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Specific desensitization of actin polymerization of bovine platelets
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 846-850
Vol. 120, No. 3, 1984 May 16, 1984
SPECIFIC DESENSITIZATION OF ACTIN POLYMERIZATION OF BOVINE PLATELETS T. Imada, T. Kikuchi, H. Shimada, Y. Inada, and Y. Saito Laboratory of Biological Chemistry, Tokyo Institute of Technology Ookayama, Meguroku, Tokyo 152, Japan Received April 2, 1984 SUMMARY: Polymerization of actin induced by activation of platelets was investigated using deoxyribonuclease I inhibition assay. When platelets were activated with ADP or 5-hydroxytryptamine, actin was polymerized quickly followed by rapid depolymerization to the initial level. Reactivation with the same agonist, however, did not cause the polymerization of actin, though with different agonists actin polymerized quite normally. The mechanism for this agonist-specific desensitization of actin polymerization was investigated by the use of a calcium ionophore A23187. It was suggested that the cause for the desensitization is the inability of platelets to mobilize Ca 2+ in response to specific agonist.
Previously we platelets.
have
They
studied
agonist-specific
desensitization
of
bovine
lost its ability to aggregate in response to an agonist when
they were preincubated with the agonist, namely they were desensitized. However, those desensitized platelets aggregated quite normally by We
also
other
agonists(1).
observed a similar phenomenon on the platelet shape change by light
scattering technique and scanning electron microscopy(unpublished data). Platelet actin is one of platelets
are
the main
activated with
components
of
cytoskeleton,
and when
agonists it begins to polymerize prior to shape
change and aggregation(2,3). In
this
communication,
polymerization
in
order
interaction with agonists. different
types
we
have
studied
to
elucidate
the desensitization
responses
Desensitization
of
platelets
to agonists
is
of after
actin the
observed with
of cells, and it might be one of the basic cellular regulatory
mechanisms(4).
ABBREVIATIONS: DNase I; deoxyribonuclease I, 5HT; 5-hydroxytryptamine ACD; acid-citrate dextrose, PRP; platelet rich p--~sma 0006-291X/84 $1.50 Copyr~ht © 1984byAcademic Press, Inc. Allrightsofreproductionin anyform reserved.
846
Vol. 120, No. 3, 1984
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MATERIALS AND METHODS ADP(disodium salt) and 5-hydroxytryptamine(creatinine sulfate) were obtained from Yamasa Shoyu Co.(Chiba, Japan) and from Tokyo Kasei Co.(Tokyo, Japan), respectively. A calcium ionophore A23187 and DNA from calf thymus(Type I) and deoxyribonuclease I(DNase I) from bovine panereas(DN-100) were purchased from Sigma Chemical Co.(St. Louis, Mo.). All other chemicals were of analytical grade. Preparation of platelet rich plasma and washed platelet suspension: Bovine blood was obtained at a local slaughter house and anticoagulated with acid-citrate dextrose(ACD). Platelet rich plasma(PRP) and washed platelet suspension in Tris-ACD buffer were prepared by successive ¢entrifugation at different speeds according to a procedure described previously(5). Platelet activation: Fifty ~i of Tris-ACD containing ADP or 5-hydroxytryptamineC5HT) were added to 450 ~i of PRP under stirring at 37°C. A23187 was dissolved in dimethylsulfoxide, and 5 ~i of the solution were added to 500 ~i of washed platelet suspension under stirring at 37°C. Measurement of polymerization of actin by DNase I inhibition assay: Specific inhibition of the activity of DNase I by monomerie actin was measured as follows. One hundred ~i of platelet samples were mixed with i00 ~i of Triton lysis buffer[2% Triton X-100, i0 mM EGTA and i00 mM Tris-HCl(pH 7.4)] and were then diluted with the same volume of Tris-ACD. Fifty ~i of the resultant lysates were mixed with 200 ~i of DNase I solution[15-30 ~g/ml in 50 mM Tris-HCl(pH 7.5) with 0.i mM CaCI2]. Two hundred ~i of the mixtures were added to 3 ml of DNA solution[50 ~g/ml in 4 mM MgSOa, 1.8 mM CaCI 2 and Tris-HCl(pH 7.5)] and the increase of the absorbance at 260 nm in the initial 2 min was recorded with a Shimadzu UV-200 spectrophotometer. All the steps above were handled within 30 seconds. The inhibition of DNase I activity by total actin was determined after depolymerizing all actin molecules with 0.75 M guanidine-HCl at 0°C for 20 to 30 min(6). The final concentration of guanidine-HCl was 9.4 mM, and it did not affect the measurement by itself. The percentage of monomerie actin against total actin was then calculated from those values of inhibition using a standard curve according to a procedure described by Fox et al.(6). RESULTS AND DISCUSSION Platelet actin is known to polymerize after activation of platelets with some kinds of agonists including thrombin(3). this
phenomenon by
Using bovine platelets
activating them with ADP and 5HT.
