Action Spectrum and Structural Changes Induction of

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1973 42: 551-555

Induction of Aggregation of Human Blood Platelets by Ultraviolet Light: Action Spectrum and Structural Changes J. C. G. Doery, R. C. Dickson and J. Hirsh

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Induction

ofAggregation by Ultraviolet

Platelets Spectrum

and

By J. Exposure

of

ultraviolet gregation. lengths

washed

light The

by

effect

between

maximal

human

is followed

response

to

platelet

ag-

and

at 248

at

225

a

J. Hirsh

enhanced

by

Platelets

exposed

show

can

the effect

and

greatly

light

with

Action

fibrinogen.

wave-

mu

m,and

is

Blood

Changes

R. C. Dickson,

platelets

occurs

302

Structural

G. Doery,

C.

oflluman Light:

be

the

ultrastructural

induced

tion ifthe

addition to

changes

independently

addition

of

ultraviolet of

of fibrinogen

which aggrega-

is omitted.

I

T HAS PREVIOUSLY been demonstrated that ultraviolet light (253.7 mz) induces aggregation of mammalian blood platelets.t Aggregation was shown to be markedly enhanced in the presence of fibrinogen and was followed by the gradual release of relatively small amounts of nucleotide and serotonin. Aggregation was inhibited by EDTA and metabolic inhibitors. These initial studies performed on pig platelets have now been extended to washed human platelets. In addition to confirming that ultraviolet light aggregates human

platelets,

the

region

of

the

ultraviolet

spectrum

capable

of

inducing

aggregation (the action spectrum) has been determined. Knowledge of the action spectrum is important to ascertain whether aggregation is being induced by a nonspecific whether activation

damaging action of electromagnetic of a specific type of chemical

of ultraviolet light the dose of radiation

on platelet required

ultrastructu has to initiate aggregation

MATERIALS

Platelet

AND

radiation on is involved.

also been determined.

platelets or The effect

investigated

and

METHODS

Isolation

Suspensions

of

was

collected

lant.

Platelets

glucose

washed

g/I00

ml)

Model

Experiments

From

were

cell

J.C.G. Hamilton, Foundation. versity.

January

Canada.

Medical by Grune

white

Dickson, Ontario. and

Recipient Ph.D.: Canada. Medicine.

Centre, Hamilton, & Stratton, inc.

Vol. 42. No. 4 (October).

1973

at 37#{176}Cas described

solution

containing

zg/ml).

Heparin

resuspended of

cell

blood

human

ml) mm.

to

3

added was

at

and apyrase Platelet

The

were less

to the

final

performed

than

I cell

g/lOO

the

first

closely

including

(25 g/ml). was

(0.35

added

Blood anticoagu-

a medium

and

counts

previously.2 acid-citrate

albumin

also

37#{176}Cin

plasma2

contamination

parts

approx-

addition

platelet using

per

ml), wash of

count

a Coulter

2000

platelets.

4 hr of venipuncture.

McMaster

revised

Research

M.Sc.:

of Pathology

Lni-ersity/973

1973:

Ontario. Hamilton.

partinents

and

of Pathology, 30.

finally g/lOO per cu

within

Doery, R.C.

were composition

commenced

the Department

Submitted

(50-100

ml), glucose (0.1 500,000 platelets

B. Red

prepared 17 parts

in a Tyrode’s

apyrase

ionic

albumin (0.2 g/l00 was approximately Counter

twice platelets

inorganic

were

anticoagulant;

and

Washed

the

platelets

human

acid-citrate

were

(0.1

mating

washed

into

(25 units/mI).

Blood.

bond

April

University,

19. 1973:

Fellow,

Department

of a Medical Research J.

Ontario.

April of

Scientist

Associate.

Hirsh,

McMaster

Hamilton,

accepted

M.D., University-

Ontario,

Pathology. Fellowship

Department

of

F.R.A.C.P., and

Canada.

20. 1973.

Director

McMaster from

the

University. Canadian

Pathology. C.R.C.P.(C): of Hematology.

Heart

McMaster Professor,

UniDe-

McMaster

Canada.

551


552

DOERY.

Exposure milliliter

to ultraviolet light aliquots of platelet

cuvettes

statically

with

at

with

platelets

was

solution

initiated

followed A

hydroxide

(moist

30

and

dialyzed

nm

the

sec

against

of

this

in

with

stirred

from

of

stirrer

HIRSH

by

the

addition

density

of

thermo-

to

a

of

irradiated

50

associatqd

Onestandard

and

transferred

37#{176}C.Aggregation

in optical

Payton

MI of

with

a

5#{176}

aggregation

recorder.

Connaught in

saline.

I

ml

NaCI

for

mI/lO IS hr

of fibrinogen

monochromator.3 radiation in

a magnetic were

at

irradiation

change

VOM-5 from

by

samples

maintained

cessation

Lomb

below the

also

preparation

0.9,,

in a high-intensity uv exposed to ultraviolet

irradiation

subsequent

obtained

concentration

clottable

after

and

solution

final

length,

The

was gel

out were

compartment

on a Bausch

Ontario).

path

fibrinogen.

fibrinogen

was carried suspension

37#{176}C.Following

sample

of human

Human

97”,,

a I-cm

controlled

aggregometer

was

AND

and Aggregation

Irradiations

quartz

DICKSON.

