The production of phosphatidylinositol trisphosphate is stimulated by thrombin in human platelets

Vol.

174,

No.

January

2, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

31, 1991

THE

Pages

PRODUCTION

OF PHOSPHATIDYLINOSITOL THROMBIN

Roger

Division

Received

of Cell Biology,

December

TRISPHOSPHATE

IN HUMAN

D. Nolan*

Burroughs

BY

PLATELETS

and Eduardo

Wellcome

IS STIMULATED

524-528

G. Lapetinaf

Co.,

Research

Triangle

Park,

NC 27709

3, 1990

Untreated human platelets labeled to equilibrium with 3*P, contained undetectable levels of 3-phosphorylated phosphoinositides. Stimulation of platelets with thrombin for 5 min resulted in an enormous increase in the amount of phosphatidylinositol3,4-bisphosphate (Nolan and Lapetina, 1. Bioi. Chem. =,2441-2445; 1990). We now report that the levels of phosphatidylinositol 3,4,5-trisphosphate are greatly elevated within 90 s of treatment of platelets with thrombin. Phosphatidylinositol3,4,5phosphate might have an important role in platelet aggregation. D 1991 Academic Press. Inc.

Much (Pls) that

recent

evidence

are phosphorylated

3-phosphorylated activated

by several during

[Ptdlns(3)P]; been (8,lO)

studies,

(8,ll)

or stimulated Using

the synthesis

platelets

which

t To whom

including

results

while

the

to a lesser extent labeled

with

that

than

correspondence

should

which

with

of platelets

by thrombin,

phorbol

results

of Medicine,

in a large

transduction

activity,

Melbourne

we investigated in thrombin-

appears in human

to constitute

$1.50

524

a

platelets.

Hospital,

Victoria

be addressed. CroPIns,

in

undetected

(9).

of Ptdlns(3,4,S)P3

and Ptdlns(3)P.

in the

However,

was either platelets

high specific

Royal

increase

are unaffected.

in stimulated

and with

in signal

(2), has also This laboratory

the formation

Ptdlns(3,4)Pz

3-phosphate

(8-12).

PI, phosphoinosititde; Ptdlns, phosphatidylinositol; phosphate; SAX HPLC, strong anion exchange HPLC.

0 1991 by Academic Press. Inc. of reproduction in any form reserved.

is

and oncogene

PI, Ptdlns(3,4,S)P3,

was Ptdlns(3,4)Pz

of the PI cycle and may be involved

Department

receptors

3-kinase,

[Ptdlns(3,4,5)Px]

levels of Ptdlns(3)P

3*P, to equilibrium

along

(Ptdlns)

(7) and platelets

and GTPyS

3-phosphorylated

Pls and report

PtdIns(3,4,5)P3,

of these

of phosphatidylinositols

activation

U46619,

intracellular polar

The synthesis

and 3,4,5-trisphosphate

analog

of the more

(1).

factor

in the formation

demonstrated

the thromboxane

University

growth

cells, such as neutrophils

have

ring

class of phosphoinositides

(2-6).

[Ptdlns(3,4)Pl];

of polyphosphorylated

address:

Abbreviations: InsP, inositol

Copyright All rights

(9,11,12)

the level

platelets. branch

kinases,

of a novel

by a phosphatidylinositol

of cell proliferation

activity,

of Ptdlns(3,4)Pz,

those

0006-291X/91

tyrosine

in nonproliferating

A23187,

* Present Australia.

of the inositol

3,4-bisphosphate

dibutyrate,

novel

protein

3-kinase

and others

treated

on the 3-position to be regulated

stimulation

detected

synthesis

the existence

Pls is thought

products,

Ptdlns

has demonstrated

glycerophosphoinositol;

3050,

Vol.

174,

No.

