Vol. 88, No. 2, 1979 May28,
BlOCHEMlCAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1979
Pages 470-476
THROMBIN INDUCED SURFACE CHANGES OF HUMAN PLATELETS K. Subbaraot
and V.V.
Kakkar*
Department of Hematology, St. Jude Children's Research Hospital, USA and Thrombosis Research Unit, King's College Hospital Medical University of London, London, UK Received
April
Memphis, School,
TN,
2, 1979
Membrane proteins of both control and thrombin-treated human platelets were labeled by (3H)-sodium borohydride reduction of Schiff bases formed between pyridoxal phosphate and protein amino groups. Fluorographic analysis of solubilised platelet proteins disclosed a substantial difference between the labeling patterns of control vs. thrombin-treated cells. Whereas the normal platelets showed a single intensely labeled protein band, cells exposed to thrombin showed about ten labeled polypeptides. When thrombin-induced serotonin release from platelets was blocked by 3',5'-adenosine diphosphate, the protein labeling pattern on the fluorograph resembled that of control platelets. These data suggest that serotonin release from platelets by thrombin involve major changes in the architecture of proteins of platelets surface. INTRODUCTION Thrombin, inducer
of platelet
showuto however membrane Part
(PALP)
be the platelet no generally
(15,16). hydride
?'Present *It activity thereby
Copyright All rights
the
reduction
and its
Despite
mainly
Research
variety the amino Service,
group
with
of high
specific
X/79/1 00470-07$01.00/O Press,
Inc.
formreserved.
470
proteins
sodium
boro-
radioactivity platelets it
VA Hospital,
should be noted that minor protein components of high may overlap within the gels with abundant but unlabeled incorrectly assign a label to a particular protein.
@ I979 by Academic of reproduction in any
phosphate
membrane
tritiated
of lysines,
of
surface
pyridoxal
including
BECEG13,
the role of platelets.
of platelet
of cells
(g-14),
inducer-stimulated that
complexes
has been
study
reaction"
groups
label
of action
to explain
specific
amino
is a potent
extensive
"release
a tritium
with
site
has emerged
of these
of a wide
address:
specificity,
We have demonstrated
incorporates
PALP reacts
(3-8).
in identifying
base with
proteins
(1,Z)
hypothesis
platelets.
a Schiff
limited
thrombin-induced
lies
Subsequent
membrane
Because
0006-291
surface
in the
on intact
(NaB3H4)
reaction"
accepted
proteins
forms
enzyme with
"release
of the difficulty
changes
into
a proteolytic
(15-20).
can be used Memphis,
to TN
specific radioproteins and
38104.
Vol. 88, No. 2, 1979
gain
information
those
BIOCHEMICAL
about
(tyrosine
the
surface
and histidine)
AND BIOPHYSICAL
accessibility
labeled
RESEARCH COMMUNICATIONS
of an amino
acid
by lactoperoxidase-catalyzed
other
than
iodination
(21-26). The purpose for
the
detection
platelets action
of this
in plasma
PALP before
of 3',5'-adenosine
was to exploit
of thrombin-induced
suspended with
investigation
alterations
(PRP).
and after
diphosphate
Platelets
thrombin
in the membrane were
treatment
(3'5'-ADP),
PALP as a chemical
a potent
examined
for
surface their
in the presence inhibitor
probe of inter-
and absence
of platelet
function
(27). MATERIALS
AND METHODS
Venous blood was obtained from normal healthy volunteers who had not taken any medication for 2 weeks preceding blood collection. One volume of 4% sodium citrate was added to every nine volumes of blood and the samples were then centrifuged at room temperature at 300 x g for 10 minutes to prepare PRP. The platelet count of isolated PRP was adjusted to 3OO,OOO/yl with autologous platelet-poor plasma. 3'5'~ADP was used as an inhibitor of thrombin-induced platelet aggregation of PRP and serotonin release (27). Commercial bovine thrombin was purified by ion-exchange chromatography (28) to a minimum specific activity of 2000 NIH units of activity per mg of protein. Thrombin-induced "release reaction" was monitored by the release of [14c] serotonin from platelets prelabeled with this amine (29). Aliquots of 0.5 ml PRP were used to study ['*c] serotonin release induced by varying amounts of thrombin (lo-100 milliunits of activity). The amount of ["c] serotonin released into the supernatant was measured and expressed as a percentage of the total radioactivity present in nonaggregated platelets. In a separate experiment, surface proteins of both control and thrombin-treated human platelets suspended in PRP were labeled by NaB3H4 reduction of Schiff bases formed between PALP and platelet protein amino The labeled platelets were solubilized in a sample buffer containgroups (15). ing 5% sodium dodecylsulfate and 1% 8-merceptoethanol (30). Platelet protein (50 pg> were separated by electrophoresis on 7.5% polyacrylamide slab gels and then stained for protein band,s with Coomassie blue (30). The gels were subsequently impregnated with 2,5-diphenyloxazole for fluorographic detection of radioactive protein bands (31). The minimum molecular weights of labeled protein bands were estimated from a calibration curve obtained with purified proteins of known molecular weights. The migration of each standard protein was determined in relation to 1% bromophenol blue marker. RESULTS The thrombin-induced dependence tested,
["c]
minute
after
on concentration
release of the
serotonin
was rapidly
the addition
of the
When PRP was preincubated
with
serotonin
of [14c] enzyme.
