Lysophosphatidic acid-induced aggregation of human and feline platelets: Structure-activity relationship

Vol.99,No.2,1981 March

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages 391-398

31,1981

LYSOPHOSPHATIDIC

ACID-INDUCED

PLATELETS:

AGGREGATION OF HUMAN AND FELINE

STRUCTURE-ACTIVITY

AKIRA TOKUMURA, KENJI FUKUZAWA, *JUNICHI

RELATIONSHIP ISOBE and HIROAKI TSUKATANI

Faculty of PhurmaceuticaZ Sciences and ‘Department of Laboratory Medicine, Tokushima University, Shomachi, Tokushima, 770, Japan

Received

January

20, 1981

SUMMARY: Lysophosphatidic acid induced aggregation of human and feline platelets, the activity depending on the structure of the sn-1-acyl moiety. A hydroxyl group at the m-Z-position was not necessary for the activity, but a wedge shaped structure was suitable for induction of aggregation. Almost all the lysophospholipids tested with a head-group attached to the phosphate portion had little or no activity, but the sn-2-acetylated analogs of these phospholipids were very active. These phospholipids with activity were suggested to induce aggregation by interaction with the same binding site on the cell surface.

Lysophosphatidic pharmacological from

effects

polyunsaturated

muscle (4,5)

from

rabbit

and feline(4)

hypotension guinea

pigs(6).

the potencies activity activity

fatty

acids:

platelets

It

for

causes

and rat and its

intravenous

that

at the sn-l-position

length

aggregation

and aggregation injection

In this acid

in rats(6,7)

communication,

of human and feline

derived smooth

acute and

of unsaturation

residue

is evaluated

and

of human

causes

and degree

of the glycerol

of lysophosphatidic inducing

of isolated

and hypertension

the chain

biological

and thromboxanes

contraction

uterus(3)

and rabbits(6)

of the vaso-effects(6).

relationship

known to have some similar

of prostaglandins

duodenum(l,2)

We found chain

is

to those

in cats(4,6)

of the acyl

its

acid(LPA)'

of LPA influenced the on the

structurebasis

of

platelets.

Abbreviations used: LPA, lysophosphatidic acid; PRP, platelet-rich plasma; GP, sn-glycero-3-phosphate; GPM, sn-glycero-3-phosphorylmethanol; GPE, snglycero-3-phosphorylethanol; GPP, sn-glycero-3-phosphorylpropanol; GPC, snglycero-3-phosphorylcholine; 12:0, 1-lauroyl; 14:0, 1-myristoyl; 16:0, lpalmitoyl; 18:0, 1-stearoyl; 18:1, 1-oleoyl; 18:2, 1-linoleoyl; 18:3, llinolenoyl; 2:0, 2-acetoyl; 3:0, 2-propionoyl; 4:0, 2-butyroyl; 6:0, 2-caproyl

391

0006-291X/81/060391-08$01.00/0 Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 99, No. 2,1981

BIOCHEMICAL

AND

CH2-O-R1

structure

I CH-O-R2

Basic

I ? CHz-O-P-O-X 0-

X:

BIOPHYSICAL

COMMUNICATIONS

1 structure

H, CH3.

of Ct$Ct$,

the

phospholipids

CHZCH2CH3,

R,:

long

chain

fatty

acyl

R2:

H or

short

chain

fatty

MATERIALS

RESEARCH

used. or CH$H$(CH&

acyl

AND METHODS

Preparation of phosphotipids 1-Palmitoyl(16:0)and 1-oleoyl(l8:1)-mglycero-3-phosphate(GP) were obtained from Serdary Research Laboratories Inc. 1-Lauroyl(l2:0)-, l-myristoyl(14:0)-, 1-palmitoyl (London, Ontario, Canada). (16:0)and l-stearoyl(l8:0)-sn-glycero-3-phosphorylcholine(GPC) were purchased from Sigma Chemical Co.(St.Louis, MO. U.S.A.). 1-linoleoyl(l8:2)and llinolenoyl(l8:3)-GP were kindly provided by Dr. T. Nakajima of Nihon Shoji Kaisha Ltd. 1-Lauroyl(l2:0)and myristoyl(14:0)-GP were prepared by the method of Long et a1.(8) from the corresponding 1-acyl-GPC by hydrolysis with phospholipase D (phosphatidylcholine phosphatidohydrolase, EC 3.1.4.4: from cabbage) and purified as described previously(6). 1-Palmitoyl-sn-glycero-3phosphorylmethanol(l6:0-GPM), 1-palmitoyl-sn-glycero-3-phosphorylethanol(l6:0GPE) and l-palmitoyl-sn-glycero-3-phosphorylpropanol(l6:O-GPP) were prepared from 16:0-GPC by transphosphatidylation with cabbage phospholipase D in the presence of 4 % methanol, ethanol and propanol, respectively, by a similar The phospholipids procedure to that used for preparation of LPA, but at pH 7.5. were purified by column chromatography on Sephadex LH-20(100 g, 3.5 x 75 cm) with The hydroxyl group chloroform-methanol mixture(l:l, v/v) as eluting solvent. at the sn-2-position of various 1-acyl-phospholipids was acetylated as follows: One ml of acetic anhydride and 1 ml of dry pyridine were added to a solution of The mixture was incubated for 2-5 25 mg of the phospholipid in chloroform. hours at 30°C, and then the reaction was stopped by adding methanol and the 2-acetylated phospholipid was purified by thin layer chromatography(Merck silica gel plate 60) with a developing solvent of chloroform-methanol-water(65:35:5, The sn-2-propionoyl-, butyroyland caproyl-analogs of l-palmitoylv/v/v). but with propionic anhydride, butyric GPC were prepared by the same procedure, anhydride and caproic anhydride, respectively, instead of acetic anhydride. l-Palmitoyl-2-acetoyl-sn-glycero-3-phosphate(l6:O-2:O-GP) and the 2-butyroyl and caproyl analogs were formed by hydrolysis of the corresponding l-palmitoyl2-short chain fatty acyl-GPC with phospholipase D by the procedure used for preparing 1-acyl-GP.

