Demonstration of platelet fibrinogen secretion via the surface connecting system

~01.3,

THROMBOSIS RESEARCH Printed in the United

DEMONSTRATION

PP* Pergamon

States

OF PLATELET FIBRINOGEN SECRETION CONNECTING SYSTEM

347-356,

1973 Inc.

Press,

VIA THE SURFACE

Jan J. Sixma*, Erik H. Mijrer and Torstein

Randi Holme,

Hovig

Electron Microscopic Laboratory, Institute of Pathology and Institute for Thrombosis R.esearch. Rikshospitalet, Oslo, Norway 14.5.1973;

in revised form 26.7.1973. Accepted by Editor J. Hugues)

(Received

Fibrin fibres were observed in the surface connecting system (SCS) in washed human platelets treated with Platelets preincubated thrombin (5 NIH units/ml). with iodoacetic acid (2 mMl and antimycin (250 ng/mll, in order to prevent the release reaction, showed no fibrin in the SCS after addition-of thrombin. These data exclude the possibility that the fibrin is formed from retained plasma fibri,nogen and indicate that platelet fibrinogen is secreted via the SCS.

ABSTRACT

Introduction Platelets tion"

respond to various

(1,2,3l, which

depending

on the strength

ristics of a secretion (3).

possibly

platelet

factor 4 (3,4,5,6)

nogen and acid hydrolases adrenaline tiated

by

and serotonin. thrombin,

and is associated

calcium,

collagen

(71. Both or

and

It has many characte-

have been discerned:

serotonin,

reac-

energy requiring,

of the stimulus.

process

Two types of release

adenine nucleotides,

stimuli with a "release

is highly selective,

with degranulation Release

I involving

glycosaminoglycans

and release

and

II involving

Release

I is induced by AOP,

release

I

latex

particles.

*On leave of absence. Div. of Haemostasis, Hosp., Utrecht, The Netherlands, 347

and

II

together

Evidence

is

fibriini-

has

Dept. of Med., Univ.

PLATELET

348

accumulated storage

indicating

organelles

acid hydrolases a-granules

FIBRINOGEN

that the dense granules

and fibrinogen

(7,81, whereas

to be located

in the

(9,101.

extrusion

morphological process

are thought

Vo1.3,No.3

are the main

for ADP, ATP and serotonin

Only very few electron showing

SECRETION

of material

evidence

have been published

from the platelet.

This lack of

may be due to the fact that the secretion

is very rapid.

Based on observations

dense tracers

as markers

White

has suggested

(10,111

micrographs

for the surface

using electron

connecting

that secretion

system

(SCSI,

may take place via

the SCS. In the present platelet

fibrinogen

paper evidence is secreted

we have made use of the chance subjected

to relatively

fibrin fibres within

is presented

in this way. observation

high thrombin

indicating

To attain

that washed

concentrations

that

this platelets

contain

their SCS.

Materials

and Methods

Antimycin, A was from Nutritional

Biochemicals

Corporation,

Cleve-

land, Ohio. EOTA, ethylenediaminetetraacetic 1111, was from E. Merck,

acid disodium

Oarmstadt,

Germany.

Iodoacetic

acid was from The British

Thrombin.

Bovine thrombin

LaRoche,

easel,

Preparation obtained

Drug Houses

Topostasine

Ltd., England.

"Roche" was from Hoffmann-

Switzerland.

of suspensions

from voluntary

used as anticoagulant Platelet

salt (Titriplex

of washed

Blood was

platelets.

donors who had used no drugs, EOTA was (2 ml O.lM EOTA,

rich plasma was obtained

pH 7.4, to 40 ml blood).

by centrifugation The platelets

(15 min at

were

isolated

room temperature,

gmax = 300 xgl.

by centrifugation

at gmax = 900 xg for 20 min at 4'C and twice

washed

by resuspending

(0.135 M NaCl, The platelet Control

0.012 M Tris-HCl,

button was finally

platelets

The platelet

the platelet

and platelets

suspensions

bolic incubator.

button

0.0015

in 25 ml buffer A

M EDTAI,

resuspended

pH .7.65, at 4'C.

in 8 ml buffer A.

treated with metabolic

inhibitors.

were shaken at 37'C in a Oubnoff

Eight hundred

~1 of platelet

suspensions

metawere

PLATELET

Vo1.3,No.3

either

incubated

solution

FIBRINOGEN

with 100 ~1 of buffer A only or 100 ~1 of a

of iodoacetic

acid in buffer A (final concentration

2 mN) and 5 ~1 of a solution concentration

250 ng/ml)

of antimycin

Thrombin

(in 96% ethanol,

final

Hundred ul of buffer

for 30 min (a).