At each
released
(60-100
471
thrombin
from platelets
enzyme and very
3',5'-ADP
from platelets
showed
concentration during
slowly,
if
at all,
!.IM) for
15 minutes
the
first
thereafter. at 37",
a
Vol. 88, No. 2, 1979
BIOCHEMICAL
thrombin-induced
["Cl
present
study,
release
affected
acrylamide
PALP was used
shown in Fig.
1.
This
disclosed
weight
a single
(Fig.
activity,
that
intensity.
produced
about
showed
moderately
caused
["c]
the appearance which
The number,
as well
related
comigrated as the
to the the
the addition
of thrombin
PEP (c.t.
amount
fluorograph
Figs.
PEP (c.f.
of
serotonin
of PBP incubated
These
472
fluorograph*: Several
(mol wt 180,000),
with
resulted
in about
of PEP also
(X, mol wt 43,000) as indicated
of proteins released
in Fig.
by PALP were
from
3',5'-ADP
indicate
IV
and IX (mol wt 54,000).
of activity) data
with
and such a prepara-
which
(X)
2A
of thrombin
2C) on the
III
band
with
(60 to 100 milliunits
Figs.
was labeled
Such a treatment
actin
of labeling
2A and 2E).
and the fluoro-
when PBP was treated
protein
intensity ['"c]
a molecular
release
(Fig.
of activity)
platelet
of control
(mol wt 69,000).
labeled
2D).
platelet
of
platelets
detected:
of
to 10 milliunits
VI (mol wt 110,000)
labeled
with
from
and VIII
also
(Fig.
of another
Accordingly,
control
were
release
with
of control
bands
(60 milliunits
serotonin
fluorograph
to be
protein
was intensely
of thrombin
(VIII)
mol wt 200,000)
release
(mol wt 90,000)
polypeptides
treated
serotonin
(II,
comparison
of PEP to 30 milliunits
V (mol wt 130,000),
amounts
80% of the
exposure
polypeptides
["c]
band
labeled
VII
band
to that
serotonin
intensely
labeled
Each of these
was similar
50% of ["c]
(mol wt 150,000),
10% of
one more protein
(mol wt 200,000),
higher
was about
However,
three
protein
(lo-100
The fluorograph
When the PEP was exposed
of such a preparation
a higher
II
labeled
2A).
there
and 2B) except
tion
intensely
2).
is proteins
to thrombin
thrombin
(Fig.
poly-
proteins
PALP (or both);however,
difference
serotonin
blue-stained
exposed
vs.
In the
A typical
platelet
of Coomassie
in control
a distinct
of 69,000
thrombin graph
of radioactivity
["c]
platelets.
were
with
(27).
thrombin-induced
pattern
or treated
blocked
of solubilized
even when the platelets
the distribution
PEP showed
if
of intact
pattern
distribution
of activity)
proteins
was completely
topography
electrophoresis
unchanged
milliunits
release
to determine
the membrane
gel
remained
serotonin
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1. found
platelets. (60 PM) prior
resembled that
on the
that
PALP interact
to of
Vol.
88,
No.