we
confirmed
As it is shown in Fig.
i(-o-), when platelets were activated with ADP, monomeric actin rapidly began to polymerize and reached to the maximum within 1 min, and quickly depolymerized to the basal level. about by
This quick change of the state
the activation of
platelets
with
of
actin was
5HT(data
also
not shown).
brought We next
examined whether the actin polymerization was affected by the preactivation with same or a different agonist. later
The second addition of ADP to platelets,
i0 min
after they had been exposed to ADP, did not cause polymerization of actin
at all(shown by -e-).
In contrast, when 5HT was used as the second activator,
the quick change
the state of actin was observed(shown by -A-), as if the
of
preactivation with ADP did not affect the second activation with 847
5HT at
all.
Vol. 120, No. 3, 1984
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
"o
,
,
a
b
~½
4o
"v"
E
g ao ~6 0
' IO
o
Incubation
~o
-time
( rain )
A~onist-specific desensitization of actin polymerization. Platelets 09/ml~ in PRP were activated with i0 ~M ADP(-o-)at time zero indicated by the arrow a. They were then activated i0 min later indicated by the arrow b either with i0 ~M ADP(-o-) or i00 ~M 5HT(-A-). At different times a portion of PRP was withdrawn and the amount of monomeric actin decreased from that at time zero, which is the % actin polymerized shown on the ordinate, was determined by the DNase I inhibition assay described in Methods.
This
agonist-specific
desensitization
of
actin
polymerization
was
observed when platelets were preactivated with 5HT; the second addition I0 min later
of
also 5HT
did not cause the polymerization of actin, whereas that of ADP did
cause the polymerization quite normally(data not shown). Polymerization of actin is apparently a very different
phenomenon,
are known to change their activities
concentration of Ca2+(8).
It is quite possible some
complex
mechanisms.
directly
receptors(10).
As
The
initiates the actin polymerization
the
interaction
0.i ~M.
of
of
agonists
with
specific
we expected, the addition of A23187 to platelet suspension
time
in
dose-dependent
manner,
as
it
is
shown
in
course for the change of the state of actin induced by
A23187 was similar to those observed with ADP and percentage
Ca2+(9).
We used a calcium ionophore A23187 in order to
without
induced actin polymerization Fig.2(-o-).
on
When platelets are activated with agonists, one
that this mobilization
mobilize Ca 2+
many
Some of
depending
of the very early events observed is the mobilization of intracellular
by
and
kinds of proteins are involved to regulate the reaction(7).
those actin-bindingproteins the
complex
5HT(data
not
shown).
The
actin polymerized reached to its maximum level of 30 to 35% with
Since platelet preparation used in this figure was different from that
used in Fig.l, values of % actin polymerized between these two figures necessarily
comparable.
