Inhibition of epinephrine-induced platelet aggregation by a derivative of wheat germ agglutinin

Vol.98,No.1,1981 January

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages

15,1981

INHIBITION

OF EPINEPHRINE-INDUCED DERIVATIVE OF WHEAT Pankaj

From

Received

Ganguly

and

PLATELET AGGREGATION GERM AGGLUTININ Nancy

G.

BY A

Fossett

the Department of Hematology, St. Jude Hospital and Department of Biochemistry, Tennessee, Memphis, Tennessee

November

297-302

Children's University 38101

Research of

29,198O SUMMARY

A nonagglutinating derivative of wheat germ agglutinin has been prepared The and used as a probe to explore the initial events in platelet activation. lectin derivative had no effect on platelet aggregation by adenosine diphosphate, collagen, ristocetin, wheat germ agglutinin or trypsin but aggregation Unlike thrombin, the induced by epinephrine or thrombin was inhibited. inhibition of aggregation by the derivative could not be overcome by The derivative did not affect increasing the concentration of epinephrine. the binding of [3H]dihydroergocryptine to platelets. A 74,000 dalton protein isolated from platelet membranes by lectin affinity chromatography strongly by thrombin but not by epinephrine. The inhibited platelet activation receptors for thrombin and for epinephrine on platelets are different but they are closely linked.

Human with

a

initial to

platelets

number step

of in

specific

of

available

on

the

primarily

between

associated

with

but

by

of

a

rapidity

host

of

which

other

To germ

from

agglutinin

aggregation was

297

is

This

paucity

little

our

these

initial

events,

affected

does

not

This

lectin

while (4).

broad

inability

metabolic

thrombin

and

information

general

capacity.

ligands

information of

and

which

The

the

the

by not

these

While

there

2).

aggregation

(3).

(WGA)

binding

of cell

interactions

explore

cell

to

incubated

(1,

binding

events

involved.

events

agents

be

known,

the

when

catecholamines

leads

of are

of

platelet

reaction

to

series

considerable

inhibited

believed

aggregation

wheat

and

surface

activation.

retains

strongly

cell

ligand-specific cell

a secretion

interactions

the

a derivative

platelets

induced

platelet

to

is

complicated

molecular

due

distinguish

tive

a

the

undergo thrombin

activation on

following

and including

platelet

characteristics

prepared

agents

receptors

secretion

is

aggregate

Further

to changes we

have

stimulate derivaaggregation studies

0006-291X/81/010297-06$01.00/0 Copyrighf 0 1981 by Academc Press, Inc. All rights of reproduction in any form reserved.

Voi.98,

No.1,1981

revealed

that

giving

rise

BIOCHEMICAL

the to the

epinephrine

and

platelet

derivative

receptors

be closely

can

possibility

thrombin for

AND

BIOPHYSICAL

block

that

platelet

there

We present

and for

COMMUNICATIONS

aggregation

may be a single

on platelets. epinephrine

RESEARCH

data

thrombin

are

by epinephrine receptor

which

for

both

show that

different

but

the

they

may

coupled. MATERIALS

AND METHODS

Blood was collected from healthy volunteers in plastic syringes utilizing 0.1 volume of 3.8% sodium citrate as anticoagulant. The red cells were removed by low speed centrifugation and the platelets were isolated as described (5). Wheat germ agglutinin (WGA) was purchased from U.S. Biochemicals (Cleveland, Ohio). Data on its purity and interaction characteristics with platelets have been published (6-8). The WGA derivative was prepared by treating the lectin in 65% formic acid with excess CNBr for 20 hr Epinephrine (1 mg/ml) in 0.15 M NaCl was purchased from Parke Davis (4). (Detroit, Michigan). Platelet aggregation was measured in a dual channel aggregometer (Payton, Buffalo, New York) (7). The experimental sample was analyzed in one channel while a control was run in the other. The amounts of stimulants shown are final concentrations. To measure secretion, platelets were loaded with [14C]serotonin (59 mCi/mmol, Amersham, Arlington Heights, Illinois) and aggregation experiments were carried out as above. Five min after the addition of the stimulant, 0.1 ml of 10% formaldehyde was added to the platelet suspension. The samples were centrifuged and the radioactivity in an aliquot of the supernatant was measured and expressed as percent of appropriate controls (7). in phosphate-buffered saline (PBS) was first Platelets suspended incubated for 10 min at room temperature with different amounts of the WGA derivative and then for another 15 min with a constant amount of C3Hldihydroergocryptine (DHEC, 24 Ci/mmol, New England Nuclear, Boston, Massachusetts) in the dark. The platelet-bound DHEC was separated from free DHEC by filtration through 1.2 u millipore filters under reduced pressure (9). The tubes and filters were then washed with 5 ml of buffer. The filters were dried and counted in a Packard liquid scintillation counter with an efficiency of 45%. Nonspecific binding was determined in the presence of 100 fold excess phentolamine (Ciba, Summit, N.J.) (10). RESULTS AND DISCUSSION Incubation

of

aggregation.

wave after

aggregation.