BIOCHEMICAL
2, 1979
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
mmlx-
x-
01 _ ..-.-
-0
E
Figure 1. Electrophoretic pattern of Coomassie blue-stained proteins from control or thrombin-treated platelets. The platelet proteins were solubilized with dodecylsulphate and separated on 7.5% polyacrylamide slab gels. About 50 pg of the platelet protein sample was applied for electrophoretic separation. The polypeptide (X) denotes the actin band of human platelets with an apparent mol wt of 43,000. The protein staining pattern remained the same as the control even when the cells were exposed to thrombin (100 mu activity) or treated with PALP (3 mM) or both. of platelet protein labeling by PALP/ (3H)NaBH4 to Figure 2. Relation depict (A) control PRP or concentration of thrombin in PRP. The fluorographs PRP exposed to thrombin activity of (B) 10 mu; (C) 30 mu; and (D) 50 mu. (E) A fluorograph of PRP preincubated with 3' ,5'-ADP (60 PM) for 15 min at 37'C before exposure of platelets to 100 mu of thrombin.
differently rest
with
or stimulated
cell
membrane
to cause
proteins
a [14C]
depending
serotonin
473
on whether
release.
platelets
are
at
Vol. 88, No. 2, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
DISCUSSION Thrombin aggregation
has been and release
has been proposed
of platelet protein
earlier
outer
these
studies of thrombin
In our
studies,
by using
amount
of thrombin,
appearance treated
of several
with
from
It
alternatives.
protein
reasonable platelet
to suggest
several
induced the
thrombin that
of labeled
of the
"release
serotonin
orientation
sensitive
labeled
reaction"
membrane
on the fluorograph
surface,
whereby
II
from
the
immediate
release
were
III
either
proteins.
was previously to be a platelet weight,
it
alterations This
is
of thrombin-treated
it
represents
thrombin-
may be suggested in the
to
of the fact
when the
evidenced
may be
corresponds
In view
observed
by 3',5'-ADP, involves
of these
(mol wt 180,000)
(3).
not
action
of serotonin
(mol wt 200,000) band
became
of PALP-building
to favor
of human platelets
was blocked
of platelet actin
band
The
enzyme on platelets
Based on themolecular
polypeptides
from platelets
surface
of thrombin-
was suggested
and the protein
release
a much lower
conditions.
subsequent
I which
the labeled
protein"
and with
band IV (mol wt 150,000)
(15,16).
M (33)
reaction".
proteolytic
at present
glycoprotein
thrombosthenin
the "thrombin that
for
a "release
availability
or from
polypeptide
as platelet
of this
(10-14).
PRP and utilized
fluorographs
have resulted
not possible
The labeled
characterized receptor
is
than
thrombin-induced
on the
the
platelets
physiological
increased
surface
appreciably
rather
on the platelet
This
iodination
platelets the
bands
PRP might
platelet
surface
to cause
under
the action
PALP.
of the enzyme on the the platelets.
occur
protein
in thrombin-treated
needed
of washed
to emerge
in the platelet
platelets
to identify
that
proteins
to react
proteins
labeled
indicate
different
accessible
able
as they might
platelets
caused
PRP instead
we were
by thrombin
and thrombin-treated
actually
both
stimulated
to differentiate
control
than
to initiate
lactoperoxidase-catalyzed
made use of washed
amounts
in platelets
changes
failed
between
higher
changes
using
surface
reaction"
conformational
proteins
pattern
at the platelet
and the "release
studies
surface
labeling
Moreover,
(3)
to involve
However,
(32).
shown to act
that
structural
by the appearance
PSP where,
80% of total
BIOCHEMICAL
Vol. 88, No. 2, 1979
[14c]
serotonin
was released
by PALP could inside
Although
labeled
the platelets
be due to the binding
to the outer
molecular
from
AND BIOPHYSICAL
surface
platelets
weights
are known
ranging
polypeptides
determined.
from
The labeling
whether
or not
other
changes
on platelets
to contain
in
to actin
several
and 160,000,
this
procedure
inducers
ZD).
The labeling during
its
of actin
exposure
from
cell.
90,000
identified
(Fig.
of coenzyme
of the
RESEARCH COMMUNICATIONS
study in
as documented
for
glycoproteins
the exact
(Figs
outlined
of platelet
membrane
identity
of the
2C and 2D) remain
this
function
study also
may help
cause
with
to be to determine
similar
surface
thrombin. REFERENCES
1.
and Pharmacology Holmsen, H. (1975) Biochemistry Foundation Symposium 35, Elsevier, Amsterdam
2.