A were then added and the incubation suspension

3-49

SECRETION

was stopped

by adding the

to glutaraldehyde.

treated

platelets

with and without

Eight hundred ul of platelet Hundred

(a) above. concentration was stopped

suspensions

ul of a thrombin

5 NIH units/ml)

metabolic

were

inhibitors.

incubated

solution

as in

in buffer A (final

were then added.

The incubation

after 1 min or 10 min by adding the incubation

mix-

ture to glutaraldehyde. Clotting

The possible

studies.

of platelet

of iodoacetic

250 ng/ml, on fibrin formation

2 mN, and antimycin, in a separate

influence

experiment:

No prolongation

poor plasma was observed

acid,

was studied

of the clotting

after 30 minutes

times

incubation

with these substances. Electron microscopy.

The platelet

9 ml 2.5% glutaraldehyde

in 0.1 N Sarensen's

pH 7.2, at room temperature mented

to a button, washed

postfixed

through

for 15 hr.

of graded

in Tyrode's

out at room temperature,

finally

in Epon 812.

with uranyl acetate Elmiskop

Ultrathin

and lead citrate,

buffer, were sedi-

buffer for 1 hr.

buffer

ethanol solutions,

dure was carried embedded

phosphate

in

buffer, 'pH 7.2, and

was then washed with Tyrode's

a series

were fixed

The platelets

with Tyrode's

in 1% osmium tetroxide

The specimen

suspensions

and dehydrated

The whole

The specimen sections

and examined

procewas

were stained in a Siemens

I electron microscope,

Results Control

platelets

that had been incubated

only, contained a-granules and dense bodies, and dilated SCS which occasionally contained material,

but was usually

Thrombin

treated

The SCS was markedly tron dense material,

empty

platelets

dilated

with buffer A

and showed

pseudopods

some amorphous

(Fig. 1). were nearly all degranulated.

and was filled with amorphous,

In many vacuoles

fibres were observed,

elec-

PLATELET

350

FIBRINOGEN

SECRETION

Vo1.3,No.3

FIG. 1 Control platelets. a-granules (GRl, dense bodies [DE!), mitochondria (MIT), glycogen (GLYl, vacuoles (SCSI.

occasionally 200-230

showing

a cross striation

A, characteristic

rence was found between minutes

the samples

(Figs. 2,3,41. incubated

of

No diffe-

for 1 or 10

with thrombin.

Platelets had a rounded, similar

for fibrin

with a periodicity

incubated swollen

iodoacetic

appearance

to the description

with monoiodoacetate

with

without

of Behnke

and potassium

acid and antimycin dilatation

of the SCS

(121 of platelets

cyanide.

treated

Vo1.3,No.3

PLATELET

FIBRINOGEN

SECRETION

FIG. 2 Platelets incubated for 10 min with thrombin. Nearly all the a-granules and dense bodies have been released. The SCS is filled with amorphous material (AM). agranules (GR), dense bodies (081, mitochondria (MIT), glycogen (GLY).

352

PLATELET

FIBRINOGEN

SECRETION

Vo1.3,No.3

FIG. 3 The same preparation as in Fig. 2 shown at a higher magnification. Fibrin fibres (FIB1 are seen in the SCS. Nitochondria (NIT), glycogen (GLYI.

Platelets incubated with thrombin for 1 or 10 minutes after preincubation with iodoacetic acid and antimycin had a swollen appearance, but no evident degranulation was observed and the SCS was empty as a rule (Fig. 5). -The platelets showed no difference from the platelets incubated with only iodoacetic acid and antimycin.

Discussion The material present in the SCS of thrombin treated platelets has the appearance of fibrin as demonstrated by the presence of the characteristic cross striation. This cross striation was,

Vo1.3,No.3

PLATELET

FIBRINOGEN

SECRETION

FIG. 4 Platelets from a suspension treated with thrombin for 10 seconds, cooled quickly in an ice-bath and prepared The specimen was embedded in Araldite further at 4’C. A fibrin fibre and sections stained with lead citrate. (FIB) with a periodicity of about 230 A is located in the SCS. Mitochondria (MIT), p;lycoEen (GLY).

353

PLATELET

354

FIBRINOGEN

FIG.

