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Immunologic
Studies
By
M
ANY
FUNCTIONS
to
plasma
mechanism
of
platelet
has
adsorbed
this
been
adsorptive
aptly
prothrombin,
factors
to
attached
be
avidly
probably
are
on
phenomenon
to
VII,’
the
factor
platelet
related
the
platelet
is not
clear;
as a sponge.5
X and
Protein
NACHMAN
proteins
described
IX,
L.
RALPH
PLATELET
directly
of Platelet
\7,2
directly
in
Many
clotting
factor
VIII,:
surface.
In
actually
form
report
a part
with
associated
platelets.
on platelet
trypsin
of the
intrinsic
is to immunologically
In
protein
platelet
characterize
addition,
will
addition,
All
glassware
was
tubing.
An
was
obtained
0.1
treated
with
from
volume
human
of
silicone
donors
a 2 per
the
been
bottle
with
Corning,
ture.
N.
The
using
a
.5
ml.
stem
cent
4 cm.
Y. ) . The
platelet
siliconized
oil bottle
button
with
Pasteur
pipettes
as
found
fibrinogen4
The
action
has
purpose
proteins
of
of
intimately
thrombin
AND
and
METHODS
( Silicad,
through
a
solution
of
15
Clay-Adams,
New
York).
Venous
gauge
needle
attached
to plastic
EDTA
was
used
as
anticoagulant.
Platelet
rich plasma
( P. R. P. ) was prepared
by differential
centrifugation
for 15 minutes
at room
temperature.
The
platelets
were
separated
from
using
the oil bottle
technic
of Green.#{176}P. R. P. was introduced
into a 100
oil
the
such
be reported.
MATERIAL
blood
on
of the
regard,
is considered
by
in the producthat
fibrinogen
structure.7’5
some
studies
this
ha’e
shown
to be intimately
associated
with
the platelet
and
some#{176}to be the primary
substrate
for the action
of thrombin
tion
of platelet
aggregation.
Recent
evidence
also
indicates
this
inThe
factors
been
may
or
membrane.14
long
with
was spun
negligible
and
a diameter
of
3.8
mm.
at 300
g.
the P. R. P.
ml. siliconized
( Corning
Glass
Co.,
at 3000
numbers
r.p.m.
for 30 minutes
at room temperaof white
and red cells was harvested
twice
at 4 C. with
Alsever’s
solution
and
washed
finally
with veronal
buffer.’#{176}
Antibodies
were
produced
in rabbits
by repeated
weekly
intramuscular
injections
of
washed
human
platelets
for a period
of 4 months.
A total number
of approximately
100 x
109 platelets
were
used.
Antifibrinogen
antibody
was
obtained
commercially
(Hyland
Laboratories,
Los Angeles,
California).
Platelets
were
trypsinized
by the method
of Schniicl,
Jackson
and
Conley.#{176} Platelet
“sonicates”
were
prepared
by separating
platelets
as described,
washing
10 times
in
veronal
buffer
to ensure
optimum
removal
of adsorbed
plasma
protein,
and sonicating
for 10 seconds
in buffer
at 20 KG tinder
ice water
using
a Branson
sonifier
at full
amplitude
(Heat
Systems,
Great
Neck,
L. I.). The sonicated
protein
was centrifuged
at 6000
g. for 15 minutes
and the clear supernate
concentrated
Cohn
fraction
1 human
fibrinogen
was obtained
minogen
free fibrinogen
was obtained
commercially
York).
ogen-free
Pooled
normal
purified
human
plasminogen
by ultrafiltration.
commercially
(Pentex).
Purified
(Mann
Research
Laboratories,
beta globulin
was
supplied
by Dr. L. Korngold,
and
by I)r. Nils Bang.
plasNew
fibrin-
From
the Department
of Medicine,
New
York
Hospital,
Cornell
Medical
Center,
New
York,
New
York.
This work
was supported
by research
grant
C-1905
from
the National
Cancer
Institute
of the U. S. National
Institutes
of Health,
and by a U. S. Public
Health
Sercice
training
grant 2A-5337
from
the National
Institute
of Arthritis
and Metabolic
Disease.
Submitted
for publication
Apr.
10, 1964;
accepted
for publication
September
3, 1964.
703
BLOOD,
VOL.
25,
No.
5
(MAY),
1965
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704
RALPH
Fig. l.-(A)
bottom
by
the
analysis of platelet sonicate.
...
filled
was
with
antihuman
arrow.
