Vol. 168, No. 3, 1990
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1292-1296
BIOCHEMICAL
May 16, 1990
COOPERATIVITY COLLAGEN
BETWEEN IN
Soichi
PLATELET-ACTIVATING
AGGREGATION
Kojimal,
Toshiyasu
OF
Pujio
FACTOR
BOVINE
PLATELETS,
Sekiyal,
Yuji
Tsukada3, and Yuji
IDepartment of Biological Sciences, Technology, Ookayama, Meguro-ku, 2Department of Materials Yokohama, Kurogane-cho,
AND II
Inada2,
Saitol*
Tokyo Institute of Tokyo 152, Japan
Science and Technology, Toin University of Midori-ku, Yokohama, Kanagawa 227, Japan
3Division of Hematologic Research, Toranomon Hospital and Okinaka Memorial Institute for Medical Research, Toranomon, Minato-ku, Tokyo 105, Japan Received March 22, 1990 SUMMARY:
When subthreshold amounts of platelet-activating factor (PAR) and collagen were added simultaneously , strong aggregation of platelets was induced. However, each agonist alone at these concentrations could not induce aggregation at all (S. Kojima -et al (1987) Biochem. Biophys. -I Res. Commun. 145, 915-920). This cooperativity was 2ybserved not only in aggregation but also in changes of intracellular Ca concentration, 47 kDa protein phosphorylation, and formation of thromboxane. These findings suggest that various steps of signal transduction pathways are Q 1990 Academic Press. Inc. enhanced during their cooperativity.
Various
agonists
mechanisms. event
It
induce aggregation
is now generally
which triggers
accepted
of platelets that
through different in many cases the initial
these responses is receptor-mediated
of phospholipases A2 and C (1). It was reported earliest events associated with PAR-induced activation phosphoinositide-specific inositol stores,
of phospholipase
*To whom all
the was
C-mediated production of which releases Ca2+ from intracellular
1,2-diacylglycerol
Collagen-induced platelet dependent on endogenously vation
that one of of platelets
phospholipase
1,4,5-trisphosphate and
activations
which activates
protein
kinase
C
(2).
aggregation is known to be essentially generated TXA2 mainly produced through acti-
A2 (3).
correspondence/reprint
We previously requests
showed that
1292
lysate
should be addressed.
The abbreviations: PAP, platelet-activating factor; ACD, acid citrate dextrose; SDS-PAGE, sodium dcdecyl amide gel electrophoresis; RIA, radioimmunoassay. 0006-291x/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
cell
TX, thromboxane; sulfate-polyacryl-
Vol. 168, No. 3, 1990
BIOCHEMICAL
from cultured bovine vascular PAF and collagen functioned together,
induced extensive
the concentrations gation of platelets
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
endothelial cooperatively aggregation
cells contained PAF and that (4). PAF and collagen, of bovine platelets even with
by which each agonist alone could not induce aggreat all. In order to clarify the mechanism of this
cooperativity, we examined here which step(s) of pathway was activated cooperatively, and found that
signal transduction at least three steps
were enhanced. MATERIALS AND METHODS Materials PAF and collagen (type I) from horse tendon were purchased from Avanti Polar Lipids, Inc. (Pelham, AL) and Hormon-Chemie, GmbH. (Munich, FRG), respectively. FuraZ-AM was obtained from Dojin Laboratories (Kumamoto, Japan). X-Ray film was from Eastman Kodak Co. (Rochester, NY). All other reagents were of analytical grade. Preparation of Bovine Washed Platelets Tris-ACD buffer was prepared from bovine as described previously (5).
