The pH dependent platelet utilization of arachidonic acid and a thromboxane A2 mimetic, in vitro

Pharmacological

Research

Communications,

Vol. 17, No.

609

7. 1985

THE pH DEPENDENT PLATELET UTILIZATION

OF ARACHIDONIC

AND A THROMBOXANE A2 MIMETIC, P.J. National

Kerry

and C.J.

VITRO

IN

Paton

Division of Blood Products, for Biological Standards Holly Hill, Hampstead, London NW3 6RB, U.K.

Institute

Received

in final

form

ACID

17 April

and Control,

1985

s-rY

The

effects

aggregation

in

observed

in

of vitro

tissue

thromboxane

A2 (TxA2)

previously

reported

contrast,

aggregation in

inhibited

importance and these

showed

the

studied

and

to

and

pH on

simulate

(PRP).

This

inflammation.

in

is

acid

proteins

in

hypothesis

that

that In

suspensions

arachidonate

conditions.

to

aggregation.

platelet

to both

of

C02-treated

similar

arachidonate-induced

response

Platelet mimetic

sensitivity effect

washed

platelet pH changes

7-oxabicycloheptane increased

of

of plasma

support

the

for

under

(CO2)

stasis

by

plasma

protein-free),

been

fluid

induced

platelet-rich

dioxide

have

aggregation

was

carbon

and TxA2 mimetic,

These

the

control

the

pH effects

(i.e.

results

confirm

of platelet are

the

function

dependent

on

proteins.

Introduction Tissue

fluid

inflammatory fell

to

pH

conditions.

5.6 during

exudates

in

Sheldon,

1971;

communication).

0031-6989/85/070609-l

has

Schade

abscess

rabbits

been

Hutchins Little

O/SOS.OO/O

(1924)

formation

and

sheep

fell

& Sheldon, is

known

measured

of

in

observed

and the to

a that

pH of

below

pH 7.0

B. Greenwood,

local

pH changes

The Italian

local

of pH

inflammatory

1972;

0 1985

number

(Edlow

&

personal occuring

Pharmacological

in

Society

610

Pharmacological

thrombotic

disorders,

one

reason

problems

of

sampling

in

However,

by

analogy

with

and

biochemical

pathological disorders

of

significant progress

of

work

pH observed

in

has

of

arachidonic

acid

with

also

circulating

pH may occur

the

ethical

considering

the

place to

during

assume

that

and may influence

relatively

the

the

to

in ---

vitro It

1984).

interaction

response

small

sufficient

& Paton,

the

of

is

changes

the

response

to

possible

arachidonic of

properties) platelets

that acid

and

arachidonic with

to

in

increase in

metabolite

of

in

binds

Granstrom

Gerrard

aqueous

albumin & Kindahl,

plasma

these

to

derivatives,

& Samuelsson,

We have

further

free

agonists

the

binding

rapidly

of

may

arachidonate 1980;

but

TxA2

the

albumin

in

Collier

&

of approximately

Svensson

react with

and

& White,

1977).

H2 (PGH2) both

than

to

solutions

(Hamberg,

prostaglandin

pH sensitive

albumin

TxA2 has a half-life

1980).

of

due

(Stuart,

protein-free

bound

to plasma

aggregation

blood

presence

Granstrom

by

taking

aggregating

acid

McDonald-Gibson,

form

situations.

a cycle-oxygenase

that

platelet

Folco,

clinical

7, 1985

affected.

control

the

and

reasonable

are

platelet

Arachidonic

seconds

and

aggregation

(Kerry

potent such

practical

and

is

that

platelet

A2 (TxAZ;

proteins, is

shown

pH may modify

thromboxane acid

in

inflammation

sensitivity

in

the

processes it

Vol. 17. No.

disease.

Recent

changes

Communications,

inflammation

changes

the

being

experimental

haemostasis,

local

Research

is

with

protein PGH2

stabilized

& Samuelsson, and the

30 in 1975;

endoperoxide, plasma

binding (Maclouf,

of

proteins TxA2 more Kindahl,

1980). investigated

the

significance

of

the

pH

Pharmacological

Research

dependent

binding

proteins,

with

Uaterials

and Methods

Preparation

of

Blood

from

Communications,

of

arachidonate

respect

platelet-rich healthy

preceeding

venepuncture,

blood.

plasma

one

part

counts

platelet

fell

were

experiments

completed

within

Preparation

of

platelets.

were

Crawford

(1981)

acid

centrifuged

and

washed

twice

di-sodium

in

Hanks

solution

adjusted PRP and Carbon

to fell

ml

1,200

the

1,200

containing

give within

the

of

of 4 ml

capacity,

weeks

centrifuge

citrate

to

9 parts

g for

10 min.