There
are
two 848
are
not
major observations we would like to
Vol. 120, No. 3, 1984
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS t¢
4C
oc
y
"O
A I I r
2£
ae
o,
o'.z
~ z'z
Concentration of A25187
( pM )
PolymeKization of actin in platelets activated with A23187 and ADP. ets(2 x lO~/ml) in Tris-ACD were activated with varied concentrations of a calcium ionophore A23187. Platelets were lysed 30 seconds later after the activation and the % actin polymerized by the activation(-o-) was determined by the DNase I inhibition assay described in Methods. The maximum extent of the % actin polymerized by the activation with ADP(20 ~M) was shown by A, and that obtained with ADP(20 ~M) plus A23187(0.24 ~M) was shown by A.
emphasize in this experiment.
First, the maximum extent of actin
polymerized
by the addition of A23187 was always bigger than that obtained by ADP(shownby A in
this
figure)
Second,
or
5HT(data
not
shown), or that obtained bybothcombined.
the maximum extent of actin polymerized with A23187
did not become any
bigger by the simultaneous addition of high concentration of ADP(shown by this
figure).
These
would
imply
that
the
initial
signal
Ca 2+
in
for the aetin
polymerization is the mobilization of intracelhlar Ca 2+, and that each mobilizes
A
agonist
from specific pools and A23187 could mobilize from all of these
pools. Taking advantage of the above conclusion, we thought about the following mechanisms
for the desensitization of aetin polymerization.
itself can not respond to the mobilized Ca 2+. A23187
to
ADP-desensitized
the
mobilization
mobilized. should
the
addition
of
Ca 2+,
The other is that the
agonist
normal
actin polymerization.
As we have already shown, the
of
the preactivation of
polymerization
of
can
not
but actin itself can polymerize if Ca 2+ is
In this case, the addition of A23187 to ADP-desensitized
cause
diminished
One is that actin
platelets should cause decreased polymerization of
actin than that in control platelets. induce
In this case,
two
platelets
Results are presented in Table i. platelets with
ADP
completely
actin induced by the second activation with 849
Vol. 120, No. 3, 1984
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table l: The effect of ADP-induced desensitization on the actin polymerization observed by the activation with A23187. At time zero, 0.24 ~M A23187 or 20 ~M ADP was added to washed platelets(2 x 109/ml) to activate them, and i0 min later A23187 or ADP was again added at the same concentrations. Thirty seconds later after each activation platelets were lysed and the % aetin polymerized by the activation was determined by the DNase I inhibition assay described in Methods. First Activation Agonist % Actin Polymerized A23187 35 ADP 22 ADP 22
ADP.
Second Activation Agonist % Actin Polymerized ADP A23187
In contrast, A23187 caused 35% of aetin
0 35
polymerized
regardless
of
the
preactivation with ADP. The
real
cause
for
the
polymerization should then be that intracellular
Ca 2+
in
response
agonist-specific platelets
This would also make
previously
agonist-specifie
change.
incapable
of
of
aetin
mobilizing
to an agonist after they are exposed to that
particular agonist. observed
become
desensitization
a
reasonable
explanation
for
the
desensitization of aggregation and shape
We are currently investigating whether
a
specific
receptor
becomes
inaccessible to an agonist. REFERENCES i.
Imada, T., Saito, Y., Shimada, H. and Inada, Y., (1983) Thrombos. Res., 31, 807-815. 2. Jennings, L. K., Fox, J. E. B., Edwards, H. H. and Phillips, D. R., (1981) J. Biol. Chem., 256, 6927-6932. 3. Pribluda, V., Laub, F. and Rotman, A., (1981) Eur. J. Bioehem., 116, 293-296. 4.~ Raff, M., (1976) Nature, 259, 265-266. 5. Saito, Y., Imada, T. and Inada, Y., (1980) Thrombos. Res., 17, 809-818. 6. Fox, J. E. B., Doekter, M. E. and Phillips, D. R., (1981) Anal. Bioehem., 117, 170-177. 7. Weeds, A., (1982) Nature, 296, 811-816. 8. Wang~ L. and Bryan, J., (1981) Cell, 25, 637-649. 9. Massini, P., K~ser-Granzmann, R. and Iflscher, E. F., (1978) Thrombos. Haemostas., 40, 212-218. i0. Mensehe, D., Israel, A. and Karpatkin, S., (1980) J. Clin. Invest., 66, 284-291.