Murer,
3.
Baenziger, Sci. U.S.
4.
Baenziger, 2723-2731
5.
Ganguly,
6.
Tollefsen, D.M., 249, 2646-2651
7.
Tollefsen,
8.
Tollefsen, 56, 241-245
9.
Ganguly,
P. (1971)
10.
Nachman,
R.L.,
11.
Phillips,
12.
Steiner,
13.
Feagler, J. Cell
14.
Hagen,
15.
Subbarao,
16.
Subbarao, K., 2, 531-534
E.H.
(1971)
Biochim.
N.L., Brodie, 68, 240-243
G.N.,
Brodie,
G.N.
N.L., P. (1974)
D.M.
Nature
Jackson,
M. (1973) J.R., Biol.
K.,
Biochim.
N.O.
Kakkar,
(1975)
246,
(1974) Biophys.
Kuchibhotla,
P.W.
Proc.
Natl.
J. Biol.
Acta
T.
Acad.
Chem. 247,
J. Biol.
Invest.
P.W.
Chem.
55, 1259-1268
(1975)
J.
(1977)
J. Biol.
Biophys.
323,
D.D.
Clin.
Invest.
Chem. 248, Acta
and Majerus, Biochim.
V.V.
352,
2928-2936
218-227
653-658
P. (1978)
and Kakkar,
475
(1974)
J. Clin.
Biochim.
and Ganguly, J.,
(1972)
B. (1973)
Chaplin,
and Olsen, V.V.
(1971)
4286-4290
A. and Ferris,
Tillack, T.W., 60, 541-553
Solum,
P.W.
and Majerus,
Chen.
P.P.
Ciba
310-315
P-W.
C.M. and Majerus,
and Agin,
p. 175,
306-307
P.W.
J. Biol.
Hubbard,
237,
and Majerus,
J.R.
and Majerus,
Acta
and Majerus,
247,
Feagler,
D.M.,
D.R.
I.,
Biophys.
of Platelets,
P.W.
Biophys. Thromb.
(1979)
(1974) Acta
Res. 2,
Biochem.
468,
l-10
1017-1029
Pharmacol.
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D.B.,
Ccmpans,
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
17.
Rifkin, 6437
R.W. and Reich,
18.
Kreibich,
19.
Cabantchik, &-em. 250,
20.
Cabantchik, I.Z., Balshin, M., Breuer, W., Markus, (1975) Biochim. Biophys. Acta 382, 621-633
21.
Berg,
22.
Phillips, D.R. 40, 284-289
23.
Bretscher,
24.
Tsan,
25.
Bretscher,
26.
Hubbard,
A.L.
27.
Subbarao,
K.,
28.
Lundblad,
R.L.
(1971)
29.
Bridges, J.M., 197, 364-366
Baldini,
30.
Laemmli,
31.
Bonner,
W.M. and Laskey,
32.
Majerus,
P.W. ami Brodie,
33.
Ganguly,
P.
34.
Cohen, I., Bohak, 10, 388-394
G. and Sabatini,
D.D.
I.Z., Balshin, 5130-5136
H.C.
(1969)
Biochim.
(1971)
M. and Berlin, M.S.
R-D.
Z.
(1977)
59, J. Cell.
J.
(1978)
Biochemistry
Nature
Brit. Z.,
J. Expt.
R.A. G.N.
A. (1975)
J. Biol.
H. and Rothstein,
A.
Biophys.
Med. 134,
Res.
Commun.
1016-1035
351-357 Biol.
55,
Biochem.
390-405
Phannacol.
27,
1193-1196
C. and Dameshek,
W. (1963)
Nature
680-685
(1974) (1972)
J. Haematol. DeVries,
2133-2138
10, 2501-2506
Fichera, 227,
6432-
65-78
58, 775-781
(1972)
M.,
183,
Biol.
and Kuchibhotla,
(1970)
Acta
Biol.
32,
Chem. 247,
W. and Rothstein,
Biochem.
(1971)
J. Biol.
Proc.
M. (1970)
J. Mol.
and Cohn,
Fed.
Breuer,
J. Mol.
(1971)
(1973)
Biophys.
and Morrison,
M.S.
U.K.
M.,
E. (1972)
Eur. J.