- 3.6x1011/1.

temperature

The and

all

of

to

pH 6.2

g for

Menashi, with

20 min.

10 min.

M citric was

4 mM KCl,

pellet

Platelet

&

pellet

The platelet and the

2.7~10~~

1.0

The

150 mM NaCl,

Weintroub

4 mM

suspension resuspended counts

comparable

to

were

that

of

on to

the

tube

(10

- 3.4x1011/1.

PRP.

CO2 at

room

PRP contained

14 mm i.d.).

PRP was measured

room

two

non-

venepuncture.

concentration range

least

2.2~10'~

20 mM HEPES.

a platelet

plasma

taken

plastic

method

adjusted

g for

to

have

at 600

at

containing

dioxide-treated

A stream surface

range

4 h of

by

at buffer

at

at into

EDTA and 10 mM HEPES, pH 7.2.

was centrifuged

to

tri-sodium

stored

washed PRP was

in

not

for

collected

the

suspensions

Platelets

known

by centrifugation

within

washed

metabolites

(PRP).

3.8% w/v

PRP was prepared

Platelet

its

drugs was

611

function.

volunteers,

anti-inflammatory

containing

and

to platelet

steroidal

tubes

Vol. 17. No. 7, 1985

with

temperature in

a new polystyrene

The pH of

a foetal

was directed

blood

test

C02-treated

PRP and control

pH analyzer

(system

RM 1306,

612

Pharmacological

Radiometer). stable

After

pH of

stopped

and

Samples

were

with

time.

Acid

treatment Washed

to

the

treated

aggregation

of

washed

platelets.

pH values

because

Platelet

of to

to

with

the

Hanks

rapidity

with

flow

of

adjusted

to manipulate

solution which

air.

pH increased

solution

(Efforts

with the

a

CO2 was

with

as the

Hanks

7, 1985

40 min.,

equilibrate

in

in

about

the

studies

0.1 M HCl.

Vol. 17, No.

the

CO2 proved

suspensions

lost

air.)

aggregation.

Platelet

aggregation

channel

aggregometer,

Samples

(200

incubated the

stage

resuspended

suspended

on exposure

co2

were

Communications.

CO2 for

PRP allowed

for

platelets

PRP to

at this

taken

platelets

difficult

of

was obtained:

6.2

different

pH of

exposure

Research

~1)

in

in

with of

37OC in

washed

the

platelet

addition were

acid

I,

of

single

1,100

r.p.m.

suspensions

Durham

transmission

a Payton

speed

polystyrene

before

light

at

a stirring

PRP or

disposable

aggregometer

Changes

was measured

of

tubes

for

were 2 min.

aggregatory

recorded

for

in

agents.

about

5 min.

Drugs Arachidonic salt.

The

experiments

of This

described

chemical Figure

Edinburgh

is structures

1.

at

is

of

the

was one

Heikes,

hereafter

was

TxA2

and

material

by Sprague,

convenience

Sigma)

7-oxabicycloheptane was synthesized

University Jones.

(grade

the of

referred mimetic

as

mimetic of

generous several

the

used

Department

Harris

TxA2

used

in

gift

of

related

Dr.

to

mimetic.

and

TxA2

are

R.L.

compounds

(1983)

TxA2

these

Pharmacology,

& Greenberg as

sodium

and

compared

for The in

Pharmacological

Research

Communications,

613

Vol. 17, No. 7, 1985

LOH bH

‘@@COOH

(

Figure 1: Chemical structure stable 7-oxabicycloC2.2.llheptane experiments (bottom).

of TxA2 mimetic

(top), compared with a of TxA2, used in these

Results Sodium

arachidonate-induced

aggreqation

in washed

platelet

suspensions.

shown

The

results

in

Figure

Washed to

sodium

from

platelets

maintained

arachidonate,

with

were

less

was

observed

suspended sensitive

primary

at

0.75

(Figure

arachidonate

was abolished, (Figure

in to

aggregation

was obtained

of

at pH 7.40

of 0.3 PM and full

Platelets

6.67

typical

three

others

are

2.

at a concentration 2a).

an experiment

2b).

2~).

Hanks

sodium

PM and At

4.5

graded

aggregation aggregation

being

at

at

pH

no aggregation

failed the

obtained

maintained

arachidonate; PM

responses

at 3 uM (Figure

solution

pH 6.41,

whereas

gave

to

give

response

to

pH 7.40

full

full 15

aggregation

uM

Pharmacological

614

A

Research

pu

Communications,

Vol. 17. No.

