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.

1296

Cooperativity between platelet-activating factor and collagen in aggregation of bovine platelets, II.

When subthreshold amounts of platelet-activating factor (PAF) and collagen were added simultaneously, strong aggregation of platelets was induced. How...
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