Washed platelet suspension in blood anticoagulated with ACD
Measurement of Platelet Aqqregation Platelet aggregation was monitored as increase of light transmission in the presence of 8 mM Ca2+ and 1 mg/ml bovine serum albumin (essentially fatty acid free, Sigma) using a Nikoh Bioscience aggregometer NKK HEMA Tracer 601 (Tokyo, Japan). Measurement of Intracellular Ca2+ Concentration The change of intracellular CaL+ concentration was determined usinq a fluorescent dve Fura2-loaded platelets as described by Pollock -et al (6). Changes in -* intracellular Ca2+ concentration and aggregation of platelets were monitored simultaneously in the presence of 1 mM Ca2+ in HEPES-Tyrode buffer by a Japan Spectroscopic Ca2+ analyzer CAF-100 (Tokyo, Japan). Measurement of Protein Phosphorylation Protein phosphorylation was measured using platelets incubated with (32Plorthophosphate (carrier free, Du Pont-New England Nuclear) according to the method of Crouch et al. (7) Labeled platelets (5 x 108 platelets/250 ul, approx. 3.6 x 15 dpm) were stimulated with PAF, collagen or their combination, and 2 min later the phosphorylation was terminated, and 40 1-11 of samples were subjected to SDS-PAGE on 10% gel. Radioactive bands were localized by autoradiography. Measurement of Thromboxane Formation The amounts of TXA2 formed 7 min after the addition of agonist was measured using TXB2 1251-RIA kit (Amersham Corp.).
RESULTS AND DISCUSSION Figure cooperative
shows changes in intracellular Ca2+ concentration during aggregation. While 1.6 nM PAF or 2 vg/ml collagen alone 2+ concentration (110 nM for could induce only a little increase of Ca PAF and 30 nM for collagen) and shape change without aggregation (curves of these resulted in increase in A, B and a, b), simultaneous addition 1
Ca2+ concentration as
represented
in two steps with by curves
C and c. 1293
shape change and strong aggregation Ca2+ Along with the shape change,
Vol.
166,
No.
BIOCHEMICAL
3, 1990
AND
BIOPHYSICAL
20 kDa--,
02 Redion
RESEARCH
71
COMMUNICATIONS
w
a
-
b
w
c
d
Time (min)
Change of intracellular Ca2+ concentration during the cooperaFIG. 1. try: aggregation between PAF and collagen. The changes in intracellular (bottom trace, A-C) and platelet aggregations Ca concentration ( uew trace, a-c) were monitored simultaneously. Curves A and a, 2 w/ml collagen + collagen; curves B and b, 1.6 nM PAF; curves C and c, 2 ug/ml 1.6 nM PAF. FIG. 2. stimulated
An with
autoradiogram of phosphorylated proteins in platelets simultaneous addition of PAF and collagen. Two proteins which showed extreme changes in phosphorylation are indicated on figure with their molecular mass. Lane a, without agonists; along lane b, lane c, 2 pg/ml collagen; lane d, 0.2 nM PAP + 2 ug/ml 0.2 l-24 PAF; ran Under this experimental condition the 20 kDa protein collagen. close to the front of the gel and it was difficult to accurately
determine the extent of phosphorylation.
concentration with
increased
rapidly
As shown in an autoradiogram lated
(190
nM) and further
increased
along
the aggregation. when platelets
in Fig.
were activated
2, some proteins
by agonists
for
were phosphory-
2 min.
tion of 47 kDa protein in particular became very notable collagen were simultaneously added at subthreshold (compare activation
lanes b and c with d). of protein kinase C (8).
This
As shown in Fig. 3, panel A, 1.7 together induced extensive formation (curve neither results addition tivity
C),
although
each agonist
may
Phosphorylawhen PAF and concentrations
represent
ug/ml collagen of TXB2 along
and
cooperative 0.8
nM PAF
with aggregation concentrations could at all (curve A). These
alone at these
produce TXB2 nor induce aggregation
suggest that phospholipases are activated by simultaneous of subthreshold amounts of collagen and PAP. This cooperain TXB2 formation is shown more obviously by adding various
amounts of PAF (0.086-5.2 nM) amounts of collagen (0.8 or 1.7
with and without the addition of fixed ug/ml). Although the formation of TxB2 during PAP-induced aggregation was reported using rabbit and human platelets (9 and IO), opposite result was also reported (11). In our study
using bovine
platelets,
PAP-induced 1294
platelet
aggregation
was
not
Vol.