850
Vol. 120, No. 3, 1984 May 16, 1984
SPECIFIC DESENSITIZATION OF ACTIN POLYMERIZATION OF BOVINE PLATELETS T. Imada, T. Kikuchi, H. Shimada, Y. Inada, and Y. Saito Laboratory of Biological Chemistry, Tokyo Institute of Technology Ookayama, Meguroku, Tokyo 152, Japan Received April 2, 1984 SUMMARY: Polymerization of actin induced by activation of platelets was investigated using deoxyribonuclease I inhibition assay. When platelets were activated with ADP or 5-hydroxytryptamine, actin was polymerized quickly followed by rapid depolymerization to the initial level. Reactivation with the same agonist, however, did not cause the polymerization of actin, though with different agonists actin polymerized quite normally. The mechanism for this agonist-specific desensitization of actin polymerization was investigated by the use of a calcium ionophore A23187. It was suggested that the cause for the desensitization is the inability of platelets to mobilize Ca 2+ in response to specific agonist.
Previously we platelets.
have
They
studied
agonist-specific
desensitization
of
bovine
lost its ability to aggregate in response to an agonist when
they were preincubated with the agonist, namely they were desensitized. However, those desensitized platelets aggregated quite normally by We
also
other
agonists(1).
observed a similar phenomenon on the platelet shape change by light
scattering technique and scanning electron microscopy(unpublished data). Platelet actin is one of platelets
are
the main
activated with
components
of
cytoskeleton,
and when
agonists it begins to polymerize prior to shape
change and aggregation(2,3). In
this
communication,
polymerization
in
order
interaction with agonists. different
types
we
have
studied
to
elucidate
the desensitization
responses
Desensitization
of
platelets
to agonists
is
of after
actin the
observed with
of cells, and it might be one of the basic cellular regulatory
mechanisms(4).
ABBREVIATIONS: DNase I; deoxyribonuclease I, 5HT; 5-hydroxytryptamine ACD; acid-citrate dextrose, PRP; platelet rich p--~sma 0006-291X/84 $1.50 Copyr~ht © 1984byAcademic Press, Inc. Allrightsofreproductionin anyform reserved.
846
Vol. 120, No. 3, 1984
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MATERIALS AND METHODS ADP(disodium salt) and 5-hydroxytryptamine(creatinine sulfate) were obtained from Yamasa Shoyu Co.(Chiba, Japan) and from Tokyo Kasei Co.(Tokyo, Japan), respectively. A calcium ionophore A23187 and DNA from calf thymus(Type I) and deoxyribonuclease I(DNase I) from bovine panereas(DN-100) were purchased from Sigma Chemical Co.(St. Louis, Mo.). All other chemicals were of analytical grade. Preparation of platelet rich plasma and washed platelet suspension: Bovine blood was obtained at a local slaughter house and anticoagulated with acid-citrate dextrose(ACD). Platelet rich plasma(PRP) and washed platelet suspension in Tris-ACD buffer were prepared by successive ¢entrifugation at different speeds according to a procedure described previously(5). Platelet activation: Fifty ~i of Tris-ACD containing ADP or 5-hydroxytryptamineC5HT) were added to 450 ~i of PRP under stirring at 37°C. A23187 was dissolved in dimethylsulfoxide, and 5 ~i of the solution were added to 500 ~i of washed platelet suspension under stirring at 37°C. Measurement of polymerization of actin by DNase I inhibition assay: Specific inhibition of the activity of DNase I by monomerie actin was measured as follows. One hundred ~i of platelet samples were mixed with i00 ~i of Triton lysis buffer[2% Triton X-100, i0 mM EGTA and i00 mM Tris-HCl(pH 7.4)] and were then diluted with the same volume of Tris-ACD. Fifty ~i of the resultant lysates were mixed with 200 ~i of DNase I solution[15-30 ~g/ml in 50 mM Tris-HCl(pH 7.5) with 0.i mM CaCI2]. Two hundred ~i of the mixtures were added to 3 ml of DNA solution[50 ~g/ml in 4 mM MgSOa, 1.8 mM CaCI 2 and Tris-HCl(pH 7.5)] and the increase of the absorbance at 260 nm in the initial 2 min was recorded with a Shimadzu UV-200 spectrophotometer. All the steps above were handled within 30 seconds. The inhibition of DNase I activity by total actin was determined after depolymerizing all actin molecules with 0.75 M guanidine-HCl at 0°C for 20 to 30 min(6). The final concentration of guanidine-HCl was 9.4 mM, and it did not affect the measurement by itself. The percentage of monomerie actin against total actin was then calculated from those values of inhibition using a standard curve according to a procedure described by Fox et al.(6). RESULTS AND DISCUSSION Platelet actin is known to polymerize after activation of platelets with some kinds of agonists including thrombin(3). this
phenomenon by
Using bovine platelets
activating them with ADP and 5HT.
