The presence of diadenosine 5′,5‴-P1,P3-triphosphate (Ap3A) in human platelets

Vol. 115, No. 1, 1983 August 30, 1983

BIOCHEMICAL

OF

DIADENOSINE 5',5"'-P',P3-TRIPHOSPHATE IN HUMAN PLATELCTS

Jtirgen

Received

17,

July

RESEARCH COMMUNICATIONS Pages

THE PRESENCE

FahrstraRe

AND BIOPHYSICAL

20,

Lijthje

Institut

fiir

and

Adaling

253-260

CApsA)

Ogilvie

Physiologische

Chemie,

D-8520

Erlangen

GFR

1983

Diadenosine

triphosphate CAp3A) has been identified and quantified in human using a coupled enzymatic assay specific for Ap3A, after fractionation of acidic extracts with high-performence liquid chromatography. Upon thrombin-induced aggregation, Ap A is released together with the homologue diadenosine tetraphosphate CAp4A 3 . Extracts of human platelets do also contain enzymatic activities that degrade diadenosine tetraphosphate as well as diadenosine triphosphate. These enzymes, however, are not released during thrombin-induced aggregation of the platelets.

platelets

intracellular

The

CAp4A)

has

level

been

cells

eukaryotic meraseo(

with

The

enzymatic

ing

lysyl-tRNA

identified

It

has

of

Ap4A

acting

main

recently

been

which

is

suggested from

the

cell

proliferation.

we

of

as

Mg

that and

the that

a potential

been

shown

in

a reaction

, and

Lysine

antagonist

by

Enzymatic assay as

izi%i;3" assa of descrrbed'bywL

was

a gift

Ap A and

A

Ogilvie

of

a role

as

Ap3A also of

DNA poly-

of

Dr.

A 9).

Ap4A The

also

is

is

mixture ADP

syn-

abundant

amounts

growth

in

of

high

has

factor

stimulation

present

ATP, Ap3A

It

(8).

additional

during

of

(6,7).

platelets

the

contain-

instead CAp3A).

Jakob

contain the

in

released Ap4A

and

an

involved

MATERIALS Rea Cla;elin;lents

of

DNA

triphosphate

platelets

being

is

is

catalyzing

With

diadenosine

human

homologue Ap3A

(5).

aggregation play

thus

activity

molecule and

2+

that

might

signal (2,3)

cells

during

tetraphosphate

proliferative

Ap4A

was

eukaryotic

platelets,

describe

platelets

Ap3A

has

ATP,

the

C3,4>.

product

Ai4A

leased

human

Ap4A

diadenosine to

potential of

of

reported

that

the

a primer

released

muscle Here

of

as

reaction in

nucleotide related

binding

synthetase,

has

unusual

directly

A target

synthesis

the

the

be

non-covalent

Ap4A

however, been

of to

Cl).

showing

thesis

been

found

re-

smooth-

amounts

aggregation.

The

in role

discussed.

& METHODS E.

Holler,

[3H]

was measured method for

Ap3A

was

from

by a coupled measuring Ap3A

Amersham

luminescence is similar

0006-291X/83

253

$1.50

Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 115, No. 1, 1983

to the kinase.

assay The

BIOCHEMICAL

of Ap4A technique

AND BIOPHYSICAL

extended by the addition has been described in

of detail

RESEARCH COMMUNICATIONS

phosphoenolpyruvate-pyruvate (7).

Isolation of platelets and extraction of nucleotides. Human platelets were prepared from fresh blood essentially as described (81, with the modification that the forces of sedimentation were increased CqOOO x g, 10 min). After the sedimentation, the platelets were resuspended in Tyrode buffer (without la% Ca 1 and quickly mixed with ice-cold TCA (IO % final cont.). Further treatment of the extract and its neutralization has been described (7,9). Release experiments. Washs$ platelets (2 x IO9 cells/ml) were suspended in Tyrode buffer (without Ca 1 and incubated at 37OC for 6 min with thrombin (2.2 U/ml). After rapid cooling, the samples were centrifuged at 12.000 x g for 5 min. The supernatant and, separately, the pellets were extracted with TCA and analyzed as described above. High-performance liquid chromatography Ch.p.l.c.1. nique of Schweinsberg and Loo (IO) was modified extract cribed (7). 200 ~1 of the neutralized c3H]Ap4A and applied onto the column. Elution Fractions of 0.2 ml were collected. The amounts mined using the absorption coefficients obtained

The reversed for our intentio were mixed with rate was constant of ADP and ATP from standard

phase techas des[ s H] Ap A and at 1 m ? /min. were deterruns.