168, No. 3, 1990
BIOCHEMICAL
A
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The Amounts of T%& (ng/108
Platelets) 0
A s 'u, .'" B
4.3
ES0 k z 1 x
Oy-F m
9.8
C
1ooL FIG. 3. Cooperativity in TXB2 formation exerted by PAF and collagen. Panel A, Aggregation patterns and the amounts of TXB formed at the end periods of aggregation. Platelet counts were 5 x l$'/ml. Curve A, 0.8
nM PAF or
1.7
pg/ml
collagen
alone;
collagen; curve C, 0.8 nM PAF + 1.7 amounts of TXBZ forned after addition (O-086-5.2
(curve
B)
1-24) or 1.7
in the absence (line A) pg/ml (curve C) collagen.
accompanied by formation note
that
conclusion,
it
transduction
concentration subthreshold interaction(s)
and the presence
(line
A).
B and C), although
was suggested
in this
pathway activated
of
of
0.8‘
PAF
ug/ml
It is interesting by increasing
the amounts of TXB2 formed is increased
of PAF added (curves induce TXB2 formation at all. signal
curve B, 0.8 194 PAF + 0.8 ug/ml collagen. Panel B, The
of various concentrations
of TXB2 at all
amounts In
ug/ml
PAF alone
study that
various
by PAF or collagen
to the
does
not
steps
of
alone at
high
appeared to be enhanced when they were added together at concentration. We are now investigating further what occurs
between each signal
transduction
pathway.
ACKNOWLEDGMENTS This on
Priority
Japan,
work was supported
Areas from the Ministry
and by a Research Grant
the Ministry
by Grants-in-Aid
of Health
for
and Welfare,
for
of Education, Cardiovascular
Scientific
Research
Science and Diseases
Culture,
(A 62-l)
from
Japan.
REFERENCES Kinlough-Rathbone, R. L., and Mustard, J. F. (1987) In Platelets in and Pathology (MacIntyre, D. E., and Gordon, J. L., Eds.) Biology Vol. III, pp.239-267. Elsevier Science Publishers B. V. (Biomedical Division), Amsterdam. 2. Kumar, R., and Hanahan, D. J. (1987) In Platelet-activating Factor and Related Lipid Mediators (Snyder, F., Ed.) pp.239-254. Plenum Press, New York. 3. Gordon, JfaLj ;~~81~IIn Platelets in Biology and Pathology (Gordon, J. L., . . , pp.l-17. Elsevier/North-Holland Biomedical Press, Amsterdam. 1.
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166, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
4. Xojima S., Hagiwara, H., Soga, W., Sekiya, F. Saito, Y., and Inada, Y. (1987) Biochem. Biophys. Res. Commun. 145, 915-920. 5. Saito, Y., Imada, T., and Inada, Y. (1980) Thrombos. Res. 17, 809818.
6. Pollock, 235, 7.
W. K.,
Rink,
T. J.,
and Irvine,
R. F.
(1986)
Biochem.
J.
869-877.
Crouch,
M. F. and Lapetina,
E. G.
(1988)
J. Biol.
Chem. 263,
3363-
3371. 8. Nishizuka, Y. (1984) Nature 308, 693-698. 9. Shaw, J. O., Klusick, S. J., and Hanahan, D. J. (1981) Biochim Biophys. Acta 663, 222-229. 10. Macconi, D., Morzenti, G., Livio, M., Morelli, C., Cassina, G., and Remuzzi, G. (1985) Lab. Invest. 52, 159-168. 11. Cazenave, J. P., Benveniste, J., and Mustard, J. F. (1979) Lab. Invest. 41, 275-285.
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