Direct evidence for intracellular divalent cation redistribution associated with platelet shape change

Vol.

71, No.

BIOCHEMICAL

1,1976

DIRECT

EVIDENCE

AND

FOR INTRACELLULAR ASSOCIATED WITH

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

DIVALENT CATION PLATELET SHAPE CHANGE

REDISTRIBUTION

G. C. Le Breton, R. J. Dinerstein*, L. J, Roth*, and H. Feinberg University of Illinois College of Medicine, Deportment of pharmacology, Chicogo, Illinois, and Department of Pharmacological and Physiological Sciences University of Chicago, Chicago, Illinois*

Received

May 4,1976

Summary: Chlortetracycline was used as a fluorescent probe to monitor shifts in divalent cation distribution when blood platelets were induced to change shape. Human platelets in their native plosma were incubated at 25’C with 50 PM chlortetracycline. It was found that when platelet shape change was stimulated by ADP or the divalent cation ionophore A 23187, a significant decrease in platelet-chlortetracycline fluoresThis fluorescence shift wos consistent with the time course for the cence occurred. change in platelet shape. ATP, which inhibits ADP-induced shape change, also inhibited the decrease in plotelet-chlortetracycline fluorescence. In response aggregation tive

and

Co2+,

be stimulated support

bute

introplatelet

this

ADP-induced

shape

change direct Using

Ca*+

were

FM),

which

chelates

for

such

metallochromic

directly

Co 2+ release (6);

with

measured

(6).

and

3.

intracellular

(8).

Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

o high

Although

the

are 2.

in the that

introplatelet

absence

shope

change

Indirect

observations

thought

to redistri-

D20,

which

blocks

reticulum

concentration

findings

by

Ca2+-sensi-

sarcoplosmic

divalent

these of

(2,3).

(4);

from

contain

suggested

which

intracellular

o mobilization

indicator

Ca2+

followed

be induced

it has been

change

change

muscle,

can

ionophores,

induceshape with

change

change

(I),

1.

change

is associated

the

include: cation,

shape

evidence

ADP

shape (500

hibits

shape

shape

like

of intraplatelet

by interfering

ADP-induced

undergo

Plotelets,

platelet

a redistribution

contention

platelets

granules.

by exogenous

divalent

chlortetrocycline

vide

blood

Since

e.g., by

contraction

inhibits

of their

proteins.

which

muscle

stimuli,

release

contractile

of external may

to various

of the cation

suggest

(5),

antibiotic

(7),

also

that

platelet

Ca 2+ , they

do not

in-

pro-

redistribution. murexide,

It was

found

362

shifts that

in the

induction

level

of

of shape

intraplatelet change

by

ADP

Vol.71,No.

1,1976

resulted

in the

sulfoxide

was

have

been

BIOCHEMICAL

formation

of intrclplatelet

required

as a murexide

altered.

On

freely

permeate

without

the

adduct

(PH

branes

Q,

(5-50

pM).

Furthermore,

brane

adduct

markedly

6.0-8.5)

vehicle,

other

hand,

membranes

10,

polar

shown,

CTC

fluorescence

(10) ll),

the (9),

the

using

red stems

Using

the

fluorescent

or

A 23187

is,

when

(9).

blood

not

cell

indeed,

associated

cations

the found

with

in the

Co-CTC that

to

platelet

platelets

biological

in

is released

low

of

mem-

concentrations cation

from

Co*’

presence

to

pH-insensitive

CTC-divalent

either

may

is known into

bound

adduct

a redistribution

CTC*

function

of the

dimethyl-

distribution

fluorescent

chelates

that

from we

be incorporated

cation

CTC

membrmes,

CTC,

can

platelet

divalent

ion

indicator

intensity

the

2+

cation

a highly

with

since

Co

divalent

interfere

Although

predominantly probe

with

COMMUNICATIONS

platelet

thus

forms

fluorescence

decreases

environment

divalent and,

chelated

does

RESEARCH

However,

normal

CTC

when and

BIOPHYSICAL

Co*+-murexide.

use of dimethylsulfoxide.

been

ADP

the

biological

9,

to a more

AND

mem-

the

membrane

or I@*+,

it has

Co

2+

and

Mg*+,

(12). shape

change

of intraplatelet

induced

by

divalent

cation.