Aggregation of platelets Human venous blood was collected from healthy volunteers into 0.1 volume of 3.8 % trisodium citrate or heparin(2 I.U./ml of blood). None of the donors had taken any drugs within the previous week. Feline blood was collected from animals under ether anaesthesia from the carotid artery into a tube containing trisodium citrate or heparin at the8concentrations described above. Platelet-rich plasma(PRP, approximately 3 x 10 platelets/ml) was prepared by centrifuging whole blood at 1509 for 10 minutes at room temperature. Platelet aggregation was monitored by continuous recording of light transmission in a platelet aggregation profiler(PAP-3, BIO/DATA). A volume of 50 pl of the test phospholipid in saline was added into 0.5 ml of PRP. The relative effects of different compounds on platelet aggregation were compared by mixing volumes of 0.5 ml of the compounds in saline with 0.5 ml of PRP, beacuse some phospholipids with little activity were poorly soluble in saline at high concentrations. Arachidonic Na2C03 and then

acid(Sigma) diluted with

and indomethacin(Sigma) saline for tests.

392

were dissolved ADP was purchased

in 0.1 M from Sigma.

BIOCHEMICAL

Vol. 99, No. 2,198l

(A)

16:0-GP

AND BIOPHYSICAL

human PRP

RESEARCH

COMMUNICATIONS

I mln I

I

J

Y5 AT110 x

15

b

30

(B)

16:0-GP

feline

PRP

&

Figure

1 Concentration-dependent aggregation of human(A) and feline(B) titrated platelets produced by 16:0-GP. Numbers represent final concentrations of 16:0-GP in pg/ml. AT=change in light transmission.

RESULTS AND DISCUSSION A low

concentration

of platelets

of 16:0-GP

in titrated

16:0-GP

induced

(Figure

1-A)

and heparinized

irreversible

being

of different

phospholipids PRP within

Feline depending about

three

a lag

time

most

gradually rapid for

60 minutes

platelets

were

times

to this

of 3-5 minutes

inducing

Higher

of platelets

in both

PRP.

platelet

after

their

of

PRP preparation. to 16:0-GP

The threshold were

with

time,

concentrations compared

using

of the blood.

aggregated

phospholipid

aggregatior

concentrations

aggregation

collection

to 16:0-GP

phospholipids

of reversible

human PRP.

in heparinized

after

extents

as shown in Figure

more sensitive

of different

similar

became desensitized

irreversibly

on the concentration,

to become desensitized concentrations

aggregation

Human platelets

desensitization

titrated

caused

by 16:0-GP 1-B.

than

titrated

with

time,

causing addition

393

with

Heparinized PRP, but as mentioned

irreversible

to titrated

a lag

time

PRP were they

tended

above.

aggregation feline

PRP were

The with

Vol. 99, No. 2,198l

BIOCHEMICAL

1.00

AND

BIOPHYSICAL

I1;1

Li 12:o GP

Figure

moiety

both

c,G-fatty

To evaluate

the

is

not

of LPA. feline

led

necessary, Next,

18:0 GP

18:l GP

18:3 GP

18:2 GP

half

that

are

of 16:0-GP,

but a wedge

shaped

the effect of 16:0-GPM

and one twenty

is

of the

acyl

Thus

favorable

the

for

one thirtieth

of that

at the

hydroxyl

platelet

was about

394

of 16:0-

chain

of the

latter

in evoking

The activity

modification

16:0-GPP

a long

of various

potencies

3-A.

in activity.

of the

of the Thus

the effects

of chemical

fifith

such as 16:0-GPE,

in Figure

structure

was 16 for

in activity.