7, 1985

B

6.67

0.75

pY

1.5 UM 2.25

t

5 i

4.5

PM

‘i

PM

a 0.75

VM

2 MIN.

3 PM

pH

7.40

Figure 2: Effect of pH on archidonate-induced aggregation of washed platelets. The recordings have been superimposed for each part of the experiment and final concentrations of reagents are given. (a) Dose-responses of a control suspension of washed platelets to arachidonate. (b) Inhibitory effect of lowered pH on arachidonate-induced aggregation in washed platelets. (c) Response of washed platelets to 15 PM arachidonate, showing full aggregation at pH 7.40 and complete inhibition of aggregation at pH 6.41. Representative of four experiments. Platelet

are

aggregation

The results

from

illustrated

in

pH 7.57, and

aggregation

With

CO2 -treated

response

transient of

co2

an experiment Figure

3.

to TxA2 mimetic

full

tion

by TxA? mimetic.

in

on platelet

mimetic

was completely

treated

PRP to

air:

reversible

atpH

PM was abolished, light

aggregation lost indeed

in as the

the

platelets

three

of

control

6.2,

(Figure the

apart

transmission.

of

aggregation

0.2 FM TxA2 mimetic

platelets,

to 0.15

increase

The dose-responses

showed

to

representative

to

others PRP, 0.15

3a).

primary from

aggrega-

a reversible

The inhibitory response

CO2 dispersed became

PM

to

0.15

effect PM TxA2

on exposure more

sensitive

of to

Pharmacological

Research

Communications,

pH

Vol. 17, No. 7, 1985

615

A

7.57

v

pH

0.15

7.80

IrM

\

i \

pH

7.44

a Y

‘. 1

PM

0.2

2

pH

7.27

pH

7.02

MIN.

dioxide on TxA2 mimetic-induced Figure 3: Effect of carbon aggregation in PRP. (a) Dose-responses of control titrated PRP to TxA2 mimetic. (b) Super-imposed recordings showing increased sensitivity of CO -treated PRP to TxA2 mimetic at pH 7.02 and illustrating the 25oss of sensitivity as the CO2 dispersed on exposure of PRP to air. Further details are given in the text. Representative of four experiments. TxA2 mimetic, to the

6.78

full

(not

illustrated).

sensitivity

aggregation

to

0.15

M HCl Unlike

showed

no

conditions. full

is

again

lost,

FM being

of

lowering

shown

in

the

reduced

in

of

at

as the

pH increased

pH increased

still

with

the

at

pH 7.80

further

original

platelet

(Figure

so

primary 3b).

suspensions

with

4. PRP to TxA2 mimetic,

sensitivity

The concentration

aggregation

obtained

pH of washed

Figure

response

increase

being As the

was

The effect 0.1

aggregation

physiological

of

to

this

TxA2 mimetic

washed agonist (0.15

pH (pH 7.40)

platelets under

acid

FM) that

gave

failed

to

give

616

Pharmacological

Research

Communications,

Vol. 17, No.

7, 1985

Figure 4: Effect of pH on TxA2 mimetic-induced aggregation of washed platelets. 0.15 PM, induced full TxA2 mimetic, aggregation at pH 7.40 (sub-maximal aggregation was observed below this concentration). Inhibition of the platelet response to 0.15 uM TxA2 mimetic was observed when washed platelets were resuspended in Hanks solution with lowered pH values: aggregation was completely inhibited at pH 6.49. Representative of four experiments. full

aggregation

with

washed

lower

pH values.

This

inhibitory

pH 6.49, shape of

aggregation

change

four

platelet action

was totally

was still

independent

suspensions

was pH dependent

abolished

evident.

These

maintained

although

results

are

at and at

a platelet representative

experiments.

Discussion Arachidonic PGH2 receptor a

specific

with

the response

respect

sensitivity sensitivity

aggregation

interaction

(Parise,

TxA2/PGH2

investigating platelet

acid-induced

to as

mimetic

effect in pH, the

of

is

Venton was

pH

rose

as pH increased

as

pH on TxA2-induced

no

to still

6.78

at and

further.

a TxA2/ 1984)

one

a biphasic pH 6.2,

then This

and

way

aggregation.

showed

aggregation

via

& Le Breton,

employed

PRP to TxA2 mimetic i.e.

mediated

of The

effect increased

gradual

loss

of

differs

from

the

Pharmacological

Research

platelet in

Communications.

response

sensitivity

the

of

from

TxA2

The pH dependent

differences

the

two

agonists

far

more

pH range

induce

difference

may again

proteins

or

arachidonic

may also

be due

products

(Aharony,

In

inhibited the

washed

platelet

importance

pH

acid

to

in

hypothesis presence

a specific on

pH to

of

was

plasma

available

or

both.