we
confirmed
As it is shown in Fig.
i(-o-), when platelets were activated with ADP, monomeric actin rapidly began to polymerize and reached to the maximum within 1 min, and quickly depolymerized to the basal level. about by
This quick change of the state
the activation of
platelets
with
of
actin was
5HT(data
also
not shown).
brought We next
examined whether the actin polymerization was affected by the preactivation with same or a different agonist. later
The second addition of ADP to platelets,
i0 min
after they had been exposed to ADP, did not cause polymerization of actin
at all(shown by -e-).
In contrast, when 5HT was used as the second activator,
the quick change
the state of actin was observed(shown by -A-), as if the
of
preactivation with ADP did not affect the second activation with 847
5HT at
all.
Vol. 120, No. 3, 1984
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
"o
,
,
a
b
~½
4o
"v"
E
g ao ~6 0
' IO
o
Incubation
~o
-time
( rain )
A~onist-specific desensitization of actin polymerization. Platelets 09/ml~ in PRP were activated with i0 ~M ADP(-o-)at time zero indicated by the arrow a. They were then activated i0 min later indicated by the arrow b either with i0 ~M ADP(-o-) or i00 ~M 5HT(-A-). At different times a portion of PRP was withdrawn and the amount of monomeric actin decreased from that at time zero, which is the % actin polymerized shown on the ordinate, was determined by the DNase I inhibition assay described in Methods.
This
agonist-specific
desensitization
of
actin
polymerization
was
observed when platelets were preactivated with 5HT; the second addition I0 min later
of
also 5HT
did not cause the polymerization of actin, whereas that of ADP did
cause the polymerization quite normally(data not shown). Polymerization of actin is apparently a very different
phenomenon,
are known to change their activities
concentration of Ca2+(8).
It is quite possible some
complex
mechanisms.
directly
receptors(10).
As
The
initiates the actin polymerization
the
interaction
0.i ~M.
of
of
agonists
with
specific
we expected, the addition of A23187 to platelet suspension
time
in
dose-dependent
manner,
as
it
is
shown
in
course for the change of the state of actin induced by
A23187 was similar to those observed with ADP and percentage
Ca2+(9).
We used a calcium ionophore A23187 in order to
without
induced actin polymerization Fig.2(-o-).
on
When platelets are activated with agonists, one
that this mobilization
mobilize Ca 2+
many
Some of
depending
of the very early events observed is the mobilization of intracellular
by
and
kinds of proteins are involved to regulate the reaction(7).
those actin-bindingproteins the
complex
5HT(data
not
shown).
The
actin polymerized reached to its maximum level of 30 to 35% with
Since platelet preparation used in this figure was different from that
used in Fig.l, values of % actin polymerized between these two figures necessarily
comparable.
There
are
two 848
are
not
major observations we would like to
Vol. 120, No. 3, 1984
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS t¢
4C
oc
y
"O
A I I r
2£
ae
o,
o'.z
~ z'z
Concentration of A25187
( pM )
PolymeKization of actin in platelets activated with A23187 and ADP. ets(2 x lO~/ml) in Tris-ACD were activated with varied concentrations of a calcium ionophore A23187. Platelets were lysed 30 seconds later after the activation and the % actin polymerized by the activation(-o-) was determined by the DNase I inhibition assay described in Methods. The maximum extent of the % actin polymerized by the activation with ADP(20 ~M) was shown by A, and that obtained with ADP(20 ~M) plus A23187(0.24 ~M) was shown by A.
emphasize in this experiment.