SECRETION

Vo1.3.No.3

5

Platelets preincubated with iodoacetic acid and antimycin for 30 min and treated with thrombin for 10 min. Slight swelling; preservation of the organelles similar in the control platelets. u-granules (GR1, (WIT), glycogen (GLY 1, (DB), mitochondria

however, in

the

often

absent,

an

observation

frequently

in platelet

canaliculi

commented

upon

literature. Fibrin

formation

demonstrated

by Droller

as releasing

agents.

fibrinogen

retained

has also been

(131 with both thrombin

This

fibrin

might

be

formed

in the SCS during washing

and collagen from

plasma

or from platelet

PLATELET

Vo1.3.No.3

fibrinogen

released

by the action

bility seems unlikely most canaliculi

ferritin

of

SECRETION

on the membrane

labelled

former

of material

possiin

This view is further

in washed platelets. of Sixma

355

The

thrombin.

in view of the scarcity

ted by the observations fibrinogen

FIBRINOGEN

suppora

(141 who found no detectable

surface

of washed

platelets

Complete

antifibrinogen.

proof,

utilizing

however,

has

not yet been obtained, The experimental conditions

under which thrombin

rin was detectable originate

set up described

iodoacetic

observed

plasma fibrinogen. after incubation

acid and antimycin

not shown).

The complete

tron micrographs

absence

of platelets

plasma fibrin.

presented a-granules

with a combination with thrombin

of fibrin

originate

Day and Solum

release of (data

in the SCS in elec-

thus treated shows that the fibrin

treatment

from retained

If fib-

it could

Negligible

and treatment

observed after thrombin

fibrinogen

cause no release.

in the SCS under these conditions,

from retained

was, however,

would

in this paper offered

of washed

platelets

does not

(151 have shown that part of the platelet

was localized

in low density

in this paper demonstrate

a-granules.

that the release

The data from the

is via the SCS.

Acknowledgements The authors express Anatomical

Institute,

for many helpful Society

their gratitude

University

suggestions.

- Landsforeningen

to dr. B. Reino Olsen,

of Oslo, and dr. H. Holmsen,

Supported

by the Norwegian

mot Kreft, by the Netherlands

Organi-

zation for the Advancement

of Pure Scientific

Norwegian

for Science and the Humanities.

Research

Council

Research

Cancer

and the

References 1.

GRETTE, K. Studies on the mechanism of thrombin-catalyzed hemostatic reactions in blood platelets, Acta physiol. stand.: 56 (suppl. 1951, 83, 1962.

356

PLATELET

FIBRINOGEN

SECRETION

Vo1.3,No.3

2.

substance HOVIG, T. Release of a platelet-aggregating (adenosine diphosphatel from rabbit blood platelets induced by saline "extract" of tendons. Thrombos. Oiathes. haemorrh. (Stuttg.1: 2, 264, 1963.

3.

HOLMSEN, H., DAY, H.J. and STORMORKEN, H. The blood platelet release reaction, Stand. J. Haemat.: Suppl. 8, 1969.

4.

STORMORKEN, H. The release Haemat.: Suppl. 2, 1969.

5.

HAGEN, I. The release of glycosaminoglycans during exposure of human platelets to thrombin and polystyrene latex partiBiochim. hiophys. Acta: 273, 141, 1972. cles.

6.

MURER, E.H. and HOLME, R. A Study of the release of calcium from human blood platelets and its inhibition bv metabolic Biochim. biophys. inhibitors, N-ethylmaleimide and aspirin. Acta: 222, 197, 1970. --

7.

DAY, H.J. and HOLMSEN, H. Concepts of the blood platelet release reaction, Ser. Haematol.: 4, 1, 1971.

6.

DA PRAOA, M., PLETSCHER, Subcellular localization mine in blood platelets,

9.

HOLMSEN, reaction 1966.

reaction

of secretion.

Stand.

J.

A., TRANZER, J.P. and KNLICHEL, H. of 5-hydroxytryptamine and histaNature (Lond.1: 216, 1315, 1967.

H. and DAY, H.J. Thrombin-induced platelet release and platelet lysosomes. Nature ILond.1: 219, 76.0,

10.

WHITE, J.G. A search for the platelet secretory pathway 31, 1970. using electron dense tracers. Amer. J. Path.: 2,

11.

Oegranulation WHITE, J.G. Path .: 68, 289, 1972.

12.

BEHNKE, 0. Effect of some chemicals on blood platelet platelet shape and some platelet functions microtubules, vitro. Stand. J. Haemat,: L, 123, 1970.

of discoid

platelets.

Amer.

J.

in

13.

DROLLER, M.J. Ultrastructural changes associated with Abstr. Proc. platelet nucleotide and calcium release. 1972. Ann. Meet. Amer. Sot. Clin. Invest., Atlantic City J. clin. Invest.: 51, 25a. 1972.

14.

SIXMA, J.J. and MOLENAAR, I. An ultrastructural study of Thrombos. adherent material to the human platelet membrane. Oiathes. haemorrh. (Stuttg.1: Suppl. 2, 21, 1967.

15.

Fibrinogen associated with subDAY, H;J. and SOLUM, N.O. cellular platelet particles. Stand. J. Haemat. In press ,1973.