lysate and measurement of enzyme activities. The sedimented platewere gently shaken for 15 min at 4oC with an equal volume of Hepes buffer (50 mM pH 7.75, MgCl21 mM) containing 1 % (v/v> Nonidet P40. After centrifugation at 12,000 x g for 15 min, the supernatant was used for measuring enzymatic activities. Hydrolysis of Ap4A, Ap3A and of ATP was followed by product analysis with thin layer chromatography as described (17). Platelet

lets

RESULTS Identification

and

human

platelets.

graphed

with

The an

identification the

determined

vely,

coeluted

both

dinucleotides

with

the

in

human

ing

the

well

with

the

optical

in

Ap4A,

ATP,

the

also

of

Ap4A

Ap,A

ADP

the Ap4A,

suggesting

fractions and

of

of in

upon

releasenucleotides

peaks the

similar

the

as

of

as

the

well

six

different

dense

254

When storage

1).

When

Ap3A

apply-

from

the

determination.

optical

The

aggregation. their

of

overestimation

from

donors

respecti-

presence

of

material.

chromatograms.

enzymati-

Ap4A,

enzymatic

therefore

with

The

asAp4Abeingpresent

corresponding

were

compared

(Table

an

allowed

Puantitation

quantitation

unidentified

thrombin-induced from

as

revealed the

Ap3A

h.p.1.c.

platelets

value

chromato-

the I).

amounts

the

same

and

(Fig.

high

was

2).

suggesting

Ap3A

however,

as

Ap3A

platelets

revealed

contained

ADP),

of

in

profile

(Fig.

peaks

that

well

as

ATP,

CAp,A)

elution

nucleotides

coefficient,

almost

measurement

concerning

lyses

almost

in

chromatogram

results

of assays

extinction

complex

(i.e.

optical

extract

platelets

human

The

standard

distinct

yielded

I).

radioactive

luminescence

integrator

h.p.1.c.

the

the

platelets

profile

of

triphosphate of

(Fig. nucleotides

as

two in

blood

analogous

Release

as

coupled

The

platelets

a mixture

appropriate

optical

major

of

diadenosine extract

system

some

run

of

neutralized

h.p.t.c. of

parallel

tally

quantitation

by peak

in

All

quantitative

the

enzymatic

ana-

contents

of

Ap3A,

listed

in

Table

are treated granules

with (II).

the

1.

thrombin, Table

2

Vol. 115, No. 1, 1983

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

T

ATP

4DP

JL ;: II

si

Y

10

20

R E T

Figure human Aliquots activity described

shows with

the thrombin.

dinucleotides

1:

High

E N T

I

0 N

T

I

H E

performance [ H] Ap

liquig chromatography A and [ H] Ap,+A were of the collecte a fractions (0.2 ml) (4, for Ap A-luminescence (01, and under MATE ;1 IALS & METHODS.

platelets.

results

of

two

Almost are

extruded

all

,I

I

P:...~....,....l....,....,

I

Q I)

independent and

to

of an acidic added as internal were measured for for Ap4A-luminescence

experiments

Ap3A a

Ap,+A

is

relatively

30

(MINUTES)

with released

platelets

being

during

higher

extract markers. tritium (0)

AMP

than

ATP

treated Both

and

even

Aq A

AP,A I

I

ADP

radioas

aggregation.

extent

ATP

from

I

Ap4A I

I

l....l....l....l....l....l,.,.l....1. 10 II E T E N T I 0 N

Figure phase cedure

20 T I n. E

2: Separation of ATP, ADP, AMP, Ap A, Ap4A and column by high-performance liquid c 2 romatography. are given under MATERIALS & METHODS.

255

30 (MINUTES) Ap

A on a etails

a

reversed of the

pro-

Vol. 115, No. 1, 1983

BIOCHEMICAL

AND BIOPHYSICAL

Table Contents

of

Diadenosine

tetraphosphate Donor

Ap3A

and

(Ap3A), ADP

in

pmol/106

Diadenosine

Human

Platelets

ATP

Ap4A (

platelets

ADP )

E.R.

0.52

0.53

40.50

30.10

Y.W.

0.41

0.36

32.48

18.12

E.H.

0.83

0.43

67.23

42.33

J.L.

0.63

0.39

36.95

24.50

H.8.

0.63

0.56

48.70

31.50

E.A.