J. Biochem. Biol.
46,
Chem. 247,
83-88 4253-4257
37, 47-51
A. and Katchalski,
E. (1969)
Eur.
J. Biochem.
BlOCHEMlCAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1979
Pages 470-476
THROMBIN INDUCED SURFACE CHANGES OF HUMAN PLATELETS K. Subbaraot
and V.V.
Kakkar*
Department of Hematology, St. Jude Children's Research Hospital, USA and Thrombosis Research Unit, King's College Hospital Medical University of London, London, UK Received
April
Memphis, School,
TN,
2, 1979
Membrane proteins of both control and thrombin-treated human platelets were labeled by (3H)-sodium borohydride reduction of Schiff bases formed between pyridoxal phosphate and protein amino groups. Fluorographic analysis of solubilised platelet proteins disclosed a substantial difference between the labeling patterns of control vs. thrombin-treated cells. Whereas the normal platelets showed a single intensely labeled protein band, cells exposed to thrombin showed about ten labeled polypeptides. When thrombin-induced serotonin release from platelets was blocked by 3',5'-adenosine diphosphate, the protein labeling pattern on the fluorograph resembled that of control platelets. These data suggest that serotonin release from platelets by thrombin involve major changes in the architecture of proteins of platelets surface. INTRODUCTION Thrombin, inducer
of platelet
showuto however membrane Part
(PALP)
be the platelet no generally
(15,16). hydride
?'Present *It activity thereby
Copyright All rights
the
reduction
and its
Despite
mainly
Research
variety the amino Service,
group
with
of high
specific
X/79/1 00470-07$01.00/O Press,
Inc.
formreserved.
470
proteins
sodium
boro-
radioactivity platelets it
VA Hospital,
should be noted that minor protein components of high may overlap within the gels with abundant but unlabeled incorrectly assign a label to a particular protein.
@ I979 by Academic of reproduction in any
phosphate
membrane
tritiated
of lysines,
of
surface
pyridoxal
including
BECEG13,
the role of platelets.
of platelet
of cells
(g-14),
inducer-stimulated that
complexes
has been
study
reaction"
groups
label
of action
to explain
specific
amino
is a potent
extensive
"release
a tritium
with
site
has emerged
of these
of a wide
address:
specificity,
We have demonstrated
incorporates
PALP reacts
(3-8).
in identifying
base with
proteins
(1,Z)
hypothesis
platelets.
a Schiff
limited
thrombin-induced
lies
Subsequent
membrane
Because
0006-291
surface
in the
on intact
(NaB3H4)
reaction"
accepted
proteins
forms
enzyme with
"release
of the difficulty
changes
into
a proteolytic
(15-20).
can be used Memphis,
to TN
specific radioproteins and
38104.
Vol. 88, No. 2, 1979
gain
information
those
BIOCHEMICAL
about
(tyrosine
the
surface
and histidine)
AND BIOPHYSICAL
accessibility
labeled
RESEARCH COMMUNICATIONS
of an amino
acid
by lactoperoxidase-catalyzed
other
than
iodination
(21-26). The purpose for
the
detection
platelets action
of this
in plasma
PALP before
of 3',5'-adenosine
was to exploit
of thrombin-induced
suspended with
investigation
alterations
(PRP).