(B)
was
filled
with
indicated
b
bottom
fibrinogen,
serum.
the
platelet
NACHMAN
1I
The
fibrinogen
line
is indicated
of normal
plasma.
Trough
at the
The
major
antigen
detected
is
analysis
antihuman
serum.
arrow.
by a modification
of the Scheidegger
technic.1’
by a modified
Ouchterlony
technic.12
Starch
performed
according
to the method
of Smithies.13
Protein
conby ultraviolet
ahsorbtion
at 280 m1z.
was
sudies
gel electrophoresis
centration
was
platelet
Immunoelectrophoretic
linmunoclectrophoresis
Immunodiffusion
L.
carried
were
was
determined
out
performed
RESULTS
Immunoelectrophoresis
At
the
of the
5 discrete
least
antiplatelet
Platelet
precipitin
antibody
Sonicate
lines
(fig.
were
1A).
evident
The
to move
only minimally
in the electrophoretic
fraction
I fibrinogen.
Immunoelectrophoretic
1B) using
the same platelet
antibody
revealed
to the
platelet
fibrinogen,
Immunodiffusion
sonicate,
beta
were
plate
using
observed
was
moving
platelet
a reaction
This
latter
a line
contaminant
antigen
of
to
of the
was
of identity
with
of fibrinogen
to
pooled
an
in
was
absent
the
in
beta
purified
in
Four
present
Cohn
an
pooled
immuno-
platelet
line
antibody
serum
in
platelet
when
2).
dense
the
antigen
present
human
present
using
appeared
and
other
a smeared
diminished
antigen
each
(fig.
formed
with
markedly
A second
antigen
sonicate
1 fibrinogen,
to
moving
Close
identity
plasma.
an
slowest
fibrinogen.
partial
antigen
platelet-poor
identical
to
fraction
antibody
The
line
Proteins
compared
antiplatelet
sonicate
plasminogen.
Cohn
were
sonicate.
identical
gave
gave
the
in the
sonicate
serum,
globulin
platelet
precipitin
field and was identical
to Cohn
analysis
of normal
plasma
(fig.
2 discrete
antigens,
one identical
probably
of Platelet
normal
human
diffusion
other
Comparison
Platelet
normal
the
in the
innermost
antigens
of the
well,
precipitin
in
obtained
fraction
globulin,
which
sonicate.
This
plasminogen-free
platelet
the
fastest
line
which
normal
serum.
from
native
1 fibrinogen,
in turn
beta
was
globulin
fibrino-
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IMMUNOLOGIC
STUDIES
OF
PLATELET
705
PROTEINS
Fig. 2.-Immunodiffusion
analysis
of the antigens
serum, Cohn fraction 1 fibrinogen, and pooled human
contained
platelet
antihuman
gen
was
used
purified
in
the
plasminogen,
contaminant
serum.
antigen
Effect
Starch
gel
well.
which
of fibrinogen
Thrombin
Platelet
of
the
the
of the
sonicate
changed
No
additional
thrombin.
diminished
clot
seen
addition
served.
The
in
was
diminution
effect
addition
of
but
however,
another
possibility
that
the
identity
concentrated
amounts
the
with
beta
globulin
also
in
thrombin
addition
The
line
sonicate;
to 100
visualized
of
the
smeared
up
to
the
of
was
platelet
protein
microscopic
clot,
the
studies
were
platelet
sonicate
obtained
from
the
repeated
blood
of
a
was
seen.
a small
double
visible.
were
platelet
To rule
gross
with
the
was
ob-
studies.
sonicate,
being
a
clot
of
line
No
diffusion
in the
using
a patient
10 units
globulin
however,
units,
of the
antigen
of
beta
a concentrated
fibrinogen
no longer
revealed
platelet
fibrinogen
did
electrophoretic
pattern
added.
platelet
disappearance
platelet
reacted
sonicate
The
The
after
was
of thrombin
10 units
not
small
calcium
a faster-moving
was
with
platelet
significantly
thrombin-treated
amounts
thrombin
the
and
the
of greater
With
of
beta
globulin
region
(fig.
