Jouas.i. Copyright ©

THE

HISTOCHEMI8TRY

OF

by The

1975

AND

LOCALIZATION

Vol.

CYTOCHEMISTRY

Histochemical

Society,

OF

23. No. 2, pp. 103-106, 1975 Printed in U.S.A.

Inc.

A THROMBIN-SENSITIVE

CALCIUM

POOL

IN

PLATELETS’ TAMIKO

SATO,

Departments

LAWRENCE

of Pathology

Downstate

Medical

HERMAN,

(T.

Center,

Welsh

S.,

L.

H.)

Brooklyn,

National

JOHN A. CHANDLER, C. DETWILER and

New

School

Biochemistry York

11203

of Medicine,

Received

(A. and

S.,

T. Inst

it ute

Cardiff

CF4

4XX,

August

STRACHER

C. D.),

Tenovus

Heath,

for publication

ALFRED

State for

University

Cancer

THOMAS

AND

of New

Research

Great

York

(J. A.

C.),

Britain

15, 1974

Electron microscopic x-ray microprobe analysis of pyroantimonate precipitates in platelets fixed in osmium tetroxide-pyroantimonate revealed calcium localization in the nucleoids of a-granules. This pool of calcium had largely disappeared within 10 sec after stimulation of platelets by thrombin. Such a rapid change suggests that this calcium pool may have a regulatory role in stimulus-response coupling.

The

essence

hemostasis

of a platelet’s

and

thrombosis

specific

stimulus

thrombin,

collagen and The responses

(12,

16).

morphologic

crucial is its

of

such

changes,

roles

response

diverse

agents

diphosphate metabolic and

secretion,

aggregation

There is considerable indirect evidence suggesting

intracellular

calcium

tion

in coupling

responses 22),

to the

and (14,

most,

of

“dense granules

perhaps

stimulus

that

is released

tion

an

and

initial

it is known

calcium

serves

some,

It has

the

releasable

other calcium poois locations associated

essential

func-

all,

of these

part

proposed is

and

potassium

pyroantimonate

cipitation

of electron-dense

all,

MA-4)

(1),

relative

in

tron

microscope

pool

of calcium

This

pool

permits

in

the

stimulation,

nucleoid

disappears

suggesting

stimulus-response

we have

quent

a possible

in

in

Thin

series

1This Health HL10099. zation,

research Service

was

Grants

J. A. C. is grateful Cardiff, for generous

supported CA06081,

by

platelets

U.S. HL14020

to the Tenovous financial support.

The

glutaraldehyde

with

in 2% aqueto establish with subse-

fixative served for comparisons (b)

1.5%

glutaraldehyde

pyroantimonate osmium

(pH

tetroxide; 2%

fixed

(c)

potassium

Public

tron

microscope

Microprobe

and

were

Organi-

sium,

establish

7.8)

2%

os-

pyroanti-

analyzed magnesium

an

in

uranyl

in a Philips

by

Epon.

acetate 300

identification

preparation

examined

using

embedded

with cation

of one

were

microanalysis

and

stained examined

samples

cells

alcohols

and

To

cipitate, disodium

of were

(20)

microscope. METHODS

20 mM

subse-

7.4) postosmicated

containing

sections

methanol

human

2%

cations

(this

fixation);

tetroxide

a graded

the

mechanism.

Platelet preparation: Washed were prepared from blood containing

(pH

were the

monate (pH 7.8); and (d) brief fixation as in a with postosmication as in c. Samples were fixed by mixing with 8 volumes of fixative, centrifuged, dehydrated in

a

thrombin role

in preof cation

of antimony.

1.5%

potassium

postosmicated

of a-granules. after

(a)

tetroxide standard 2%

of

precipitation

were:

antimony

mium

results at sites

retention

and

buffer

containing

in elecidentified

maximal

used

ous osmium a morphologic

of the

elements

for

cacodylate

(11, cat-

techniques particles

(11, 18, 19). Platelet samples a variety of fixatives to establish

to binding

fixatives

(EM-

measurement

specimens,

quent

trans-

microscopy

of individual

quickly

procedure

may represent secretory but little is known about

electron

which

amounts

accumulation exposed to

or

stored

or their intracellular with stimulation.

analytical

21,

stimula-

Using a combination of pyroantimonate 19) which forms precipitates with various ions,

of thrombin.