First, the maximum extent of actin
polymerized
by the addition of A23187 was always bigger than that obtained by ADP(shownby A in
this
figure)
Second,
or
5HT(data
not
shown), or that obtained bybothcombined.
the maximum extent of actin polymerized with A23187
did not become any
bigger by the simultaneous addition of high concentration of ADP(shown by this
figure).
These
would
imply
that
the
initial
signal
Ca 2+
in
for the aetin
polymerization is the mobilization of intracelhlar Ca 2+, and that each mobilizes
A
agonist
from specific pools and A23187 could mobilize from all of these
pools. Taking advantage of the above conclusion, we thought about the following mechanisms
for the desensitization of aetin polymerization.
itself can not respond to the mobilized Ca 2+. A23187
to
ADP-desensitized
the
mobilization
mobilized. should
the
addition
of
Ca 2+,
The other is that the
agonist
normal
actin polymerization.
As we have already shown, the
of
the preactivation of
polymerization
of
can
not
but actin itself can polymerize if Ca 2+ is
In this case, the addition of A23187 to ADP-desensitized
cause
diminished
One is that actin
platelets should cause decreased polymerization of
actin than that in control platelets. induce
In this case,
two
platelets
Results are presented in Table i. platelets with
ADP
completely
actin induced by the second activation with 849
Vol. 120, No. 3, 1984
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table l: The effect of ADP-induced desensitization on the actin polymerization observed by the activation with A23187. At time zero, 0.24 ~M A23187 or 20 ~M ADP was added to washed platelets(2 x 109/ml) to activate them, and i0 min later A23187 or ADP was again added at the same concentrations. Thirty seconds later after each activation platelets were lysed and the % aetin polymerized by the activation was determined by the DNase I inhibition assay described in Methods. First Activation Agonist % Actin Polymerized A23187 35 ADP 22 ADP 22
ADP.
Second Activation Agonist % Actin Polymerized ADP A23187
In contrast, A23187 caused 35% of aetin
0 35
polymerized
regardless
of
the
preactivation with ADP. The
real
cause
for
the
polymerization should then be that intracellular
Ca 2+
in
response
agonist-specific platelets
This would also make
previously
agonist-specifie
change.
incapable
of
of
aetin
mobilizing
to an agonist after they are exposed to that
particular agonist. observed
become
desensitization
a
reasonable
explanation
for
the
desensitization of aggregation and shape
We are currently investigating whether
a
specific
receptor
becomes
inaccessible to an agonist. REFERENCES i.
Imada, T., Saito, Y., Shimada, H. and Inada, Y., (1983) Thrombos. Res., 31, 807-815. 2. Jennings, L. K., Fox, J. E. B., Edwards, H. H. and Phillips, D. R., (1981) J. Biol. Chem., 256, 6927-6932. 3. Pribluda, V., Laub, F. and Rotman, A., (1981) Eur. J. Bioehem., 116, 293-296. 4.~ Raff, M., (1976) Nature, 259, 265-266. 5. Saito, Y., Imada, T. and Inada, Y., (1980) Thrombos. Res., 17, 809-818. 6. Fox, J. E. B., Doekter, M. E. and Phillips, D. R., (1981) Anal. Bioehem., 117, 170-177. 7. Weeds, A., (1982) Nature, 296, 811-816. 8. Wang~ L. and Bryan, J., (1981) Cell, 25, 637-649. 9. Massini, P., K~ser-Granzmann, R. and Iflscher, E. F., (1978) Thrombos. Haemostas., 40, 212-218. i0. Mensehe, D., Israel, A. and Karpatkin, S., (1980) J. Clin. Invest., 66, 284-291.
850