4).
gel but remained
at the origin.
in intensity
was
a line
antibody
Sonicate
electrophoresis
platelet
antiplatelet
3).
single
line in
not move
into
bovine
The
gave
(fig.
on the
in platelet
sonicate,
normal
beta globulin. The center well
there
sonicate;
out
the
trapped
highly
concentrated
with
thrombocytosis.
in
a
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RALPH
706
Fig. 3.-Immunodiffusion
analysis of the antigens
fraction 1 fibrinogen, pooled human
beta globulin
The
center
Two
well
units
contained
of
thrombin
immunologically
concentration
(fig.
creased
The
fibrinogen
of
was
treated
sonicate,
due
fibrinogen.
added
untreated
5).
number
sonicate
line
high
this
sonicate.
were
It
It
of
the
of platelet
lines
were
seen,
line,
however,
then
of
the
in
split.
splitting
was
sonicate
evident
to be
the
concentration
inner
in platelet sonicate, Cohn
and purified plasminogen.
of platelet
lines
plus
5 antigen
small
to
appeared
antigens
NACHMAN
serum.
aliquot
Six antigen
to the
The
platelet
were
an
to
sonicate.
antihuman
L.
protein
untreated
platelet
is possible
that
the
fibrinogen
line
in
protein.
with
compared
equal
little
In
or
disappeared
after
thrombin-
the
no
change
the
inthis
in
the
addition
of
thrombin.
The
Effect
of Try psin
Sonicates
were
these
platelets
then
divided
phoresis
100
units
the
not
into
2
using
the
of
thrombin.
an
fibrinogen
(fig.
was
6A).
aliquots,
one
clot
was
The
which
was
The
to
the
antibody.
pattern
the
detectable
of
addition
(fig.
The
the
studied
the
of
6B).
aliquot
clot-free
untreated
a large
obtained
amount
of
sonicate
by
was
immunoelectrowas
treated
material
sample
fibrinogen
sonicate
Aliquots
concentrated
other
formed;
compared
antifibrinogen
Following
of
platelets.
trypsinized
thrombin.
antibody.
A small
no longer
washed
by
antiplatelet
immunoelectrophoretic
platelets
Sonicate
from
clumped
sonicate
using
Platelet
prepared
were
thrombin-treated
diffusion
on f/ic
line
by
was
from
of
with
from
this
immuno-
present
in
trypsinized
thrombin,
this
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IMMUNOLOGIC
STUDIES
OF
PLATELET
707
PROTEINS
Fig. 4.-Starch
gel electrophoretic
of (A) normal
serum,
(B) platelet sonicate,
(C) platelet
sonicate
plus
thrombin.
pattern
Platelet
sonkate
thrombin
+
Fibrinogen
0
Platelet-sonicate
0
Thrombin
Fig. 5.-Immunodiffusion
analysis of the antigens in highly concentrated platelet
sonicate,
sonicate
plus
thrombin,
Cohn
fraction
1 fibrinogen,
and
thrombin.
The arrow
indicates
the antigen
in the platelet
sonicate
which
disappeared
following
thrombin
action.
The center well contained
antihuman
platelet
serum.
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708
RALPH
Fig.
6.-(A)
platelets.
human
Immunoelectrophoretic
The
fibrinogen
platelet
line
serum.
(B)
Cohn
fraction
1 fibrinogen,
trypsinized
platelets
plus
An antigen
identical
to
platelets,
and
The
present
by
following
of
the
Immunodiffusion
sonicate
thrombin.
fibrinogen
disappeared
analysis
is indicated
a
sonicate
arrow.
The
from
trough
of the
analysis
L.
NACHMAN
trypsinized
contained
fibrinogen
anti-
antigen
in
from
trypsinized
platelets,
and
sonicate
from
The center
well contained
antihuman
fibrinogen.
was
present
in the
sonicate
from
trypsinized
the addition
of a large
amount
of thrombin.
DISCUSSION
demonstrate
experiments
some
of which
were
not detected
normal
serum,
but was greatly
plasma.
coagulation
It is conceivable
process.
One
clottable by thrombin
platelet fibrinogen
and
more
troversy
ture
fully
and
was
studied
of
the
platelet
immunologic
first
in
as to whether
Utilizing
that
of the
it was
most
released
platelet
whether
technics,
forms
it
1948
Johnson
an
is only
Salmon15
platelet
antigens,
by
et
integral
found
Fahey
there
part
on
demonstrated
during
Although
the
and
is still
of the
plasma
fibrinogen
the
is a protein
to fibrinogen.
Ware,
al.,’4
platelets
antigens
identical
in
by
from
prominent
described
1952
5 discrete
One of these
antigens
was found
in serum
derived
from
platelet-
immunologically
fibrinogen
or
least
in plasma.
diminished
in
poor
at
Seegers4
some
interior
construc-
membrane.
in
bovine
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STUDIES
IMMUNOLOGIC
platelets
after
tracellular
OF
7 saline
fibrinogen
fibrinogen
clottable
fibrinogen
is an
protein
firmed
the
and
treated
observations
to note
of
thrombin,
digested.