Medical

Research

0.9#{176},,NaCI

was

fibrinogen

at room

in the

Laboratories

adsorbed

solution),

temperature.

solution

with

clarified On

was

(Downsview,

twice

adjusted

the

by

basis

of

(e

to 5 g/l

aluminum

centrifugation the

OD

at

l3.6).

#{176}=

280

It was

thrombin.

Dosinzetrv In determining the

incident

day

was

light between

radiation of

Using 250

mz

maintained

was

over

dosage

platelet

2 and

time

radiation

the was

the

wavelength.

a

aliquot

I-mI

at

230

mM,

2.1

I ml

was

suspension per

l0

ergs

l0

platelets.

the

(0.5 Dosage

106

of

x

cells/cu

was

on

Ir-

Control

intensity

mm), value

2-p at

the

in

a given

photons).

reported. the

corresponding

on

1017

studies

adjusting

calculated

photons

aggregation

6.02 the

by

x

The

number

full

Einstein throughout

achieved

platelets.

total

to give

(lii

at 5 mm

I .9 ergs per

spectrum required

platelets.

time

platelet

uv

dose

maintained

irradiation of

to

the The

per

being

constant

at each

through

constant.

4pEinsteins

constant,

corresponds

1.6 and

response

of

the

Einsteins

m

300 basis

of

at

equals incident

light. Preparation

of

Fixation shape

in

and

was

added

pH

7.4)

to

osmium

viscocity

3 ml

of

incubated

by

centrifugation,

embedding

was associated

and

tetroxide

Electron

for

glutaraldehyde

ultrastructure

separated I,,

Samples

carried

in 0.15 medium

out

with

glutaraldehyde at

twice

ability

of ultraviolet

light

platelet

buffer

sectioning

order

was

I hr.

carried

AND

out

by

the

medium was

medium.

When

to induce

the

the basis of other studies2 to be the structural and metabolic integrity could not be reduced further.

0.1

M

medium,

according

of

and embedding

to standard

platelet

suspension

phosphate

fixation,

Dehydration.

platelet

absorption

was measured in the same carried out, the absorbance

(Fig. 3). This was found to be largely due The level of albumin used in these studies,

in

loss

platelet

buffer

platelets then

were

postfixed in Spurr

in low-

techniques.

DISCUSSION

2). This reduced response at lower wavelengths crease in the effective dose of radiation reaching pending irradiation

the

warm

glutaraldehyde suspending

for

prevent

of the

aggregation

lengths was tested from 313 to 220 mz. Aggregation tion at 313 mjz but occurred from 302 to 225 mz sponse was observed at 248 mhz, and fell off sharply

sorption

to

aliquot

Following

in

RESULTS

The

37#{176}Cin A I-mI

(2.5#{176},,glutaraldehyde

I hr.

M phosphate and

at

chilling. fixative

37#{176}Cfor washed

Microscopy

at specific

wave-

was not induced by irradia(Fig. 1). The maximum rebelow 230 mz (Figs. I and

was the

probably platelets

spectrum

related to a deas a result of abof

the

platelet

sus-

quartz cuvettes in which the platelet increased sharply below 242 mz

to the protein content of the medium. namely, 0.2 g/lOO ml, was shown on

minimum necessary for of platelets in washed

the maintenance suspension and

of so


248mg

Fig.

Aggregation of washed human platelets following irradiation by 2-M Einsteins of ultraviolet light over 5 mm. Fibrinogen was added 30 sec after completion of irradiation. Aggregation did not occur when fibrinogen was added to nonirradiated platelets. The wavelength of the incident radiation is shown at the end of each aggregation tracing.

23OmM

1.

a

and

2. Effect as degree

4

5

(mns)

i’

.qqEa’c’ ci

Fig. shown

3

Tjme

..(..(

230

2

1

220mp

5

cqq;eqaici

cc ii*i

240

of wavelength of aggregation

on ultraviolet-light-induced platelet aggregation. Results are of irradiated platelets observed at mm (broken line) and 5 mm (solid line) after addition of fibrinogen. These results were obtained using the same platelet preparation, each point representing the mean of duplicate observations. This experiment is one of three similar experiments performed. Total dose of incident irradiation was 2.M Einsteins per ml platelet suspension given over 5 mm.

4

3. platelet

Fig.

the

determined W#{149}n1.ngth

(n

cm path

Absorbance suspending in quartz

length.

read

spectrum medium, cuv#{149}ttes with

against

air.

of as 1-


DOERY.