The

induced

by epinephrine

trypsin

was

inhibition perturbation

with

The aggregation

a secondary to

platelets

not of

There

some delay. WGA derivative

affected

platelets

amounts

of

was characterized

while

under

the

by

by a primary

inhibited

by collagen, same conditions

aggregation the

epinephrine

was no detectable

strongly

aggregation

epinephrine-induced of

small

WGA derivative.

was

led

to

cell

wave followed shape

change

platelet

by prior

aggregation

ADP, WGA, ristocetin (Fig.

1)

(4).

not

due

to

The

inhibitory

Thus,

and the

a nonspecific effect

was

Vol.98,No.1,1981

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

b.

1

,

I

1

1 MINUTE

1 MINUTE

A Fiq. 1. (A) Inhibition of epinephrine-induced aggregation of human platelets in plasma (3 x 108/ml) by the derivative of wheat qerm aqqlutinin. Pattern (a). shows the control while in patterns (b) and -(c) piatelets were preincubated with 100 pg/ml and 150 pg/ml of the lectin derivative respectively. Aggregation was initiated with 10 pg of epinephrine added at the arrow to a final volume of 0.5 ml of the platelet suspension. (B) Lack of effect of the lectin derivative on ADP-induced platelet aggregation. This experiment was done as in (A) by adding 1 x 10 5 M ADP at the arrow. Pattern (a) shows the buffer control while in pattern (b) the sample was preincubated with 100 pg/ml of the derivative.

more

on

prominent

aggregation

by

platelets the

we

conditions from

serotonin

release

overcome One

with this on

is

not

the

epinephrine-induced phenomenon

that

of

of

or

(c)

of

only the

the C3H]OHEC to

its of

to

2).

299

the sample

Under release

(a)

release.

different

the

platelet

receptor

of

showed This

inhibi-

could

not

derivative

activation on

87%

be

that

WGA the is

by

platelets.

concentrations The

suggesting by

platelets.

WGA derivative

platelets.

platelets

aggregation

4%

blocks

to

effect

1,

from

epinephrine.

WGA derivative binding

from

Fig.

secondary

secreted

aggregation,

had by

Since

materials

serotonin

In

secretion

the

DHEC

with

blocked

(b)

of

aggregation.

inhibited.

measured

platelet (Fig.

release

for

binding

binding

associated

concentration

epinephrine

the

been

or

the

primary

derivative

sample

we

on

also

aggregation

point,

derivative affect

was

increasing

than

the WGA

while

possibility

competing explore

the

platelets

platelet by

has

measured

when

serotonin

of

secondary

epinephrine

(ll),

tion

the

To of

derivative inhibition a post-binding

the did of

Vol. 98, No. 1,198l

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

6-

I0

2

I 6

4

I 8

I IO

, 12 DERIVATIVE

LECTIN

I I 14 16 pg/ml

I I8

I 20

Fiq. 2. Effect of increasing concentration of the derivative of wheat germ agglutinin on the binding of [3H]dihydroergocryptine to platelets. Platelets in PBS were first incubated with different amounts of the lectin derivative for 10 min at room temperature and then a constant amount (1 nM) of DHEC was added. The amount of DHEC bound to platelets was determined after 15 min at room temperature by millipore filtration.

The

above

derivative two

results

are

different

different of

of

isolated

glycoprotein

tested.

Similar

the

glycoprotein

The

glycoprotein

conditions

be

platelet

did

not

of

and

different

caused

led

aggregation

the

The by

collagen

by

epinephrine

only

by

on

solubi-

isolation effect

of of

several

ADP,

induced

WGA or

of

to

(4).

by

the

molecules

chromatography

induced

aggregation

platelet

two

74,000

aggregation

affect

epinephrine

WGA derivative

aggregation

blocked

on

weight

platelet

for

Affinity

the

molecular

to

located

molecule.

utilizing

on

receptors

this

agents or

was

trypsin, (Fig.

thrombin

3).

under

these

(4).