We
conclude
platelet
structure
as
sensitive
qualitative
immunologic
the different
may
explain
trypsinized
The
platelet
platelets
that
did
forms
being
of
fibrinogen
studies
We
form
intricate
to
technics
used
results
obtained
the
in
have
have
con-
demonto fibrinoIt is im-
aggregates
fibrinogen
an
adsorbed
that
anti-
identical
platelets.
not
membrane
the view
to extract
concluded
Our
investigators.
in-
detect
a fluorescent
presence
immunologically
from
trypsinized
fibrinogen
as
that
the
antigens.
The
in the
was
part
plasma
of
probably
the
internal
membrane.
The
in this study
to detect
by other
investigators
of
sonicate,
a series
fibrinousing
Cohn
platelets
suggests
that
studies
fraction
studies,
on
of
with
purified
is one
demonstrated
1 fibrinogen
Schmid,
platelets
of platelet aggregation,
that
which
Jackson
is
the
is also
and
of
the
the
beta
present
Conley6
substrate
viscous
metamorphosis,
Grette,7 studying
the release of intracellular
constituents
of thrombin
on
hydrolytic
cleavage
platelets, concluded
of a nonfibrinogen
production
reacts
plasminogen
in
is plasminogen.
of extensive
the surface
fibrinogen
to washed
strongly
immunodiffusion
contaminant
platelet
After
antibody
plasminogen,
globulin
that
that
well
and
the
to
favored
was able
Using
the
these
trypsinized
indicating
to trypsin,
Grette7
fibrinolysin.
of
unable
platelets.
observation
fibrinogen-free
the
these
by
a part
were
platelets,
or
protein
obtained
presence
it was
who
of platelets.
reported
reported
portant
gen
that
trypsinized
trypsin
strated
that a thrombin-clottable
gen was present
in sonicates
al.6
of the platelets
of the platelets.
Yunis8
with
that
et
constituent
Gokcen
platelets
concluded
Schmid
from
intracellular
technic,
washed
and
platelets.
after
exposure
is on the surface
thrombin
body
of
709
PROTEINS
washings
structure
platelet
that the
PLATELET
of
concluded
thrombin
and
clot
following
that
the
thrombin
substrate
categorized
in
the
retraction.
the
action
effect
follows
the
as a “yet unknown
protein.”
studies
Our
demonstrate
that
clearly altered the immunologic
platelet protein.
changed
in
The
beta
study
reported
and
globulin
in starch gel following
a similar
in addition
the
to its action
electrophoretic
on fibrinogen,
of platelet
portion
thrombin
characteristics
sonicate
of
2
fractions
another
was
It is of interest
electrophoresis.
disappearance
of
that
grossly
Salmon’6
a platelet
of
soni-
cate following
the additicn
of thrombin.
The effect
of thrombin
on the platelet sonicate was also demonstrated
in the immunodiffusion
studies. Fibrinogen
in the thrombin-treated
sonicate diminished
platelet antigen disappeared.
The amount
immunologic
er than that
of only
while
2
studies
nificance
and
cf the second
nonfibrinogen
to act on the platelet
fibrinogen
units of thrombin,
no visual
These
these
character
required
change
do
appreciably.
of thrombin
this unidentified
in fibrinogen
not
allow
any
of the
nonfibrinogen
effect
other
investigations
that
lines were
conclusions
platelet
(fig.
platelet
antigen
was small5). In the presence
antigen
disappeared,
noted.
regarding
of thrombin
thrombin
In addition,
a second
necessary
to alter
the
on
acts
on
the
platelets.
platelet
physiologic
sig-
It is clear
from
fibrinogen.
It
is
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710
RALPH
conceivable,
however,
that interactions between
protein besides fibrinogen
may
play
a role
in
metamorphosis
and
clot
L.
NACHMAN
thrombin
and other platelet
platelet
aggregation,
viscous
retraction.
SUMMARY
Antigens
ma.
were
One
leased
tected
demonstrated
of these
antigens
during
the
in platelets,
structure.
least one
process
and
in platelets
was
Antigenos
esseva
Un
de
ille
in
esseva
detectable
in plas-
was
presumed
to be
re-
Plasminogen
appears
to be
and
part
fibrinogen
were
of the intracellular
de-
IN
demonstrate
antigenOs
not
and
found
to act not
platelet
protein.