554

Ultra.structural

Before

Effect

absent in the veloped

to

disc shaped occasionally

to these

membranes aggregates

of adjacent occasional

ultraviolet

light

with abundant present and

the

The

washed

granules degranulated

irradiated

platelets

platelets platelets

was

and

platelets

study, indicating that ultraviolet but that it does not produce

by

pre-

Pseudopods consistently

aggregation,

opposed (Fig. which appeared

and

the

4C). Within the to have lysed.

that while gradual release light-induced aggregation observation was confirmed

of platelactate in the

light may initiate the platelet significant lysis as defined by

release loss of

LDH. exact

mechanism

of

action

of

ultraviolet

light

on

platelets

certain. The ultrastructural changes in the platelet membrane violet light may act primarily at this site. This is supported ultraviolet

appeared

microtubules. platelets were

followed

became closely were observed

However, in an earlier report’ it was shown let serotonin was associated with ultraviolet dehydrogenase (LDH) was retained. This

platelet

HIRS

(Fig. 4A). Following exposure to 2-M Einsteins (over a period of 2 mm) absence of fibrinogen, hence prior to aggregation, most platelets depseudopods which tended to be short and thick (Fig. 4B). Addition of

fibrinogen

present reaction

AND

at 253. 7 m,u

oflrradiation

exposure

dominantly were only

DICKSON.

light

increases

the ion

permeability

of

human

suggest by the red

blood

is still

un-

that report

ultrathat

cell

mem-

Fig. 4. Electron micrographs of human platelets without treatment A. following irradiation by ultraviolet light without the addition of fibrinogen B. and following addition of fibrmnogen to irradiated platelets C. Dose of ultraviolet light was approximately 2- M Emstems per 1 ml of platelet suspension given over 2 mm.


AGGREGATION

branes.5

OF PLATELETS

light

Ultraviolet

teins6

and

555

to

initiate

has been

reported

evidence that membrane sulphydryl let aggregation.8 It is suggested, aggregation this

by

theory

a direct

effect

cystine absorbs radiation low, with a peak around by

Everett

disulphide

exchange.7

groups therefore,

on

is the observation

Studies

to cleave

sulphydryl-disulphide

In

the

over the 250 mz.9

bonds.

disulphide

region

et al.’#{176} indicate

in prothere

are involved in ADP induced that ultraviolet light may

sulphur-containing

that

bonds addition,

of

that

Consistent

bond-containing the

spectrum

with

amino

of

a significant

is

plateinitiate

300

quantity

mz of

acid

and

be-

ultraviolet

light at wavelengths at least as low as 290 mz is able to penetrate the human dermis to the level of the capillary blood vessels. Since light of this wavelength is active on platelets, it is possible that prolonged exposure to sunlight or other ultraviolet vivo.

light

sources

may

produce

damage

to platelets

ACKNOWLEDGM This

investigation

was

Foundation. The technical

supported

assistance

by

of Miss

the

and

blood

cells

in

ENT

Canadian

R. Samudre

or other

Heart Mr.

Foundation

and

T. Bistricki

of this

studies

kindly

the

Ontario

Heart

is gratefully

department

acknowledged. The

high-intensity

uv monochromator

H. E. Johns,

Department

Fisher

same

of the

used

of Medical

department

Biophysics,

is also

in these University

was

made

of Toronto.

The

available

assistance

of

by

Dr.

Mr.

G.

acknowledged.

REFERENCES 1. Dickson

RC,

Ultraviolet tion

of

Doery

A new

light: platelet

JCG,

Lewis

stimulus

aggregation.

for

the

Science

6. Jagger

AF: induc-

172:1140,

N.J.,

1971

Photobiology.

Prentice-Hall,

7.

Eager

photooxidation VI. A study

sponsive

interchange

to

3.

Johns

of high photobiology

Comparison

HE.

4:673,

Sober

chemistry.

with

platelets

AM:

Theory

and

design

u.v. monochromators for photochem istry. Photochem 1965

HA

(ed):

Cleveland,

Handbook

of

Chemical

Rubber,

Bio1968.

p C38 5.

Tosteson

DC:

Regulation

by sodium and potassium man iF (ed): The Cellular brane Prentice-H

Transport. all,

Englewood 1963,

p 3

of

cell

transport. Functions

volume in Hoffof Mem-

Cliffs,

N.J.,

light

2:25,

Research

in

Cliffs,

p 102 WE:

of amino acids of the initiation by

Photobiol

(in press)

Rauth

intensity and

Photobiol 4.

ADP:

Br J Haematol

Savige

to

Englewood

1967,

JE.

2. Doery JCG. Hirsh J. Mustard iF: Energy metabolism in washed human platelets rein plasma.

J: Introduction

Ultraviolet

Photolysis and of

irradiation.

and

peptidesdisulphide Photochem

1963

8. Aledort LM, Troup SB. Weed RI: Inhibition of sulfhydryl-dependent platelet functions by penetrating and nonpenetrating analogues of parachloromercuribenzene. Blood 31:471, 1968 9. Asquith RS, Hirst L: The photochemical degradation of cystine in aqueous solution in the

presence

184:345, 10. Olson violet

of

air.

Biochim

Biophys

Acta

1969 Everett RL: rays.

MA, Penetration Photochem

Yeargers

E,

Sayre

RM,

of epidermis by ultraPhotobiol 5:533, 1966