and after
diphosphate
Platelets
thrombin
in the membrane were
treatment
(3'5'-ADP),
PALP as a chemical
a potent
examined
for
surface their
in the presence inhibitor
probe of inter-
and absence
of platelet
function
(27). MATERIALS
AND METHODS
Venous blood was obtained from normal healthy volunteers who had not taken any medication for 2 weeks preceding blood collection. One volume of 4% sodium citrate was added to every nine volumes of blood and the samples were then centrifuged at room temperature at 300 x g for 10 minutes to prepare PRP. The platelet count of isolated PRP was adjusted to 3OO,OOO/yl with autologous platelet-poor plasma. 3'5'~ADP was used as an inhibitor of thrombin-induced platelet aggregation of PRP and serotonin release (27). Commercial bovine thrombin was purified by ion-exchange chromatography (28) to a minimum specific activity of 2000 NIH units of activity per mg of protein. Thrombin-induced "release reaction" was monitored by the release of [14c] serotonin from platelets prelabeled with this amine (29). Aliquots of 0.5 ml PRP were used to study ['*c] serotonin release induced by varying amounts of thrombin (lo-100 milliunits of activity). The amount of ["c] serotonin released into the supernatant was measured and expressed as a percentage of the total radioactivity present in nonaggregated platelets. In a separate experiment, surface proteins of both control and thrombin-treated human platelets suspended in PRP were labeled by NaB3H4 reduction of Schiff bases formed between PALP and platelet protein amino The labeled platelets were solubilized in a sample buffer containgroups (15). ing 5% sodium dodecylsulfate and 1% 8-merceptoethanol (30). Platelet protein (50 pg> were separated by electrophoresis on 7.5% polyacrylamide slab gels and then stained for protein band,s with Coomassie blue (30). The gels were subsequently impregnated with 2,5-diphenyloxazole for fluorographic detection of radioactive protein bands (31). The minimum molecular weights of labeled protein bands were estimated from a calibration curve obtained with purified proteins of known molecular weights. The migration of each standard protein was determined in relation to 1% bromophenol blue marker. RESULTS The thrombin-induced dependence tested,
["c]
minute
after
on concentration
release of the
serotonin
was rapidly
the addition
of the
When PRP was preincubated
with
serotonin
of [14c] enzyme.
At each
released
(60-100
471
thrombin
from platelets
enzyme and very
3',5'-ADP
from platelets
showed
concentration during
slowly,
if
at all,
!.IM) for
15 minutes
the
first
thereafter. at 37",
a
Vol. 88, No. 2, 1979
BIOCHEMICAL
thrombin-induced
["Cl
present
study,
release
affected
acrylamide
PALP was used
shown in Fig.
1.
This
disclosed
weight
a single
(Fig.
activity,
that
intensity.
produced
about
showed
moderately
caused
["c]
the appearance which
The number,
as well
related
comigrated as the
to the the
the addition
of thrombin
PEP (c.t.
amount
fluorograph
Figs.
PEP (c.f.
of
serotonin
of PBP incubated
These
472
fluorograph*: Several
(mol wt 180,000),
with
resulted
in about
of PEP also
(X, mol wt 43,000) as indicated
of proteins released
in Fig.
by PALP were
from
3',5'-ADP
indicate
IV
and IX (mol wt 54,000).
of activity) data
with
and such a prepara-
which
(X)
2A
of thrombin
2C) on the
III
band
with
(60 to 100 milliunits
Figs.
was labeled
Such a treatment
actin
of labeling
2A and 2E).
and the fluoro-
when PBP was treated
protein
intensity ['"c]
a molecular
release
(Fig.
of activity)
platelet
of control
(mol wt 69,000).
labeled
2D).
platelet
of
platelets
detected:
of
to 10 milliunits
VI (mol wt 110,000)
labeled
with
from
and VIII
also
(Fig.
of another
Accordingly,
control
were
release
with
of control
bands
(60 milliunits
serotonin
fluorograph
to be
protein
was intensely
of thrombin
(VIII)
mol wt 200,000)
release
(mol wt 90,000)
polypeptides
treated
serotonin
(II,
comparison
of PEP to 30 milliunits
V (mol wt 130,000),
amounts
80% of the
exposure
polypeptides
["c]
band
labeled
VII
band
to that
serotonin
intensely
labeled
Each of these
was similar
50% of ["c]
(mol wt 150,000),
10% of
one more protein
(mol wt 200,000),
higher
was about
However,
three
protein
(lo-100
The fluorograph
When the PEP was exposed
of such a preparation
a higher
II
labeled
2A).
there
and 2B) except
tion
intensely
2).
is proteins
to thrombin
thrombin
(Fig.
poly-
proteins
PALP (or both);however,
difference
serotonin
blue-stained
exposed
vs.
In the
A typical
platelet
of Coomassie
in control
a distinct
of 69,000
thrombin graph
of radioactivity
["c]
platelets.
were
with
(27).
thrombin-induced
pattern
or treated
blocked
of solubilized
even when the platelets
the distribution
PEP showed
if
of intact
pattern
distribution
of activity)
proteins
was completely
topography
electrophoresis
unchanged
milliunits
release
to determine
the membrane
gel
remained
serotonin
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1. found
platelets. (60 PM) prior
resembled that
on the
that
PALP interact
to of
Vol.
88,
No.