Materials

from pared

and

Methods

Human blood, anticoagulated with acid-citrate-dextrose commercial blood banks within 4 hours of bleeding. by centrifuging the blood at 164 x g for 15 minutes.

solution, Platelet-rich

was obtained plasma was

pre-

CTC was incorporated into the platelet by incubating platelet-rich plasma at 25% with the antibiotic (obtained from Sigma Chemical Co., St. Louis, MO.) dissolved in normal saline. One ml of plasma was layered over 0.075 ml silicone oil (Ii’ parts Dow 220; 4 parts Dow 550). The platelets were pelleted by centrifugation at 7000 x g for 1 minute (Fisher Model 59) (13). The platelet pellet was transferred to a depression milled in a Plexiglass slide, and the fluorometric spectra was determined with a photon counting microspectrofluorometer developed by Dinerstein, Ferber and Roth (14). Platelet shape change was separately determined in CTC-treated platelet-rich plasma by monitoring the intensity of scattered light at 90” (15).

*CTC

= chlortetracycline

363

Vol.

71, No.

Results

and

When ment

uptake

of CTC the

of

platelets

were

intensity

25 minutes

the

reached

a plateau,

suggesting

CTC,

rate absolute

concentration

of

with

50

5 pM served antibiotic

Fig. lets CTC and 530 CTC four and

pM

CTC.

CTC

levels

BIOPHYSICAL

exhibited

volume

was substantial,ly

plasma not

RESEARCH

time

for

an equilibration

COMMUNICATIONS

the

activity

incubation

of platelet-free

plasma

both

antibiotic the

CTC with

same

Thus,

then

platelets

fluorescence containing

membrane

change. enhanceIn approx-

1).

concentrations

the

for

concen-

shape

(Fig.

was directly

fluorescence

to platelet

observed

medium.

of platelet

platelet

with

a progressive

for

was

pellet

intensity

bound

was

intensity

of the platelet

at 25%

fluorescence,

of the

in fluorescence

10 x-greater

incubated inhibit

of incubation

fluorescence

in the

was

significantly

analyzed

of the

contained

Furthermore,

in an equal

and

fluorescence CTC

did

with

of increase

the

which

isolated

imately

the

Platelet-rich

CTC:

(5 or 50 PM)

of fluorescence

though

AND

Discussion

Platelet trations

BIOCHEMICAL

1, 1976

Al-

platelets.

5 pM

or 50 pM

proportional

to the

platelets

treated

incubated was

CTC,

50-fold indicating

with that that

obthe

components.

Platelet fluorescence of chlortetracycline as a function of time. Human plate1. in their native plasma were incubated at 25% with 5 FM CTC (dashed line) or 50 PM (solid line). The platelets were pelleted through silicone oil at 5 minute intervals the pellet fluorescence determined in a microspectrofluorometer (400 nm excitation; nm emission). The number of counts observed for the platelets incubated with 50 PM has been divided by a factor of 10. The above values represent data obtained from separate blood donors; error bars indicate standard error of the mean. At 15 minutes 35 minutes the values for the two concentrations are superimposed.

364

VoL71,No.

BIOCHEMICAL

1,1976

Caswell occurs

(9)

when

and

CTC

binds

Thus, the increase of the Ca2+

to platelet

and/or

Mg2+

(Fig. Ca-CTC

associated

the

that

membranes

which

are

RESEARCH

significant only

fluorescence

membranes

the

described emission

adduct. with

demonstrated

to biological

systems,

been

2) that

BIOPHYSICAL

fluorescence

if divalent

observed associated

COMMUNICATIONS

cations

with with

enhancement

time

are

must

a divalent

present.

reflect

binding

cation,

e.g.,

.