0.94

0.51

19.50

13.40

Neutralized Ap4A, tography

I

triphosphate ATP

(Ap4A),

RESEARCH COMMUNICATIONS

ATP

acidic and ADP, and

of

extracts

from

human

respectively, enzymatic assays

by as

Table Release from

of human

Experiment

diadenosine platelets Treatment

platelets

means described

of

uere

and

other

thrombin-induced Ap3A

Ap4A

(Extracellular total

for

Ap3A,

liquid chroma& METHODS.

II

triphosphate after

measured

high performance under MATERIALS

nucleotides aggregation

ADP

ATP

content,

% of

amount)

Buffer

5.2

n.m.

1.3

1.3

Thrombin

98

88

63

29

Buffer

31.5

14.1

16.7

12.6

Thrombin

78.5

90.5

76.7

48.1

I

II

human platelets (about 2 x 109/ml) suspended in Tyrode buffer Cuithout 1 were treated with thrombin as described under MATERIALS & METHODS. A buffer-treated platelet suspension served as a control. After incubation, the The adenine nucleotides of the pellet probes were cooled and centrifuged. (cellular content) and of the supernatant (extracellular content) were determined. The sum of the corresponding pellet and supernatant contents was set at 100 X. Wa&ed

Ca

256

Vol.

115,

ADP

(Table

lets

of

the

2).

The

platelets,as

realized

pLateLets

and

ATP,

was

tested

100

pM

With

in

Ap3A

as

the

a concentration

of (Fig.

from in

paration

of

not

shown). are

We

also

the

3b).

the

crude

This

measured

results

KM for

Ap3A

be

for

using

at

100

the

who

not

a different the

wash

Liquid

with

the

three

that

the and

activities

enzymatic not after

0.25

and

cases

AMP.

substrate

con-

being present . was only partial-

pM

of

find

of

Ap3A,

all

Ap4A

are

degradation

method

detected

of

nucleotide

hydrolysis

did

using the

Ap4A,

of Ap3A

Measuring

membrane-bound

hydrolysing

Ap3A

& Klenow

platelets (8).

obtained

whereas

and

was in

degradation

obtained

Flodgaard

suggests or

the

3a)

platelets

Each

with

could

result

100 of

Ap,A

degradation

platelets.

effect

human

of

dissolves

of

a remarkable

enzyme

intracellular

and

human

kinetics

human

and

rapidly

was

The

activities

washed that

the

from

product

(Fig.

aggregation

Ap,A

we observed

of

from

partial

degradation pM

report

extracts

hydrolyzing

tes

3.6

plate-

buffer-treated

to

degrade

from

5 show

lysates

major

complete

that

a detergent

3.6

The

substrate,

ly metabolized

no

ATP.

from

was due

prepared

P 40, 3 -

RESEARCH COMMUNICATIONS

examination.

were

concentrations,

Almost

Ap4A

Lysates

in

of

II

activities

% Nonidet

respectively, two

centration.

different

enzymatic

Figures

at

nucleotides

microscopic

(12).

case

of

experiment

by

0.5

membrane

Ap4A

in

cellular

containing

AND BIOPHYSICAL

release

contain

Total

AMP.

cellular

of

partial

individual

buffer

at

BlOCHEMlCAL

1, 7983

another

Human to

No.

for the

activities

'in with

thrombin-induced

pre-

platelets,

substrates

a contamination

the

(data the

lysaplasma.

aggregation.

a

. 30

60

QO

30

min

60

90

Figure 3a: Kinetics of Ap3A hydrolysis by a lysate of human platelets and product analysis. The degradation of L3H] Ap A (3.6 CIM) by a lysate of human platelets was folloued by withdrawal o T aliquots at different times and chromatography on poly(ethylene-imine)-cellulose thin layers. The development of the plates was performed with O.SM,lithiun chloride. The sum ADP (A) and AMP (0) was of radioactivity found in Ap A (01, ATP (A), 3 set at 100 X. Figure 3b: Kinetics of two different substrate described for Fig. 3a. (0). One unit of tritium

Ap3A hydrolysis by a lysate of human platelets at concentrations. The method nas identical with that The Ap3A concentrations were 3.6 fl (0) or 100 WM radioactivity was equivalent to IO3 cpm/pl assay. 257

min

Vol. 115, No. 1, 1983

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

5 ” ” ’ n” s ” or* 30

Figure product human

4:

platelets

Figure CA),

3a. The sum of and AMP CO) was

Figure duct

of

The

5: analysis.

platelets activity

No activity all

Kinetics

analysis.

on

activity,

was

gives

to

the

degrade

hydrolysis

b

degradation

measured

the set

a lysate

of

analogously

to

radioactivity at 100 X.

the

found

(a),

ATP

for

CA),

of ATP hydrolysis in a lysate of The degradation of [3H] ATP CO.25 KM) was determined as described for Figure 3a. found in ATP CO), ADP (A), and AMP CO) was

human platelets by a lysate of The sum of the set at 100 X.