adrenergic,

receptors

p-adrenergic

catecholamines

in

evidence

the

the

may same

apparent

Catecholamine

through

the

membranes

a glycoprotein

that

which

sites

/ platelet

lized

indicate

that

or

aggregation

receptor

prepared

a

explore

the

cells

by

although

the unclear

derivative events

in

to

response.

platelets

remained

have

according

cellular

human

receptor

nonagglutinating initial

a of

have

different

dopaminergic

elucidating

an or-adrenergic platelet

on

of

platelet

classified

the

potency

There

is

catecholamines identity (11,

WGA activation

300

been

and 12, and

used (4).

of

exact

indirect mediated

properties

We it

be

have as

This

a

CY-

various

some may

13).

as

of recently

probe derivative

to

BIOCHEMICAL

Vol.98,No.1,1981

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

1 MINUTE

I 1 1 MINUTE

B

A

Fiq. 3. (A) Lack of on epinephrine-induced Pattern (a) is the incubated for 10 min Aggregation protein. arrow to a final platelet aggregation ment was done as in the arrow. Pattern was first incubated ture before addition

blocked

platelet

induced

by

on

number

to

in

exerts

a

action study

through

a

the

isolated

are

by

two

blocked

effect

on

platelet

aggregation

protein

concentration.

Thus,

tion leads

by

epinephrine to

signal

by generation.

it

only

agent

the

observations

301

derivative inter-

results

unlike

platelet

of

the

at

inhibits

processing suggest

could

and

(b) but

ten

fold

platelet of

the

derivative

a

this

thrombin,

activation

even

derivative

with

the

The

by

act

specificity

epinephrine-platelet

(a)

was

may

of

concentration

epinephrine

lectin

These

that

thrombin.

thrombin-induced

study

epinephrine

unlikely

epinephrine

by

aggregation

This

degree

aggregation

the

interfering

and

differences:

platelet

the

(4).

high

on

except

important

increasing

glycoprotein

The

effect

while

affected thrombin

made

other

epinephrine

not

whether

reaction

any

by

receptor.

epinephrine-induced

reversed

was

hypothesis

inhibitory

there

and

agents

inhibition

that

thrombin

single

affecting

of be

other

the

without

inhibition not

of

nonspecific

show

by

explore

platelets

observed

of the isolated receptor for the lectin derivative aggregation of platelets in plasma (3 x lOa/ml). buffer control while in pattern (b) the ligand was first at room temperature with 4 pg/ml of the 74,000 dalton was initiated with 10 ug of epinephrine added at the volume of 0.5 ml. (13) Inhibition of thrombin-induced by the 74,000 dalton protein of platelets. This experi(A) but with 50 mU (1 nM) of thrombin which was added at (a) is the buffer control while in (b) the thrombin sample with 1.2 pg/ml of the protein for 10 min at room temperato the platelets.

stimulation

a

undertaken

effect

the had higher

aggrega-

the

receptor

that

the

which thrombin

no

BIOCHEMICAL

Vol.98,No.1,1981

receptor

and

closely

linked.

epinephrine

AND

receptor

on

BIOPHYSICAL

platelets

are

RESEARCH

COMMUNICATIONS

different

but

they

may

be

and

by

ACKNOWLEDGMENTS This

study

was

supported

by

grants

HL

16720

and

CA 21765

from

NIH

ALSAC. REFERENCES

1.

2. 3. 2 6. 7.

O'Brien, J. R. (1963) Nature 200, 763-764. Sidhu, P., Sutherland, S. B., and Ganguly, P. (1979) Am. J. Physiol. H353-H358. Ganguly, P. (1974) Nature 247, 306-307. Ganguly, P., and Fossett, N. G. (1981) Blood, in press. Ganguly, P. (1972) J. Biol. Chem. 247, 1809-1816. Ganguly, P., and Fossett, N. G. (1979) Biochem. Biophys. Res. Commun. 1154-1160. and Sidhu, P. (1979) Biochim. Biophys. Ganguly, P., Fossett, N. G.,

237,

89, Acta

586, 574-583.

8. 9. 10. 11. 12. 13.

Ganguly, P., 261. Ganguly, P., Alexander, R. 61, 1136-1144. Mills, D. C. 195, 715-722. Grant, J. A., Rossi, E. C., 286-294.

and

Fossett,

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G.

and H.,

Sonnichsen, Cooper,

B.,

W. J. (1976) and Handin,

B.,

Robb,

and Scrutton, Louis, G.,

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(1980)

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Roberts,

C. (1979) Zeller, E.

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Haematol. BI r. J. R. I. (1978) J. G.

C.

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34, Clin.

291-301. Invest.

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Nature 277, 659-661. A. (1979) J. Lab.

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