In membrane have

(12)

in platelet-CTC

drug

adducts

others

AND

wavelengths as 530

spectrum

Therefore, the

nm and

520

emission

nm,

of CTC-treated

the

formation

of peak

for

respectively platelets

platelet-CTC This

Ca-CTC

(9, coincides

fluorescence

of Co-CTC.

the

must

contention

and

12,

We found

16).

with

Mg-CTC

that

of the

be predominantly

is consistent

with

the

relative

Human Excitation and emission spectra of chlortetracycline-treated platelets. Fig. 2, platelets in their native plasma were incubated at 25°C with 50 FM CTC for 40 minutes. The platelets were pelleted,md the excitation and emission spectrum of the pellet was determined in a microspectrofluorometer; for the excitation spectrum the l/2 bandwidths were: 2.25 nm excitation and 6 nm emission; for the emission spectrum the l/2 bandNote: Since a glass objective widths were: 6 nm excitation and 2.25 nm emission. was employed, the spectrum below 350 nm is not accurate.

distribution clpproximately

of platelet 21%

Ca2+ of the

total

and

h2+ platelet

as determined Ca2+

365

was

by lipid

Steiner bound

(17) (i.e.,

who about

found 2 mM).

that

Vol.

71, No.

Since

the

mately

platelet

does

Mg2+

530

nm than

(16) I and does

Thus,

Change in

CTC-treated

Ca*+

has a 3-fold-greater

with of ADP

platelet

can

88??

of the

seconds,

A 23187,

and

with

(3 mM)

the

ratio

total

ratio

Ca*+/Mg*+

fluorescence

ADP

or the

shape divalent

was equilibrated to inhibit

with

platelet

of the

analyzed

to Mg-CTC [(I .67)

for

CTC

than at

fluorescence

x (3)

x

be contributed

by

Shape

change

change: cation 50 pM

aggregation.

platelet-rich

for

fluorescence

as:

would

is approxi-

affinity

relative

be estimated

platelet

pellets

the

of Co-CTC,

with

samples

platelet

(18),

RESEARCH COMMUNICATIONS

has a 1 .&fold-greater

therefore

intensity

plasma

EGTA or

Ca-CTC The

platelets

supplemented

220

environment,

in fluorescence

platelet-rich

and

mM

(12).

about

Specifically,

addition

is 1.2

in membranes,

intact

AND BIOPHYSICAL

concentration

Mg-CTC

nm in the

or 7.54.

duced

/@*+ In a lipid

1.67.

at 530

BIOCHEMICAL

1, 1976

plasma fluorescence

ionophort CTC.

Ca-CTC. was

in-

A 23187. The

Subsequent were

(l.Wl,

taken

plasma

was

to the at 60,

130

intensity.

Relationship between platelet shape change induced by 10v5M ADP and Fig. 3. fluorescence of platelets containing chlortetracycline. Human platelets were incubated at 25% with 50 pM CTC for 40 minutes. The plasma was supplemented with 3 mM EGTA to prevent aggregation. Platelets from a portion of the plasma were then pelleted through silicone oil and the control pellet fluorescence determined in a microspectrofluorometer (400 nm excitation: 530 nm emission). ADP (lO”5M) was added to the remaining plasma and samples were pelleted and analyzed for fluorescence at 60, 130 and 220 seconds (dashed line). The fl uorescence values at each time point represent the average of 16 samples from three different blood donors; error bars indicate stcndard error of the mean. Platelet shape change in response to 10m5M ADPwas separately determined The change in platelet shape (solid by measuring the intensity of scattered light at 90”. line) represents a typical platelet response in the presence of 50 pM CTC.

366

Vol.71,No.

1,1976

BIOCHEMICAL

AND

BIOPHYSICAL

Time

RESEARCH

COMMUNICATIONS

(see I

Fig. 4. Relationship between platelet shape change induced by 10m6M ADP and fluorescence of platelets containing chlortetracycline. Conditions were as stated in Fig. 3 with the exception that the fluorescence values at each time point represent the average of 25 samples from three different blood donors; dashed line: fluorescence, solid line: shape change.

