From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
The
Development
Latex
Particles-An
By
B
in
the
and
in 1959
that
concentration
generation
of
Platelet
Factor
Interaction
1. HoRown’z
HERBERT
reported
‘
of size
of
a relatively
inert
are free
and
The
was
primarily
ing
groups.
ity
dependent
The
standing
protein.
of this
incubation
in
with
plasma
latex
particlesf
compound
size
j,
0.264
0.365
detergent,
the
per
3-10
saline
shaking,
or
nine
parts
free
of
of
j.,
composition
of
solids.
Prior
),
( lBS
serially
saline
to
particles
by
no
polymers
and
3 activity*
of
specific
react-
gain
some
under-
platelet
only
of
phosphorus,
to
that
cephalin
factor
independent
particles
range
brain
are
platelet
undertaken
latex
defined
and
particles
apparent
with
a sharply
factor
after
3 activ-
a period
of
constituents.
p., 0.557
cent
I)uffered
and
by
MARCUS
platelets
that
was
It became
Activity
Plasma
containing
implied
particle
association
in
for
These
report
MATERIALS
Polystyrene
particles
substitute
investigation
phenomenon.
develop
would
on
present
J.
AARON
thromboplastin.
phenylethylene,
of lipid
latex
could
blood
AND
3
with
AND
the
following
1.171
and
which
to
use,
diluted
directly
prepare
buffered
not
the
at
centrifugation
at
The
be
particles
rpm
10,000
in
were
2.64
could
centrifuged
METHODS
diameters
lBS.
evaluated:
were
ascertained,
at
were
washed
and
either
a
resuspended
Imidazole
The
suspending
25,000
rpm
in
0.188
in
lBS
7.3,
model
L
vigorous
mixed
could
a
of
imidazoleby
was
detergent
t,
in
range
once
fresh
pH
a Spinco
p,
suspended
concentration
in
buffer,
saline.
0.088
particles
be
with
obtained
Preparative
Ultra-
centrifuge.
Human
brain
cephalin
was prepared
by the method
of Bell
and
Alton.2
Stable
product
I was prepared
from
rabbit
serum
and
plasma
by the
method
of Spaet.3
Russell’s
viper
venom
( Stypven
) was obtained
from
Burroughs
Welcome
Company,
Tuckahoe,
N. Y.; the
venom
was
diluted
1 : 10,000
either
in the manufacturer’s
diluent
or normal
saline,
further
(hinted
to
CaCl2.
“Exhausted
mg/mi.
1:50,000
of
celite
platelet
in four
1.
clotting
or
following
2.
saline
was
and
finally
by
prepared
(Filter-Cel,
Johns-Manville
at 37 C. after
adjustment
factor
3 activity
mixed
with
contact
an
Corporation,
of the pH
of latex particles
equal
activation
was
of
volume
intact
Lampoc,
to
of
plasma
Calif.)
0.025M
with
15
followed
by
7.0.
compared
with
appropriate
controls
systems:
Recalcified
glass
isotonic
for 5 hours
incubation
The
in
plasnm”
clotting
siliconized
time5-particles
tubes,
and
were
the
mixture
added
clotted
to
platelet-poor
with
plasma
0.025M
CaCI2
(PPP)
in
immediately
or
incubation.
Thromboplastin
were
From
generation
as the platelet
used
the
the
Hematology
Supported
a grant from
Submitted
#{176}This will
Departme’nt
of
Section,
in part by
the
July
be
coagulation
factors
Kindiy
provided
New
Grant
New
York
10,
1963;
defined
in
by
test
reagent
with
or
were
without
suspended
prior
the
formation
in
buffer
and
Medical
Center,
and
Health
Service
and
incubation.
New
York
Hospital-Cornell
York V. A. Hospital,
New York, N. Y.
H 5746 from the United
States
Public
Association.
for
accepted
J. W.
#{176}-particles
test,
Medicine,
Heart
as
the
(TGT)
in this
publication
contribution
of
Vanderhoff,
of
a prothrombin
I)ow
Aug.
6, 1963.
platelets
to
the
interaction
of
plasma
activator.
Chemical
Co.,
Midland,
Mich.
178
BLOOD,
Vot..
23,
No.