BIOCHEMICAL
2, 1979
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
mmlx-
x-
01 _ ..-.-
-0
E
Figure 1. Electrophoretic pattern of Coomassie blue-stained proteins from control or thrombin-treated platelets. The platelet proteins were solubilized with dodecylsulphate and separated on 7.5% polyacrylamide slab gels. About 50 pg of the platelet protein sample was applied for electrophoretic separation. The polypeptide (X) denotes the actin band of human platelets with an apparent mol wt of 43,000. The protein staining pattern remained the same as the control even when the cells were exposed to thrombin (100 mu activity) or treated with PALP (3 mM) or both. of platelet protein labeling by PALP/ (3H)NaBH4 to Figure 2. Relation depict (A) control PRP or concentration of thrombin in PRP. The fluorographs PRP exposed to thrombin activity of (B) 10 mu; (C) 30 mu; and (D) 50 mu. (E) A fluorograph of PRP preincubated with 3' ,5'-ADP (60 PM) for 15 min at 37'C before exposure of platelets to 100 mu of thrombin.
differently rest
with
or stimulated
cell
membrane
to cause
proteins
a [14C]
depending
serotonin
473
on whether
release.
platelets
are
at
Vol. 88, No. 2, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
DISCUSSION Thrombin aggregation
has been and release
has been proposed
of platelet protein
earlier
outer
these
studies of thrombin
In our
studies,
by using
amount
of thrombin,
appearance treated
of several
with
from
It
alternatives.
protein
reasonable platelet
to suggest
several
induced the
thrombin that
of labeled
of the
"release
serotonin
orientation
sensitive
labeled
reaction"
membrane
on the fluorograph
surface,
whereby
II
from
the
immediate
release
were
III
either
proteins.
was previously to be a platelet weight,
it
alterations This
is
of thrombin-treated
it
represents
thrombin-
may be suggested in the
to
of the fact
when the
evidenced
may be
corresponds
In view
observed
by 3',5'-ADP, involves
of these
(mol wt 180,000)
(3).
not
action
of serotonin
(mol wt 200,000) band
became
of PALP-building
to favor
of human platelets
was blocked
of platelet actin
band
The
enzyme on platelets
Based on themolecular
polypeptides
from platelets
surface
of thrombin-
was suggested
and the protein
release
a much lower
conditions.
subsequent
I which
the labeled
protein"
and with
band IV (mol wt 150,000)
(15,16).
M (33)
reaction".
proteolytic
at present
glycoprotein
thrombosthenin
the "thrombin that
for
a "release
availability
or from
polypeptide
as platelet
of this
(10-14).
PRP and utilized
fluorographs
have resulted
not possible
The labeled
characterized receptor
is
than
thrombin-induced
on the
the
platelets
physiological
increased
surface
appreciably
rather
on the platelet
This
iodination
platelets the
bands
PRP might
platelet
surface
to cause
under
the action
PALP.
of the enzyme on the the platelets.
occur
protein
in thrombin-treated
needed
of washed
to emerge
in the platelet
platelets
to identify
that
proteins
to react
proteins
labeled
indicate
different
accessible
able
as they might
platelets
caused
PRP instead
we were
by thrombin
and thrombin-treated
actually
both
stimulated
to differentiate
control
than
to initiate
lactoperoxidase-catalyzed
made use of washed
amounts
in platelets
changes
failed
between
higher
changes
using
surface
reaction"
conformational
proteins
pattern
at the platelet
and the "release
studies
surface
labeling
Moreover,
(3)
to involve
However,
(32).
shown to act
that
structural
by the appearance
PSP where,
80% of total
BIOCHEMICAL
Vol. 88, No. 2, 1979
[14c]
serotonin
was released
by PALP could inside
Although
labeled
the platelets
be due to the binding
to the outer
molecular
from
AND BIOPHYSICAL
surface
platelets
weights
are known
ranging
polypeptides
determined.
from
The labeling
whether
or not
other
changes
on platelets
to contain
in
to actin
several
and 160,000,
this
procedure
inducers
ZD).
The labeling during
its
of actin
exposure
from
cell.
90,000
identified
(Fig.
of coenzyme
of the
RESEARCH COMMUNICATIONS
study in
as documented
for
glycoproteins
the exact
(Figs
outlined
of platelet
membrane
identity
of the
2C and 2D) remain
this
function
study also
may help
cause
with
to be to determine
similar
surface
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