I T HAS. PREVIOUSLY been demonstrated that ultraviolet light. (253.7 mz) induces aggregation of mammalian blood platelets.t. Aggregation was shown to.
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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
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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
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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-
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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.
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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