2 (FEBRUARY),
1964
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PLATELET
3. Product
test,
in
been
4. Stypven
I to
the
is an
the
available
The
the
viper
venom
thromboplastin
prepared
generation
generation
from
test,
and
substrate
in a dilution
time
of
the
serum
used
plasma
to
to
been
of 1:100,000
was
containing
added
plasma
requiring
has
the
is
platelet-poor
clotting
time
I,
and
accelerate
which
latex
substitute.
thromboplastin
Stypven
of
ability
of
latex
PPP
particles
platelet
found
of
procedure
induce
with
time
with
PPP
cubating
to
incubated
thromboplastin
typ(’
normal
a platelet
recalcified
of
product
to
factor
vary
used
3 for
inversely
like
product
latex
particles.
optimal
with
coagulation
the
concentration
phospholipid.8’9
ability
partial
is a modification
thromboplastin
of
as
incomplete
acceleration;
of
time
added
time-Russell’s
accelerate
Stypven
the
179
PARTICLES
intermediate,
of
clotting
have
test
protein
reagents
recalcified
particles
time7-this
a stable
plasma
BY LATEX
ACTIVITY
I substrate
which
adsorbed
the
3
FACTOR
of
exhausted
saline.
The
tests
been
found
to
has
surface
latex
activation
particles
in
plasma.
were
in
by
tubes
values
out
activated
evaluated
glass
Control
carried
measure
was
siliconized
were
XI
shorten
the
obtained
siliconized
factor
comparing
to
glass
by
in-
tubes.
in a specific
This
fashion.4
RESULTS
Findings
of
with
0.188
the
were
ii
particles
to the
in
3 activity
Acceleration
of
a mixture
other
tests,
less
plastin
when
tested
ficult
to
that
without
latex
particles
even
small
shortened
with
not
latex
the
particles
the
mixture
suits,
a plasma
37
On
C.
was
for
as
optimal
as compared
the
other
showed
incubated
a similar
great
as
as platelet
substitutes
hand,
considerable
period.
that
produced
concentration,
plasma-latex
When
incubation,
system,
but
in
the
mixtures
the
over
plasma
was
It
plasma,
since
test
system
in the
TGT
owing
to
hand
when
in an
1, indicate
that
platelet-like
activity
and
concentration
of
presence
of
non-specifically
of
(see
thrombin.
to interact
below)
yet
reagent.
plasma
otherwise
dif-
activity
to be able
as platelet
) 3-adsorbed
Al( OH
the
elaboration
found
activity
used
proved
platelet-like
the
was
thrombo-
concentrations
components.
whole
reagent
suhoptimal
of
with
platelet-like
figure
in the
range
the
other
TGT
in
1 and
a wide
evaluate
time
develop
incubated
derivative,
test
control.
at
1. Particles
cent.
without
to
plasma
as a platelet
in table
in any
with
on the
and
per
incubated
and
system
clotting
accelerate
were
inert
sizes
of whole
used
shown
all
incubation
particles
table
tested
as
time
the
in
of 2.6
cephalin.
plasma
greatly
mixture
hour
in
incubation
substrate
summarized
mixtures
of
test
following
the
itself
at
prior
amounts
1
cephalin
of
a TGT
Al (OH)3-adsorbed
added
completely
test
devise
detected
clotting
and
were
generation
be
saline
activity
than
particles
the
for
recalcified
plasma
platelet-like
definitely
Latex
with
plasma-saline
the
of
and
incubated
to
are
at a concentration
could
of plasma
compared
systems
tests
to plasma
mixtures
activity
clotting
these
added
factor
activity
plasma-latex
by
four
were
platelet
Ho
the
used
for
normal
following
1 hour
TOT.
found
and
The
incubation
was
did
Therefore
rewith
in
particle
mixtures.
The
most
influence
readily
of time
studied
Stypven
time
of 0.088
p latex
sively
during
as an
index
and
an
of incubation
in the
hour’s
product
of platelet
plasma.
Maximal
incubation
I time
factor
and
3 activity
was
activity
at 37 C.
of the
Stypven
was
found
at a concentration
tests.
In
particles
was
figure
2 the
followed
in mixtures
to develop
progresof 2.6
per
cent
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
180
HOROWITZ
Table
Test
1.-Effect
of Incubation
with
Plasma
Activity
of Latex
Particles
Unincubated*
Latex
(sec.)