2, 1991

BIOCHEMICAL

AND

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MATERIALSAND

RESEARCH

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METHODS

Blood from human donors was collected in trisodium citrate and platelets were prepared as described previously (8). After labeling with 32P, (8), platelets were resuspended in buffer without EDTA and incubated with a-thrombin for the times indicated on figures. Incubations were terminated with CHCl5ICH30HIHCI (100:200:2) and phospholipids were extracted and deacylated to form glycerophosphoinositides (CroPIs), which were separated by strong anion exchange (SAX) HPLC as previously described (8). [azP]labeled GroPls were identified by coelution with [3H]labeled GroPl standards (8). After separation by HPLC, the individual GroPls were chemically treated to remove the glycerol moiety and the resulting inositol [32P]phosphate (InsP) head groupswere identified by cochromatography with standard [jH]lnsPs on SAX HPLC (8). [52P]orthophosphate was purchased from ICN K&K Laboratories, Inc. and [3H]inositol phospholipids and phosphates were from DuPontNew England Nuclear and Amersham. a-Thrombin was a gift from Dr. John Fenton II, Division of Laboratories and Research, New York State Department of Health, Albany, NY.

RESULTS The deacylated by SAX HPLC.

Pls from

We previously

[32P]-labeled identified

GroPlns(3,4)Pz,

and GroPlns(4,S)Pz

reported

in which

more

here, polar

GroPlns

deacylated

polyphosphate

human

platelets

glycerophosphoinositol

in samples Pls from (peak

treated

(GroPlns),

of thrombin-treated a-thrombin-treated

X) was eluted

with

after

platelets platelets the CroPIns

a-thrombin

were

GroPlns(3)P,

GroPlns(4)P,

(8). were

separated

In the experiments separated

bisphosphates

by HPLC, a (Fig.

1). The

, 6000 e--GroPlns(3,4)P, II I\ ‘I I; ‘I ‘I ‘I ‘I

ELUTION Figure

1.

HPLC

analysis

of [32P,llabeled

TIME (seconds)

liPids from human

olatelets

Platelets were labeled with [QP,] for 90 min and stimulated with a-thrombin (5 U/ml) for 5 min. The phospholipid fraction was deacylated and the GroPlns phosphates from unstimulated platelets (broken line) and platelets stimulated with a-thrombin (solid line) were separated by SAX HPLC as described in Materials and Methods. The elution timesof [sH]lns-labeled internal standards, GroPlns(3,4)Pz. and GroPlns(4,5)P2 are indicated. Unknown peak X was collected for further analysis

525

Vol.

174,

No.

2, 1991

400

BIOCHEMICAL

AND

BIOPHYSICAL

-

RESEARCH

[3HIlns(l

COMMUNICATIONS

,3,4,5)R,;

II

200 -

8000 ELUTION Figure

2.

HPLC

TIME

of the water-soluble

analvsis

(seconds)

head qroup

of unknown

phosoholipid

X

Deacylated phospholipid peak X was collected and deglycerated asdescribed in Materials and Methods. The resulting polar head group was subjected to SAX HPLC and its elution time was compared to that of [sH]lns-labeled inosltol phosphate internal standards (arrowed). The resultant single peak of [QPlradioactivity (solid line) coeluted with standard [3H]lns(1,3,4,5)P, (broken line).

inositol

phosphate

glycerol

moiety,

head

group

coeluted

exactly

Ins(l,3,4,5)P4-containing

with

phospholipid,

standard

levels

which

over

increased increase

peaked

within

after

thrombin

platelets

90 s, as distinct

a slowertime

course

(Fig.

formed

[3H]lns(1,3,4,5)P4

PI in thrombin-treated

GroPlns(3,4,5)P3

by a slight

of this unknown

by chemical

removal

on SAX HPLC (Fig.

was therefore

to the previously

3). GroPlns(4,S)Pz

identified

2). The as Ptdlns(3,4,5)P3.

identified

showed

of the

GroPlns(3,4)Pz,

an initial

decrease

followed

addition.

GroPlns(4,5)Pz

2

GroPlna(3,4,5)P3

2

7 9

2

P

51 L0

0 0

200

1200

0

200 TIME

Fiqure

GroPlns integrated

3.

Time course stimulation

of formation with a-thrombin

of CroPIns

1200

0

200

(seconds) polvphosohates

in [QPI-labeled

olatelets

polyphosphates were separated by SAX HPLC as in Fig. 1. Peaks of [QPlradioactivity to give amounts of each GroPlns polyphosphate produced at each incubation

526

followinq

were time.

1200

Vol.

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No.

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2, 1991

AND

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RESEARCH

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DISCUSSION We and others Ptdlns(3,4)Pz

previously

(8-12).