SUMMARIO
plasma.
were
in serum
of coagulation.
the fibrinogen
Thrombin
was
other
unidentified
which
detected
only
on
fibrinogen
but
also
on
at
INTERLINGUA
plachettas
detegite
que
in sero,
non
esseva
detegibile
presumitemente
in
in conse-
quentia de su relaxation durante le processo del coagulation. Plasminogeno
e
fibrinogeno
esseva detegite in plachettas, e le fibrinogeno
pare esser parte
del
structura
super
intracellular.
fibrinogeno
sed
etiam
Esseva
trovate
que
super
al minus
un
thrombina
altere
age
non-identificate
non
solmente
proteina
plachettal.
REFERENCES
1.
Bounameaux,
de
2.
3.
4.
5.
6.
coagulation
Y.:
Dosage
conten
des
facteurs
us dans
l’atdes plaquette
Etudes
din
mosphere
plasmatique
humaines.
Rev.
Franc.
et biol. 2:52, 1957.
Mann,
F. D., Hum,
M., and Magath,
T. B.: Observations
on the conversion
of prothrombin
to thrombin.
Proc.
Soc. Exper.
Biol. & Med.
66:
33, 1947.
-:
Reactivity
of hemophiliac
plasma
to platelet
thromboplastin.
J. Lab. &
Clin.
Med.
48:51,
1958.
Ware,
A. C., Fahey,
J. L., and Seegers,
W. H.: Platelet
extracts,
fibrin formation and interaction
of purified
prothrombin
and thromboplastin.
Am. J.
Physiol.
154:140,
1958.
Adelson,
E., Rheingold,
J. J.,and Crosby,
W. H.: The platelet
as a sponge:
A
review.
Blood
17:767,
1961.
Schmid,
H. J., Jackson,
D. P., and
Conley,
C. L.: Mechanism
of action
of thrombin
on platelets.
vest. 41:543,
1962.
7. Grette, K.: The Mechanism
Catalyzed
Hemostatic
Oslo, Norwegian
Uni1962.
8. Gokcen,
M., and Yunis,
E.: Fibrinogen
as part
of platelet
stnicture.
Nature
200:590,
1963.
9. Green,
I., and Solomon,
W.: Separation
of human
lymphocytes
and monocytes
using
an
‘oil bottle.’
J. Clin.
Path.
16:180,
1963.
10. Sequira,
M., and Nelson,
R. A.: Platelet agglutination
by immune
complexes
and
its possible
role
in hypersensitivity.
J. Immunol.
86:516,
1961.
11. Komgold,
L., van
Leeuwen,
C., and
Engle,
R. L.: Diagnosis
of multiple
myeloma
and macroglobulinemia
by
the Ouchterlony
gel diffusion
technique.
Ann. N. Y. Acad. Sci. 100:203,
1962.
12. -:
The distribution and immunochemical properties
of human
tissue and
tumor
antigens.
Ann.
N. Y. Acad.
J. Clin. In-
Reactions
Platelets.
versity
Press,
Sci. 69:681,
13.
of Thrombin
Blood
Smithies,
starch
in
serum
1957.
0.:
gels:
proteins
Zone
Group
electrophoresis
variations
of
normal
in
in the
human
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
IMMUNOLOGIC
STUDIES
OF
adults.
Biochem.
J. 61:629,
1955.
14. Johnson,
S. A., Smathers,
W. M., and
Schneider,
C. L.: Platelets
and their
plasma cofactor
activity in the activation of purified
prothrombin. Am. J.
15.
711
PROTEINS
PLATELET
Arch.
16.
-,
Physiol.
170:631,
1952.
Salmon,
J.,and Bounameaux,
tion
du
Ralph
fibrinogene
L.
Laboratories,
Cornell
Y.: Situadecele dans les
Nachman,
New
University
M.D.,
York
1957.
and
quettes
gene.
mt.
isoles
Biochem.
-:
Etudes
des
bovins
et en
Thromb.
particulie
et Diath.
1958.
Director,
Hospital,
Medical
Physiol.
thrombocytes
Clinical
Hematology
Instructor
Center,
New
in
York,
Medicine,
N. Y.
et laves.
65:502,
antigens
pladu fibrino-
Hem.
2:93,
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1965 25: 703-711
Immunologic Studies of Platelet Protein
RALPH L. NACHMAN
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