System
Recalcified
time-glass
Recalcified
227
276
70
89
72
313
305
92
287
95
in
90
90
90
90
7
90
90
31
59
9
47
64
14
63
8
29
29
14
30
8
in
plasma
time
time
#{176}Tested
immediately
Incubated
$Substrate
after addition
with plasma
time after 6
with
lesser
for
activity
per
9.7
t;
of 0.088
at
1 hour
minutes
of
at
thromboplastin-promoting
37
C. prior
for
for
coalesce
and
form
masses
viscous
lower
0.088
3.65
which
metamorphosis,
to
testing.
or
higher
were
;
p;
3.2
and
per
9.7
BR
ATE
TIME
concentration.
at
the
cent
for
per cent
tested.
particle
superficially
similar
latex
noted
Maximal
following
0.188
,i;
for 0.557
concentra3.0
p
per
clumps
blood
of
platelets
platelets.
LATEX
.-.j9H405
PLASMA
40
-
20
SECONDS
INCUBATION
Fig. l.-.--Platelet
generation
test.
factor
3 activity
of 0.188
TIME-
p latex
cent
particles.
When
viewed
by phase
suspensions
seemed
to
resembled
to human
UNINCUBATED
80
90
latex.
incubation.
properties
cent
solids
per cent
p diameter
Larger
particles
were
inert at all concentrations
microscopy,
both
the
active
and
inactive
undergoing
(see.)
I substrate
Stypven
tions:
2.6
for 0.264
Cephalin
Control
(see.)
Saline
clotting
Al(OH)3
solids,
Platelet-like
Incubatedt
Latex
(sec.)
Saline
(sec.)
MARCUS
clotting
time-silicone
T.C.T.f-latex
buffer
T.G.T.-latex
Product
on
AND
particles
MINUTES
in the
thromboplastin
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
PLATELET
3
FACTOR
ACTIVITY
BY
LATEX
iai
PARTICLES
40
35
STYPVEN
TIME-
25
SECONDS
20
‘51-
101
30
5
INCUBATION
Fig.
2.-Change
in
Stvpven
time
45
TIME
on
60
MINUTES
-
incubation
of
0.088
p
latex
particles
with
plasma.
In
order
to
detergent
determine
whether
of unknown
or absence
of platelet
which
been
had
Washed
particles
whereas
the
The
factor
free
retained
diluent
factor
normal
of other
final
Stypven
plasma
shortened
incubation.
he
found
in the
was
still
In
addition,
would
absent
not
such
ings
do
change
indicate
ticles
so
coated
In an
these
Stypven
precipitate
again
an
and
effort
or
in
during
cannot
further
the
a
presence
on
particles
used
factor
plasma-latex
alone.
3 activity,
fresh
plasma,
this
so
would
a mixture
to 22.6
return
and
the
particles
and
during
interact
plasma
The
further
3 activity
had
constituent
that
not
activity
incubation.
factor
which
It is possible
with
nature
a period
could
plasma.
after
pres-
of
platelet
plasma
the
activity
supernatant
not
that
influence
seconds
centrifugation,
developed
some
was
not
activity
with
fresh
particles.
of platelet
factor
3 activity
process.
the
mixtures
testing,
plasma
39.4
did
with
further
out
was
platelet
before
in the
already
the
carried
the
incubation
at 37 C. In the data
was diluted
1:10 and added
to
latex
had
interaction
to define
by
speed
and
which
to
diluent
generate
from
high
resuspension
consumed
to
circumstances
time
would
not induce
that
the development
involves
parently
are
on
or
in the
Following
particles
were
immediately
Under
latex
studies
material,
developed
factors
the
1 hour
this
( presumably
medium
con tributed
inactive.
substrate
of
suspending
particles
over
2 hours
of
plasma-latex
mixture
coagulation
time.
the
the
ability
was
3 activity
platelet-poor
ence
of
the
alone
labile,
slowly
disappearing
presented
in table
2, the
or
3 activity,
washed
platelet
)
identity
by
supported
These
latex
which
the
latex
findparis
particles
plasma.
of the
plasma
component
required,
ap-
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182
HOROWITZ
Table
2.-Loss
of PF-3
Activity
Centrifugation
by
and
AND
______
Plasma-Latex
Resuspension
Suspension
Stypven
after
Time
Tjnincubated
Incubation
Mixture
39.0
35.5
2.