Bovine thrombin was purified by the method of Glover and Shaw (8) to a specific activity of 1900 units/mg. Electron microscopy preparation: Application of

of platelet

that

calcium

unit/ml

that

thrombin

been

bodies,” which (2, 3, 13, 22),

circum-

(6, 7, 9, 17,

a major

following

15).

as

adenosine include

and contraction. stantial and

citrate and 1 mM ethylenediaminetetraacetate as anticoagulants and suspended in a Tris-NaC1-citrate solution, pH 7.4. Details of platelet preparation, washing and final suspension have been published (5). The platelet suspensions (about 5 x 10. platelets! ml) were brought to room temperature and samples were fixed either before or 10 sec after addition of 1

in

the

to

in

electron of pre-

of antimonate-

electron

AEI-EMMA-4

probe

x-ray

analytical

elec-

(1).

analysis for calcium, and

103

Downloaded from jhc.sagepub.com at UNIV OF NORTH DAKOTA on June 4, 2015

of thin

sections:

antimony, zinc;

significant

Specimens

sodium,

potasrecordings

SATO

104 were

obtained

only

tions

of

probe

current

Counts

for

crystal

spectrometers.

for calcium

analysis

were:

0.1 Mamp calcium

and

antimony.

accelerating

and

and

probe

were

kV, zm.

80

diameter

antimony

0.2

recorded

Pentaerythritol

obtained,

Condi-

voltage

on

crystals

were

chosen with one spectrometer set on peak position for the calcium K line and then set slightly off this calcium wavelength peak for background count. Counting times were usually 100 sec per area analyzed

and

all counts

were

corrected

ET AL.

by subtracting

Na

while

or

level

of Mg2t

is in large area

The

diameter, few

to the

sections

Thus, granule

the

in

of the

granules

without

cleoid)

or of other

morphologic

platelets

(embedding

mixtures substantial

ing film) antimony

Unfortu-

background.

only

fixed directly in osmium-antimonate (fixative c in Methods) contained amounts

of

nately,

antimonate

the

latter

preservation,

precipitate.

fixative

gave

a frequent

poor

problem

with

fixation in osmium antimonate microtubules, microfilaments extent,

mitochondria

mode rations presumed (2),

and

also failed to be the

although

occasionally 10) following

and

similar

primary

The where

et

1),

al.

(18).

(Fig.

2).

10

deposits

experiments.

This

tion

a functional

suggests

precipitation

rapid

was

of the essential

platelets platelets,

higher

content

were

of

composition by EMMA-4

a-granules 3).

nucleoids

For

with control

were

microanalysis; tional

calcium

to

tates

within

first

observed

of

antimonate

the

mean for

calcium

at

random

x-ray

count of

394

172

is

thrombinwith the sufficient

of

by

of

which

have

a-granules.

to

been

demonstrated

results

here

They

neutralize

acid

be present

to

in

experiments

that

antimonate

sulfate

mucosubstances

of the

demonstrate

nucleoid

identifi-

antimonate-reac-

the

of

The

because

conjectural.

serve types

were

but

technique,

that

might some

(18),

remained

suggested

in

the

precipinucleoids

et al.

Spicer

cation

cation

antimonate

a-granule

most,

de-

if not

precipitate

all,

is due

of to

calcium.

x-ray

is

Seventy

to

eighty

known

to

be

thrombin release

(propor±

had

foci

of the

esters

the

with

for

control

of

70% indicated and thrombin-

precipitate

platelet

tentatively

nucleoids

35 granules

the

for

ratio

calcium

in the granules

nonspecificity

cation

tive

inde-

precipitates microanaly-

without

platelets,

chosen a

mass)

and

the x-ray

a Ca/Sb

because, only those of

granules

count

in granule

of

Electron-dense

after

cation composito evaluate its

of

and

24

DISCUSSION

were

an

of x-ray

to

sec

that

analysis a mean

than

counts

the of a

to be counted.

scribed

The cation was determined (Fig.

calcium

10 sec that

a manifestation

decrease greater

by

significance.

sis

much

treated treated

antimonate

but so

The

(18)

5 sec in some in cation loca-

change

specific,

actually

a consistent

the

role,

is not

pendent determination tion of the deposit

was only

0.04.