Ap3A,

the

Ap4A

or

ATP

could

remained

no

evidence

extruded

be

detected

extractable for

release

nucleotides

in

from of

(data

the

ADP and prohuman radio-

supernatant.

pellets.

hydrolyzing not

of

described

Ap,+A

ALmost

Therefore,

enzymes

min

and

UN) in a lysate

procedure

in

90

platelets

human

[ r H] Ap,+A (3.6

of

Kinetics

however,

experiment

Ap4A

60

the

that

are

able

shown).

DISCUSSION Human

platelets

contain

as

homologue

amounts

its

and

quantitation

of

for

this

(7).

lets

purpose

is

in

raised in

the

the

that

blood

extracted tides

are

Diadenosine upon

blood

platelets

also

present

triphosphate

thrombin-induced

nules

which

releasable ta,

i.e.

ATP

(15).

also

is

Located contain

(results in

contain

total

data

other was

other our extrusion

ADP

The

found

of

in and

Ap4A Our

more

Ap4A

not

This

suggests

given).

high

identification

FLodgaard pool

platelets.

the

developed human

data,

have

KLenow

that

and

plate-

is

present

however,

Ap3A

than

that

the

show can

be

dinucleo-

constituents. completely

are Ap4A

released that

releasable

of

and

(13,14).

suggesting

results

ATP

similarly

methodology

2 - 3 times

blood almost

(8).

complete in

in

a specific

of

the

aggregation

nucleotides,

almost

whether

extracts

from

using

published

to

(Ap3A)

tetraphosphate

performed

concentration

with as

circulating

was

The

accordance

triphosphate

diadenosine

Ap3A

question

whole

diadenosine

Ap3A

constituents in (8)

258

good and

accordance partial

from is

the

platelets

stored

(II).

in

dense

Concerning with

release

graother

published of

ADP

daand

Vol. 115, No. 1, 1983

Human

platelets

specific from

contain have

(20). suggest

that

the

that

for

From

studies

with

lets,

however,

Flodgaard occur

(211, sine

are

has

and

since

will

shown be

necessary

triphosphate

act a poor to

in

look

the

synthetic

devoid that

of the

the

together effects gave

of

the

no

evi-

extracellular

space.

dinucleotides

machinery

protein

di-

function

released

the

the

investigated

however,

synthetases protein

If

possible

into

phos-

are

(5).

synthetic

biosynthesis

Plateactivity.

of

Ap4A

might

megakaryocytes. inhibit

interferes

might be

the

suggested

to

limit

activities

almost

i.e.

found Ap3A

to

be

therefore

cells,

triphosphate been

of to

to

20)

dinucleotides

(II)

are

-

concentrations

We have

experiments,

aminoacyl-tRNA

known

been

order

aggregation

enzymatic

purified

precursor

Ap3A

those

side-products

8 Klenow in

Since

sine

be

of

in

The

the

enzymes.

enzymes

Highly (17

specific

variable

granules

unclear.

those

nucleotides

at

Ap4A. Ap4A

less

different

inactive remains

for

of

Ap3A

by

as

and

degradation and

degraded

aggregation

well

as

(16) enzymes,

the

Ap4A

metabolically

degradation.

a release

to

for

platelets

during

corresponding by

believed

in

Ap3A

specific

of are

in'human

possibility

dence

It

stored

enzymes

nucleotides

responsible

degradation

RESEARCH COMMUNICATIONS

Ap3A

for

those

nucleotides

are

those

has

of

both

nucleotides

with

be

kinetics

hydrolyze

described

Besides

could

The

AND BIOPHYSICAL

that

been

tissues.

phodiesterases

the

enzymes

hydrolases different

of

BIOCHEMICAL

the with

as

an

primer for

binding the

of

DNA synthesis

further

Ap4A

metabolism

antagonist of

of

to

of

Ap4A.

DNA polymerase

Ap4A

(221,

diadeno-

Furthermore, when

physiological

compared functions

6(

Ap to of

A itself 3 Ap4A (4).

diadeno-

platelets. ACKNOWLEDGEMENT

We thank Herbert Brljnner for critical discussion. meinschaft SFB 118.

for This

skillful work

was

technical supported

assistance by the

and Deutsche

Prof. W. Kersten Forschungsge-

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RESEARCH COMMUNICATIONS

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260