Plasma-latexf
39.4
22.6
3.
Plasma-latex
4.
Fresh
5.
Plasma
2,
plasma,
for
and
latex
from
#{176}Testedimmediately
tincubated
fresh
after
1 hour
Stypven
test
in
various
37
with
PPP
by
allowing
mixtures
were
to 0.1
were
added.
As seen
was
not
Latex
but
The
they
not
latex
in untreated
activation
52.0
40.7
21.3
to clot
This
plasma
the
buffered
tubes.
finally
factor
difference
since
the
plasma
not
be
activity
not
developed
heated
glass
is shown
tubes.
in table
The
4.
inability
of
the
attributed
to
could
readily
in
particles
na-
heparinized
5 minutes.
of intact
which
the
develop
in
at 60 C. for
of PPP
Cl2
in plasma
in prothrombin-rich
activation
activation
dilutions
Stypven-Ca
same
but
surface
of
could
or plasma
induce
ml.
at
ob-
incubation
of the
developed
3 activity
not
ml.
3 activity
factor
of contact
The
0.1
0.2
plasma
did
of platelet
were
incubated
and native
serum
saline,
and
)3-adsorbed
degree
development
Particles
serum,
platelet
the
particles.
in silicone-coated
3, platelet
the
latex
),
OH
PPP
serum.
p
in EDTA
particles
increase
47.6
42.5
evaluate
Al(
1 : 10 with
Al( OH
serum.
plasma,
PPP
of
to
0.088
derivatives.
of normal
in
content
prothrombin-poor
used
with
in table
found
protbrombin
tive
ml.
of
plasma
diluted
added
but
was
native
were
45.0
to testing.
suspension
adsorbed
then
44.3
39.0
latex
cent
3 activity
tamed
latex
at 37 C. prior
per
PPP,
2
addition.
factor
C.
from
fresh
sixth-tenths
modified
2
resuspended
plasma,
6. Fresh
the
(see.)
Plasma-saline
centrifuged,
$Two
Ineubatedt
(see.)
1.
from
MARCUS
PPP
nor
be
achieved
could
to
induce
did
contact
DIscussIoN
In the
lets
early
interact
stages
with
of blood
plasma
This
contribution
of the
The
ethanolamine
and
lets
are
capable
in certain
brain2’13
platelet
available
in
or
factor
for
platelets
3 activity.
interaction
in the
platelet
it has
factor
a certain
form
However,
with
platelet
the
the
of lipoprotein.’15
3 activity
micellular
defined
factor
platelet
plasma
phenomenon
function
Recent
derived
from
3 activity
of
lipid
plate-
activator.
factor
isolated
clotting
from
a prothrombin
as platelet
systems.5’1#{176} Phospholipids
of phosphatides14
can
mechanism
for this availability
tivity
in vivo may be a collective
occur
be
to form
phosphoglycerides
the
clotting
sources
phospholipids
factors
may
serine
of replacing
vitro
other
coagulation,
coagulation
3 activity.9
human
whole
plateplatelets
from
ervthrocytes,11’12
be shown
to possess
also
seems
factors,
to be
more
though
the
readily
precise
is unknown.
Platelet
factor
3 acof all the platelet
lipids
or it may
studies
can be a property
of
configuration,16
particle
have
indicated
any
phospholipid
size16 and surface
that
provided
charge.’7
the
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
PLATELET
3
FACTOR
ACTIVITY
______
Table
3.-Effect
BY
LATEX
of incubation
with
Activity
183
PARTICLES
Plasma
of Latex
or Serum
on
Platelet-like
Particks
Stypven
Time
Unincubated5
Incubated?
Latex
Incubation
Mixture
Latex
(sec.)
Saline
(see.)
Plasma
52
57
28
50
Serum
50
51
42
43
Al( OH
)3 plasma
61
65
33
63
Native
senim
52
51
64
47
#{176}Tested immediately
flncuhated
Table
Saline
(see.)
(sec.)
after
2 hours
4.-Lack
at
addition.