Ptdlns(3,4,5)P3

Although

or showed We now

treatment.

basis that

its polar

shown

a small

report

thrombin

agonist,

which

stimulation

between

in resting

would

result

Ptdlns(3,4,5)P3

and Rittenhouse

muscle

cells the same

(9).

that

4-kinase.

(10,15)

that

before

no

isa dramatic is that

the addition

in

of

less dramatic

and Ptdlns for the Ptdlns

Ptdlns(4,5)P2

is synthesized

be activated

is synthesized

enzyme

Ptdlns(3,4)Pz

the data

postulated

that

respectively

3-kinase

(2).

slowly

by a Ptdlns(3,5)Pz

(2).

is Ptdlns(3,4,5)P3

than

because

the it

in platelets. via a Ptdlns(3)P

the Ptdlns

4-kinase

be that

Alternatively,

can also be synthesized

more

in smooth

It may

in platelets.

than

of

of Ptdlns(3)P,

(4,5)Pz,

are different

by a different that

with

for the synthesis

Ptdlns(4)P.

and

to thrombin

consistent It has been

is responsible

Ptdlns,

may

there

in response

for this is unclear.

of Ptdlns(4)P

4-kinase

(9), we found

3-kinase.

It appears

Ptdlns(3,5)Pz

has not

described.

platelet

course

of production

aggregatory

has been (14.16).

ruled

response.

out because

Ptdlns(3,4,5)P3

recently

of Ptdlns(3,4,5)Ps

The contribution

of the

relative

may function,

demonstrated

that

polymerization

of actin

in stimulated

charged

phospholipid,

the highly

response.

actin-binding

in vitro

reports

protein,

platelets

in recent profilin,

in platelet

aggregation

In summary,

in human

in uiuo because

(20, Fig. 3). Also,

it may be involved

3) which course

in regulation

that

of Ptdlns(3,4,5)P3

Pls to phospholipase

closely

demonstrate

that

after

Ptdlns(4,5)Pz

of Ptdlns(3,4)Pz in vitro

of appearance

agonist appears

within

of the platelet

527

cytoskeleton,

tightly

However,

to be too slow

in aggregating which

evidence

of actin to the Ptdlns(4,5)Pz

in neutrophils

l-2 min of addition

of Ptdlns(3,4,5)P3

the

providing

binds

(19). binding

eta1

of the cytoskeletal

in the regulation

(18) and neutrophils

C

Eberle

parallel

curve,

in regulation

to be involved

of the

of lns(1,3,4,5)P4

phospholipid.

formation

is involved

shown

a mediator

to the production

as did the dose response

platelets

occurs

it being

of 3-phosphorylated

it is metabolized

the formation

(Fig. the time

been

with

asa membrane-bound

Ptdlns(3,4,5)P3, have

not be involved

resistance

neutrophils,

Polyphosphoinositides

polymerization

is consistent

of Ptdlns(3,4,5)P3

therefore,

the kinetics

that

that

Ptdlns(3,4)Pz

substrates

Ptdlns(3,4,5)P3

The time

may

than

3-kinase,

has demonstrated

The Ptdlns(3)P

unlikely

quickly

good

Ptdlns(3,4)Pz

This laboratory

studies

and a much

to 5 min for Ptdlns(3,4)Pz,

from

on the

has been

for this discrepancy

factors

to

of agonist.

more

Ptdlns

explanation

of Ptdlns(3,4,5)P3

Pls are equally

of phosphorylation

is possible

addition

as Ptdlns(3,4,5)Ps

to thrombin

by endogenous

levels

The explanation

enzyme,

not all of those

kinetics

basal

90 s compared

and Ptdlns(3,4,5)P3

However,

(17)

upon

within

Kucera

been

in higher

in response

to earlier

in response

1). A possible

be activated

platelets

on SAX HPLC, which

In contrast

However,

(Fig.

could

is synthesized

it peaks

Ptdlns(3,4)Pz,

platelets.