Figures or by

3 and

10m6M

nm is observed. inhibited

ADP,

4 illustrate, a significant On

by pretreatment

the

that

when

decrease

platelet in the

shape intensity

other

hand,

when

shape

change

with

10W5M

ATP,

no significant

change

is induced

by

of platelet

fluorescence

in response

to 10m6M

decrease

in fluorescence

10m5M

ADP

at 530 ADP

is in-

Inhibition of ADP induced shape change and platelet fluorescence change by Fig. 5. 3 with the exception that the platelets TOs5M ATp. Conditions were as stated in Fig. The fluorescence values at each time point represent were pretreated with 10m5M ATP. the average of 16 samples from two different blood donors; dashed line: fluorescence, solid line: shape change.

367

Vol.

71, No.

tensity A

occurs

23187,

course the

for

BIOCHEMICAL

(Fig.

5).

the

time

course

the

change

decrease

on the in

1, 1976

causing

Furthermore, for

the

of the Ca2+

ADP

CTC

redistribution

platelet

in platelet

to shape This .

may

unrelated

RESEARCH

shape shape

fluorescence

relative results.

BIOPHYSICAL

when change

in platelet

in fluorescence

basis

AND

(Fig.

change

be due

to shape

change

is induced

is consistent 6).

than

by

with

In this

is larger

to the

COMMUNICATIONS

the

case,

might

nonspecificity

10d

M

time

however, be expected

of the

ionophore

change.

Relationship between platelet shape change induced by 10B6M A 23187 and Fig. 6. fluorescence of platelets containing chlortetracycline. Conditions were as stated in Fig. 3 with the exception that the fluorescence values at each time point represent the average of 8 samples from two different blood donors; dashed line: fluorescence, solid line: shape change.

In order addition the

were

platelet

fluorescence in ed.

to establish not

Thus,

fluorescence Since

merely

pellet,

shape

in response

fluorescence

that

physical

observed platelet

changes

associated

with

change to 10e5M

intensity the

the

still

fluorescence

ADP

the

intensity

an alteration

inhibited

occurred

change upon

was

in fluorescence

was even

in platelet

with

500

when

addition

of ADP.

is almost

exclusively

pM

shape

CTC

(5)

found

change

is not responsible

due

ADP

scattering

It was

measured.

shape

368

in light

upon

to CTC

or A 23187

properties and that

of

the platelet a 12%

decrease

was substantiallyblockfor

the

associated

decrease

with

in

mem-

Vol. 71, No. 1,1976

brane-bound

divalent

change

cation

in fluorescence

divalent

cation

polarity

of the

to the

more

cence

membrane cytosol

for

efficiency

is not results

A 23187,

therefore,

of divalent

cation

Although

by ADP

we

determined

adduct

is wholly

Mg-CTC

nm)

ratio

(520

that

which

change

in fluorescence

change

in the

for

the

before 1 Xl

two ADP

and

platelet

220

exhibit

other

species. addition seconds

fluorescence

the

to the nm)

decrease

will

We found and after (530

at the and

of two

different

1.175

alter

the

ratio

*

0.003, A change

were

almost

The

with with

ADP

or

a redistribution

1.171

*

in ratio entirely

is the

of peak

at intervals

thereafter.

have

and

would due

to

and

not

for

the

was done

Mg-CTC

A

of a corresponding at

(i.e.,

1.178

*

be

detected

Ca-CTC

Ca-CTC

shape.

activity ratio

the

This Ca

absence

question

emission

e.g.,

in the 0.003

for

a characteristic

in the

induc-

this

emission

wavelength

species,

primary

intensity

to approach

of peak

of fluorescence change

Co-CTC

In order

will species

no substantial

ADP). nm)

maxima, of the

ANS

aggregation.

in fluorescence

wavelength

oddition

of one

its fluoresthat

fluorescence

that

of Ca2+.

emission

intensity species

ADP

and

has

environment.

observed

fluorescence

complex 8-aniline-l

alters

reported

change

in

This probe

is associated

indicates

at the

composed

different

polar

redistribution

before

shape

change

probe

rather

They

spectrum

of fluorescence

a spectrum

adducts

to a more

that

to the

relative

adduct basis

follow

due

the

(530

on the

not

but

in CTC-platelet

shape

cation

properties.

conformation.

platelet

a shift

CTC

a

of the

represent

fluorescent

cation,

undergo

fluorescence

the

membrane

divalent

a decrease that

could

used

total),

environment

membrane-bound

have

platelet

platelets

an apolar

platelet

it need

ed

when

demonstrate

from

(19)

of membrane

suggest

the

et al.

