICATIONS

corresponding

ADP,

Ap

of

of

act

Ap 3A was

towards

properties.

done

of

comparing

however,

released

process

in

to be

by

dinucleotides

the

a variety

of seen

RESEARCH

(13).

functions

considered

from

were,

disaggregating

extracellular been

be

known

which

PRP

platelets

and on

BIOPHYSICAL

concentration can

the

platelets

ADP, Ap4A

in

differences

The

effects

possible

rate

5.

conclusion,

stored

plasma.

same

of

are

AND

Ap3A

as

sensitivity

platelets In

the

5. These

4 and

Figs.

the

of

differences,

different in

potency

measured,

quantitative in

BIOCHEMICAL

3, 1984

for have

(7). also

ope-

function. ACKNOWLEDGEMENTS

We thank Prof. W. Kersten for critical discussion cellent technical assistance. We are also indebted ticular to E. Hornemann. This work was supported schaft (SFB 118) and Fonds der Chemischen Industrie.

and Herbert Briinner for exto all blood donors, in parby Deutsche Forschungsgemein-

REFERENCES 1. 2. 3. 4. 5. 6.

7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

Ogilvie, A. & Jakob, P. (1983) Anal. Biochem. 134, 382-392 LDthje, J. E Ogilvie, A. (1983) Biochem. Biophys. Res. Commun. 115, 253-260 Rapaport, E. & Zamecnik, P.C. (1976) Proc. Natl. Acad. Sci. U.Sx.73-,3984-3988 Grumnt, F., Waltl, G., Jantzen, H.-M., Hamprecht, K., Huebscher, U. E Kuenzle, C.C. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 6081-6085 Rapaport, E., Zamecnik, P.C. E Baril, E.F. (1981) J. Biol. Chem. *,12148-12151 Zamecnik, P.C., Rapaport, E. & Baril, E.F. (1982) Proc. Natl. Acad. Sci. U.S.A. 2, 1791-T794 Varshavsky, A. (1983) Cell 34, 711-712 Flodgaard, H. 8 Klenow, H. n982) Biochem. J. 208, 737-742 Harrison, M.J., Brossmer, R. & Goody, R.S. (19m FEBS Letters 2, 57-60 Born, G.V.R. Nature 194, 927-929 (1962) Nachman, R.L. & Ferrx B. (1974) J. Biol. Chem. 2, 704-710 Ogilvie, A. (1981) Anal. Biochem. l& 302-307 Gerrard, J.M. (1982) in Methods in Enzymol. 86, 642-654 (eds. E.M.Lands d W.L. Smith) Academic Press, New York Benveniste, J., Cochrane, C.G. 6 Henson, P.M. (1972) J. Exp. Med. 136,1356-1377 Chignard, M., LeConedic, J.P., Tence, M., Vargaftig, B.B. E Benveniste, J. (1979) Nature 279, 799-780 Ohlrogge, J.B.3982) Trends in Biochem. Sci. July, p-235 Sillero, M.A.G., Villalba, R., Moreno, A., Quintanilla, M., Lobaton, C.D. E Sillero, A. (1977) Eur. J. Biochem. 76, 331-337 Jakubowski, H. B Guranowski, A. (1983) J. Biol. Chem. 258, 9982-9989 Ogilvie, A. F, Antl, W. (1983) J. Biol. Cheer. 258, 4105709 Vallejo, C.G., Lobaton, C.D., Quintanilla, M.7’ Srllero, A. E Sillero, M.A.G. (1976) Biochim. Biophys. Acta 9, 304-309

709