PtdIns(3,4,5)P3

identified

Ins(l,3,4,5)P4

of lnsP4 (14).

of Ptdlns(3,4,5)Ps

the platelets

with

is lesswell

to detect

(9).

in human

has been

exactly

failed

activation

and

alsosynthesize

of Ptdlns(3,4,5)P3 either

after

Ptdlns(3)P

cell typescan

of Ptdlns(3,4,5)P3

the isomers

of Ptdlns(3,4,5)P3

treatment;

in Ptdlns(3,4,5)P3

coelutes

to synthesize

the production

polyphosphoinositide

group

in the amount

experiments

other

of Pls in platelets

stimulation

This novel head

Ptdlns(3,4,5)P3

increase some

increase

are able that

activation, studies

the potent

to distinguish

detectable

Previous

platelets

established

to receptor

in platelets.

(8,10,11),

that

it is well

in response

characterized

showed

results

(17) and

for it to be involved of thrombin platelets in platelet

(21). indicates shape

Vol.

174,

change

No.

2, 1991

and release

group,

the possible

which

initiates

would

act in concert

response,

namely

BIOCHEMICAL

of secretory actions

granule

increased

contents.

of Ptdlns(3,4,5)Pj

the polyermization with

AND

the known cytosolic

of actin

Because

may

include

and platelet

responses calcium

BIOPHYSICAL

it has a highly complexing

shape

to thrombin, concentration

RESEARCH

negatively-charged

of actin-binding

change. which

COMMUNICATIONS

Such a response contribute

and protein

head proteins, to thrombin

to the platelet

phosphorylation.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

Whitman, M., Downes, C.P., Keeler, M., Keller, T., and Cantley, L. (1988) Nature 3321644-646. Auger, K.R., Serunian, L.A.,Soltoff,S.P., Libby, P., and Cantley, L.C. (1989)Cell 57- 167-175. Varticovski, L., Druker, B., Morrison, D., Cantley, L., and Roberts, T. (1989) Nature x,699-702. Coughlin, S.R., Escobedo, J.A., and Williams, L.T. (1989) Science=, 1191-l 194. Sugimoto, Y., Whitman, M., Cantley, L.C., and Erikson, R. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 2117-2121. Fukui, Y., and Hanafusa, H. (1989) Mol. Cell. Biol. 9, 1651-1658. Traynor-Kaplan, A.E., Harris, A.L., Thompson, B.L., Taylor, P., and Sklar, L.A. (1988) Nature 334, 353-356. Nolan, R.D., and Lapetina, E.G. (1989) 1. Biol. Chem. =,2441-2445. Kucera, G.L., and Rittenhouse, SE. (1990) J. 8iol Chem. 265, 5345-5348. Yamamoto, K., and Lapetina, E.G. (1990) Biochem. Biophys. Res. Commun. m,466-472. Sultan, C., Breton, M., Mauco, G., Grondin, P., Plantavid, M., and Chap, H. (1990) Biochem. J. X&831-834. Gutkind, J.S., Lacal, P.M., and Robbins, K.C. (1990) Mol. Cell. Biol. a, 3806-3809. Menniti, F.S., Oliver, K., Nogimori, K.G., Obie, J.F., Shears, S.B., and Putney, J.W., Jr (1990) J. Biol. Chem. 265. 11167-l 1176. Serunian, L.A., Haber, M.T., Fukui, T., Kim, J.W., Rhee,S.G.. Lowenstein, J.M., and Cantley, L.C. (1989) J. Biol. Chem. 264, 17809-17815. Yamamoto, K., Graziani, A., Carpenter, C., Cantley, L.C., and Lapetina, E.G. (1990) J. Biol. Chem. 265, in press. Lips, D.L., Majerus, P.W., Gorga, F.R., Young,A.T., and Benjamin,T.L. (1989) J. Biol. Chem. 264, 8759-8763. Eberle, M., Traynor-Kaplan, A.E., Sklar, L.A., and Norgauer, J. (1990) J. Biol. Chem. 265, 16725- 16728. Goldschmidt-Clermont, P.J., Machesky, L.M., Baldassare, J.J., and Pollard, T.D. (1990) Science 247, 1575- 1578. Stossol, T.P. (1989) J. Biol. Chem. 264, 18261-18264. Billah, M.M., and Lapetina, E.G. (1982) J. Biol. Chem. 257, 12705-12708. Lapetina, E.G., Schmitges, C.J., Chandrabose, K., and Cuatrecasas, P. (1977) Biochem. Biophys. Res. Commun. 3 828-835.

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