1% of the

in the

in environment of the

membra-re-bound

altered

which

change

to examine

as a function

fluorescence

component,

Home

constitutes

modification

or liberation

(ANS)

specificity

present

.

RESEARCH COMMUNICATIONS

fluorescence

represent

This

itself

AND BIOPHYSICAL

platelet

must

complex.

sulfcnate

direct

(native

intensity

CTC

polar

naphthalene no

BIOCHEMICAL

peak

emission

1.176

* 0.004

0.004

cmd the

at 60, if the decrease

in

Vol.

71, No.

1,1976

fluorescence ratio the

with

would same

BIOCHEMICAL

not

ADP occur

In either

ated

with

associated

results

using

movements This National

are

associated work

now with

of Mental

BIOPHYSICAL

fluorescence case,

the

a shift

therefore,

in progress platelet

and

in Ca-CTC

divalent to further

the

Mg-CTC

fluorescence

demonstrate

RESEARCH

On

to Co-CTC.

of intracellular

was supported

Institute

due

with

CTC,

a redistribution

Experiments

also

if Co-CTC

proportion.

be predominately Our

were

AND

other

hand,

COMMUNICATIONS

a change

fluorescence

decrease

in

decreased

induced

by

in

ADP

would

fluorescence. that cation,

investigate

platelet

shape

i.e.,

change

presumably

intraplatelet

is associcalcium.

divalent

cation

function.

by grants

from

the

Chicago

Heart

Association

and

the

Health.

References 1. 2. 3. 4. 5. 6. 7.

a. 9. 10. 11. 12. 13. 14. 15. 16. 17.

18. 19.

Born, G. V. R. (1970) J. Physiol., 209, 487-511. White, J. (1969) Sccmd. J. Haematol., 6, 236-245. Holmsen, H. (1974) Platelets: Production, Function, Transfusion and Storage, pp. 207-220, Grune and Stratton, Inc., New York. Kinlough-Rathbone, R. L., Chahil, A., Packham, M. A., Reimers, H. and Mustard, J. F. (1975) Thrombosis Res., 7, 435-449. Kaminer, B., and Kimura, J. (1972) Science, 176, 406-w. Le Breton, G. C., Sandler, W. C., and Feinberg, H. Thrombosis Res. Accepted for Publication January 1976.

J.,

mljedal, I. (1974) Biochim. Biophys. Acta, 372, 154-161. Le Bretcn, G. C., and Feinberg, H. (1974) Pharmacologist, 16, 699. (Abs.) Caswell, A. H., and Hutchison, J. D. (1971) Biochem. Biophys. Res. Commun . , 42, 43-49. Caswell, A. H. (1972) J. Membrane Biol., 7, 345-364, Caswell, A. H. (1972) in The Molecular Basis of Biological Transport, pp. 99-l 11, (Wossner, J. F. and Huijing, F., Ms.) Academic Press. Hallett, M., Schneider, A. S., and Carbone, E. (1972) J. Membrane Biol., 10, 31-44. Feinberg, H., Michel, H., and Born, G. V. R. (1974) J. Lab. Clin. Med., 84, 926-934. Dinerstein, R. J., Ferber, A., and Roth, L. J. (In Preparation). Michel, H., and Born, G. V. R. (1971) Nature New Biol., 231, 220-222. Caswell, A. H., end Hutchison, J. D. (1971) Biochem. Biophys. Res. Commun. , 43, 625-630. Steiner, M., and Taleishi, T. (1974) Biochim. Biophys. Acta, 367, 232-246. Marcus, A. J., and Zucker, M. D. (1965) The Physiology of Blood Platelets, p. 1, Grune and Stratton, Inc., New York. Home. W. C., Guilmetter, K. M., and Simons, E. R. (1975) Blood, 46, 751-759.

370