37
C.
of Contact
prior
to
Activation
to Test for
testing.
by Latex
Activation
Particles
Product
Using
Recalcified
“Exhausted
Clotting
Time
PPP-Saline
91.0
93.2
60’
95.2
96.8
Biochemical
studies
shown
the
dition
to
the
of
the
diester
form,
of
phospholipids
glycerides
the
phosphatides
saturated
or
the
these
structural
phatides
from
remaining
of
66 per
uncertain
cent
development
an
or
of platelet
factor
the particles
with
platelets
report
the
the
period
after
for
pre-incubation
clot-promoting
appears
his
phenomenon
of
replace
platelets
parable
since
maximal
ported
some
found
to be
developing
The
the
activity
the
3 activity
precise
inert
precise
nature
ethanolamine
34
are
in
a
phosphomay
clotting
should
activity
take
is labile,
to be
with
in the
When
somewhat
is
not
have
of
these
phosbio-
has
been
particles
absence
of
activity
with
plasma
basis
before
our
of
on
of our
they
(0.144
tested
0.09
p
incubation
in order
be
which
us.
diameter,
shown
plasma
to
com-
he
However
a
it
found
he
required
for
re-
size
which
we
but
capable
of
this
interaction
pre-inctibation.
constituent
of
for
strictly
not
with
wtih
size
pre-incubation
could
by
further
observations
of
are
is)
and
plasma
type
studies
on
the
activity
obtainable
Boyles1
did
not
with
the
an adsorpplasma.
It
disappearing
some
plasma
particles
of the
On
obtained
particles
dependent
particles
Admittedly
be
latex
compared
to
incomplete.
involved
diameter
not
by
of interaction
(probably
and a component
in the
and
and
TOT.
could
considerable
ad-
Only
as plasmalogen,
sources19
It appears
probably
particles
the
activity
The
in
platelets
present
to the
platelets
to develop.
methods
in
human
as
of these
properties
that
of
animal
at 37 C.
centrifugation.
and their
concentration.
or phospholipid
it
need
molecule.
have
acid
consideration.
incubation
incubation
the
from
shown
to be dependent
upon
some
type
tion phenomenon)
between
the particles
requires
in
being
than
activity5
fatty
function.5
or
clotting
unsaturated
component
properties.
Conclusions
various
sources
other
into
one
phosphoglycerides
synthetically18
findings
with
and
serine
ethanolamine
obtained
chemical
platelet
of one
ethanolamine
cent
The
of
presence
per
class
(sec.)
PPP-Latex
Unincubated
Incubated
Plasma”
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184
HOROWITZ
with
polystyrene
plasma,
EDTA.
particles
heat-labile,
Plasmas
significantly
perhaps
that
clear,
in
not
from
less
of
the
factor
however,
the
require
further
elucidation.
plasma
constituent
untreated
latex
did
plasma
particles
in
inhibited
by
contained
normals,2#{176}
lipids
MARCUS
is present
Al( OH
and
acanthocytosis
than
with
It
)
adsorbed
by
with
congenital
is associated
that
coagulation
will
completely
four
patients
AND
suggesting
or $-lipoproteins.
cannot
substitute
It is
for
platelets
tests.
SUMMARY
Platelet
absent
factor
when
3 activity
the
Following
such
optimal
An
component(s)
has
polystyrene
were
incubation,
activity.
of activity
of
particles
tested
the
particles
interaction
during
the
been
postulated.
incubation
Esseva
trovate
con
non
le
activitate
typo
de
lahil
plasma
which
rise
be
sub-
some
plasma
type
to a labile
de
particulas
de
sin
incubation
anterior
es testate
le periodo
ha essite
but
and
gives
to
plasma.
INTERLINGUA
le particulas
durante
de acivitate
found
with
considerable
particles
factor-plachettal-3
quando
was
incubation
plasma.
Post tal incubation,
le particulas
disveloppa
un optimal
grado
de activitate.
Un interaction
inter
componentes
un
que
es absente
the
period
IN
particles
prior
developed
between
SUMMARIO
polystyrenic
latex
without
de
un considerabile
le particulas
incubation
con
latex
sed
e certe
le resultato
de
postulate.
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3 ACTIVITY
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1964 23: 178-185
The Development of Platelet Factor 3 Activity by Latex Particles−−An
Interaction with Plasma
HERBERT I. HOROWITZ and AARON J. MARCUS
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