The

to

induce

aggregation

higher

lipoxygenase

protein-free and

co-

two control

the

platelet

TxA2

mimetic

was

was no indication

increase

the

these

the

with

of

PRP.

The

sensitivity

agonists

confirms

of platelet

pH effects

of the

function

reported

are

proteins.

TxA2 mimetic

TxA2 -induced

for

observed

the

that of

of

mimetic

in

to

quantitative

affinities

and there

to

proteins

although

PM TxA2 mimetic This

arachidonate

TxA2

the

1982).

conditions

aggregation

on the

changes

to

a reduction

the

0.2

inhibitory

aggregation

PRP,

view.

PM) was necessary

necessary

& Silver,

on

exudates,

conversion

of

by

binding

effect,

platelets,

acid

response

of plasma

supports

by

this

inflammatory

than

low

to

sites

protein

support

relative

synthesis

Smith with

The use of of

the

response of

dependent

to

under

biphasic

their

TxA2

binding in

aggregation).

arachidonic

in

inability

and

to

contrast

suspensions

full

relatively

acid of

PRP (less

reflect

the

concentration

in

for

150-300

1984).

PGH2 (generated

qualitative

(typically

aggregation to

in

a similar

arachidonate

sufficient

1980)

encountered

exhibited

full

al.,

increase

may be related

acid,

and TxA2,

the

& Paton,

response

acid)

et

only

pH (Kerry

arachidonic

and PGH2 (Maclouf,

Over

elicit

of

arachidonic

proteins.

lowered

in platelet

affinities

platelets

PRP, where

in

at

difference

relative

plasma

to arachidonate

was observed

The qualitative

617

Vol. 17, No. 7, 1985

to

investigate

aggregation

the has

effect

certain

616

Pharmacological

not

limitations, two agonists.

and

increased

its

if

effect

Acknowledgement We are most grateful

stable

J.B.

P.J.

GRANSTRGM,

W.J.

(1971).

MACLOUF, J., KINDAHL, Eur. J. Biochem. 109,

W.H. (1984).

C.J.

of (1982).

Sot.

B.

(1972). Br.

(1975). Proc.

J.

J.

Biol A--. Med

Sot.

Pharmac.

SCHADE, H.

71,

J.B., INGERMAN, 58, 1119-1122.

med. C.

Wschr.

& SILVER,

M.J.

82,

Proc.

H. & CRAWFORD, N. (1981).

Munch.

Physioi.,

FEBS Letters

BRETON,

SMITH, Invest.

Biochim. -------

exp.

H. (1977).

VENTON, D.L. & LE PARISE, L.V., J. Pharmacol. exp. Ther. 228, 240-244. (1924).

in

Edinburgh,

81,

J.

Natl. -

exp.

Biol. -

125-130.

H., GRANSTR?jM, E. & SAMUELSSON, 561-566.

MENASHI, S., WEINTROUB, 256, 4095-4101.

acid

factor

(1980).

Proc.

E. & KINDAHL,

G.M. & SHELDON, 623-627. & PATON,

significant

M.J.

HAMBERG, M., SVENSSON, J. & SAMUELSSON, Acad. Sci. 72, 2994-2998.

KERRY,

arachidonic

in haemostasis.

& SILVER,

W.H.

the

comparable

193-200.

EDLOW, D.W. 61 SHELDON, 137, 1328-1332.

HUTCHINS, Med. 140,

of

are

to

a

COLLIER, H.O.J. & MCDONALD-GIBSON, Lond. 308, 93-94P.

FOLCO, G., 321-324.

mimetic

platelets be

stability

to Dr. R.L. Jones, University the gift of TxA2 mimetic.

for SMITH, 718,

in

Vol. 17, No. 7, 1985

CO2 (and pH) on platelet

of

well

behaviour

Communications,

difference

of

could

platelet

References AHARONY, D., Biophys. Acta

the

sensitivity

metabolites

influencing

the

by TxA2 and the

induced

the

being

However,

aggregation then

least

Research

(1980).

B. Biol.

G.C.

Chem. (1984).

l-4. (1976).

J. Clin. -----

SPRAGUE, P.W., HEIKES, J.E., HARRIS, D.N. 61 GREENBERG, R. (1983). In: Advances in Prostaglandin, Thromboxane and Leukotriene Research, vol. 11. (Eds. B. Samuelsson, R. Paoletti & P. Ramwell). New York: Raven Press, pp. 337-343. STUART, 418-423.

M.J.,

GERRARD,

J.M.

& WHITE,

J.G.

(1980).

Blood

55,