fixed

confirmed

24

±

and above

calcium

significantly

was

118

reported

these

after

only

areas support-

of platelets

gave

Fig.

localized

Within

This

of ±

for

not

calcium;

precipitate

0.19

osmi-

antimony

with

absent

in

was

observation, mostly

prepa-

organelles

as previously

thrombin,

absent striking

of

precipitation (Fig.

of

deposits

deposits

in

calcium

(their

platelet

reduction with

Hardin

fixation

only

precipitation

nu-

nonbiologic

of thrombin

decreased

of

lacking

Formvar

counts

analysis

addition

by this

to preserve dense bodies, major repository of calcium bodies

“nucleoid”

and

such

dense

a-granules,

largely

hands

Greene

large

by Spicer addition

in our

Spicer,

containing the

all destroyed

observed

um-antimonate.

primary

(10). Glycogen, and, to a lesser

were

of fixation,

after

the

a reliable Analysis

(those

carbon

x-ray were

was

that

to be

surrounding

per the

Ca/Sb so

of calcium.

medium,

Microprobe

morphologic

ratio

precipitate

gave that

precipitate.

0.03),

appears

amount

with or (fixaadequate

but

entire

±

of precipitate

in

so that

calcium counts In contrast to

the

(0.14

the

100 nm

thickness,

the

calcium,

Cacodylate-buffered glutaraldehyde without osmium antimonate postfixation tives a, b and d in Methods) provided preservation,

section

include

indicator

counts

of sectioning;

mean value for is a low estimate.

constant

amount

in calcium

to factors

is approximately

equal

K4.,

a detectable

variability

due

detectable

no

occasionally

of precipitation

nearly RESULTS

was

only

part

variability

background.

there

and

Zn’

was

(5)

(14, has

using

per

cent

released 15),

and

been

studied

exactly

the

Downloaded from jhc.sagepub.com at UNIV OF NORTH DAKOTA on June 4, 2015

of platelet

after the

time

in detail same

calcium

stimulation

by

course

of

this

biochemically

conditions

for

pre-

.,.‘

$

.1

3, FIGs. 1 AND 2. Human platelets before (Fig. 1) and 10 sec after (Fig. 2) exposure to thrombin. Note heavy antimonat.e precipitates within nucleoids of a-granules in Figure 1 and greatly reduced precipitation in thrombin-treated platelets in Figure 2. Platelets were fixed in osmium antimonate (fixative c in Methods) and stained with uranyl acetate. x28,000. FIG. 3. Human platelet illustrating areas subjected to EMMA-4 microprobe anlaysis. This is a control platelet (no thrombin) as in Figure 1. a-Granules appear as homogeneous moderately dense round to oval bodies with some containing eccentric nucleoids filled with coarse dense precipitation representing focal accumulations of antimony-cation complexes. Circled regions indicate areas of analysis: area a contains antimonateladen nucleoid and gave high counts for both calcium and antimony, while area b gave counts that were not above background for these elements. x72,000. 105

Downloaded from jhc.sagepub.com at UNIV OF NORTH DAKOTA on June 4, 2015

SATO

106 paring

and

incubating seconds

platelets

here.

Ten

such

a releasable

calcium

leased

whereas,

as demonstrated

the calcium this time.

apparently

secretory to

suggested

that

70-80%

of

“dense

bodies”

dense

in

4,

that

platelets

were

in platelets

in

with

representing

has

highly

not

the

a small

in

Although

we

pools

did

not

of calcium

experiments. major calcium pool

of the

cannot

be excluded

(mitochondria, served,

even

were the

leaching

of

excluded. antimony primary

The failure to precipitation sites glutaraldehyde-antimony

glutaraldehyde antimonate loss has dense

been body

antimonate to permit

during

fixation postfixation, extensive. was

fixation, EMMA-4

sites

not

pre-

well

possibility

fixation

of

cannot

be

observe cationfollowing either fixation, or by

suggests Although with

these analysis.

other

possible

followed

observed

this pool a possi-

appreciable

because

if preserved

calcium

plate-

organelles,

membranes)

and

possi-

total

observe

in other

preosmium-

let calcium. The quick discharge of after stimulation by thrombin suggests ble role in stimulus-response coupling. amounts

in

electron-

a-granule part

glutaraldehydetoo

infrequent

ACKNOWLEDGMENTS

We

are

cal

grateful and

to Anthony

Bernice Labissiere

Martin, for

Alan techni-

assistance. LITERATURE

1. Chandler electron

JA:

Recent

1973

Feinman RD: Kinetics of the thromrelease of adenosine triphosphate by

Comparison

with

release

of

CITED

developments in analytical J Microsc 98:359, 1973 2. Costa JL, Murphy DL, Tanaka Y: Storage of calcium in human platelets: estimation of storage-packet size. Fed Proc 33:269, 1974 3. Costa JL, Reese TS, Murphy DL: Serotonin storage in platelets: estimation of storage-packet

calcium.

secretion Nature

1974

8. Glover G, Shaw E: The purification of thrombin and isolation of a peptide containing the active center histidine. J Biol Chem 246:4594, 1971 9. Grette K: Studies on the mechanism of thrombincatalyzed hemostatic reactions in blood platelets. Acta Physiol Scand 56: suppl 195, 1962 10. Herman, L, Sato T, Hales CN: The electron microscopic localization of cations to pancreatic islets of Langerhans and their possible role in insulin secretion. J Ultrastruct Res 42:298, 1973 11. Komnick H: Elektronenmikroskopische lokalisation von Na und Cl- in zellen und geweben. Protoplasma 55:414, 1962 12. Marcus AJ: Platelet function. New Engl J Med 280:1213, 1969 13. Michal R, Firkin BG: Physiological and pharmacological aspects of the platelet. Ann Rev Pharmacol 9:95, 1969 14. Murer EH: Thrombin-induced release of calcium from blood platelets. Science 166:623, 1969 15. Murer EH, Holme R: A study of the release of calcium from human blood platelets and its inhibition

mide 16.

by

and

metabolic

aspirin.

1970 Mustard

JF,

inhibitors,

Biochim

Packham

Biophys MA:

Factors

platelet function: adhesion, release, tion. Pharmacol Rev 22:97, 1970 17. Robblee LS, Shepro D, Belamarich Platelet calcium flux and the release Haematol Spicer

N-ethylmalei-

Acta

222:197,

influencing

and

aggrega-

FA, Towle C: reaction. Ser

6:311, 1973 Greene WB,

Hardin JH: Ultrastructural localization of acid mucosubstance and antimonate-precipitable cation in human and rabbit platelets and megakaryocytes. J Histochem Cytochem 17:781, 1969 19. Spicer SS, Hardin HH, Greene WB: Nuclear precipitates in pyroantimonate-osmium tetroxide-fixed tissues. J Cell Biol 39:216, 1968 20. Stempak JG, Ward RT: An improved staining method for electron microscopy. J Cell Biol 18.

Lieberman

12:282,

TC,

249:172,

osmium

that calcium an occasional

were

Biochemistry

the platelet 1971 of the thromby platelets.

Biochemistry 12:2462, 1973 7. Feinman RD, Detwiler TC: Platelet induced by divalent cation ionophores.

been

adequately

these be the

4.

bin-induced platelets.

including

directly

thus

6. Detwiler

is stored

13),

fixed

mixtures would

It

calcium,

3,

of at a-

re-

it could

material,

1974

gone from

of

represent

although release.

platelet (2,

antimonate Dense bodies pool

itself, early

total

directly

183:537,

most

been

here,

secretable

organelles

served

bly

the

had

Science

Day HJ, Holmsen H: Concepts of release reaction. Ser Haematol 4:3, 5. Detwiler TC, Feinman RD: Kinetics bin-induced release of calcium (II)

15%

is already of calcium not

size.

described only

store

does

process

contribute

as

stimulation,

in the a-granules Therefore, loss

granules the

after

ET AL.

microscopy.

22:697,

SS,

1964

21.

Stormorken H: The release reaction of secretion. Scand J Haematol suppl 9, 1969 22. White JG: The Circulationg Platelet. Edited by SA Johnson. Academic Press, New York, 1971, p 46

Downloaded from jhc.sagepub.com at UNIV OF NORTH DAKOTA on June 4, 2015

Localization of a thrombin-sensitive calcium pool in platelets.

Electron microscopic x-ray microprobe analysis of pyroantimonate precipitates in platelets fixed in osmium tetroxide-pyroantimonate revealed calcium l...
660KB Sizes 0 Downloads 0 Views