relative

and elongation

decrease

of the s)z-

of unsaturation

we tested

Their

shown

length

to

aggregation.

lyso-form

acyl-GPs.

to progressive

The activity

lysophospholipids,

fatty

increase

platelet

length

optimal

in the degree

slight

for

l-acyl-GPs(LPAs)

The chain

and the

increase

of the

of various

platelets.

in only

platelets

we examined

platelets

potencies

potency,

required

influence

of feline

sn-2-position

the hand,

is

chain

was about

relative

resulted

region

1-palmitoyl-2-short

2:0-GP

other

residue

hydrophobic

the

influenced

On the

acyl

aggregation

16:O GP

of human and feline

greatly

species.

chain

2 shows

aggregation

1-acyl

14:o GP

2 Relative potencies of various 1-acyl-sn-glycero-3-phosphates in inducing aggregation of human platelets(black bars) and feline platelets(white bars). The mean threshold concentration of 16:0-GP is 9 rig/ml for human platelets and 0.7 pg/ml for feline platelets. n=5

compared. produce

COMMUNICATIONS

-

0.01

Figure

RESEARCH

group aggregation

phosphate of 16:0-GP

on human platelets.

and 16:0-GPC,

were

portion

all

on

Other inactive.

Vol. 99, No. 2,1981

BIOCHEMICAL

1.00

AND

BIOPHYSICAL

(A)

1.00

RESEARCH

COMMUNICATIONS

.

(B)

s 5 5 g s 0 %

0.10

0.10

-

0.01 16:0

16:0 2:o GP

GP

Figure

However, fatty strong

analog

l-position

also

aggregation

depended

influenced

16:O 3:o GPC

16:O 4:o GPC

16:O 6:0 GPC

Possibly,

the

in determining balance

between is

Recently, basophils

the potency.

a decisive the

factor

platelet

found

resulted

of the

acyl

at the sn-

in producing

in Figure

4.

Figure

group

The potency

attached

to the

of ethyl

group

property

phosphate with

5

of the head-group the structure

of the chemical

group

in controlling

the

factor(PAF)

to be 1-O-alkyl-2-acetoyl-GPC(9,lO).

395

also portion,

feline

and human

was not

of sn-1-acyl

potency released

critical

moiety,

at the sn-Z-position

and

of activity. from

IgE-sensitized

Its activity

in

The sn-

1-palmitoyl-2-acetoyl-phospholipids

Like

activating

chain

in Figure 3-B.

length

as shown

was that

chemical

the sizes

phospholipids

and human platelets.

chemical

a short

of 1-acyl-2-acetoyl-GPCs

platelets,

activity

with

as shown

The chain

of different

of the for

platelets,

active.

of feline

size

at the sn-2-position

the potencies

potencies

on the size

head-group

GPC

16:O 2:o GPC

of choline-containing

on feline

was the most

aggregation

platelets.

rabbit

activity

relative

and the optimum

the

16:0

group

series

of human and feline

inducing

factor

in the

aggregatory

P-acetoyl

in

of the hydroxyl

anhydride

shows the

16:O 6:O GP

3 Relative potencies of various 1-palmitoyl-Z-acyl-sn-glycero-3-phosphates (A) and 1-palmitoyl-2-acyl-sn-glycero-3-phosphorylcholines(B) in evoking aggregation of feline platelets. The mean threshold concentration of 16:0-GP is 0.7 pg/ml, and that of 16:0-2:0-GPC is 4 pg/ml. n=5

acylation

acid

16:O 4:o GP

in

Vol.99,No.2,1981

BIOCHEMICAL

AND

1.00

E 5 : f

0.01 II

14:o 2:o

12:o

2:o GPC

producing

4

GPC

16:O 2:o

18:O 2:o

GPC

GPC

of washed

rabbit

platelets

was about

the corresponding

1-acyl

analog,

indicating

that

chain

to the

glycerol

residue

influences

shown

in this

hydrocarbon factors

Figure

COMMUNICATIONS

Relative potencies of various 1-acyl-2-acetoyl-sn-glycero-3-phosphorylcholines in producing platelet aggregation in human PRP(black bars) and feline PRP(white bars). Threshold concentration: 16:0-2:0-GPC, 22 ug/ml(human PRP); 18:0-2:0-GPC, 3.5 ug/ml(feline PRP) n=4

aggregation

structural

RESEARCH

I

0,lO

E 1

Figure

BIOPHYSICAL

5

thirty

times

the mode of binding the

potency

that

of

of a long

as well

as the

study.

16:0

16:0

2:o

2:o

16:O 2:o

16:O 2:o

16:0 2:o

GP

GPN

GPE

GPP

CPC

Relative potencies of producing aggregation platelets(white bars). ug/ml(human platelets)

various 1-palmitoyl-2-acetoyl-phospholipids of human platelets(black bars) and feline Threshold concentration: 16:0-2:0-GPE, and 0.4 pg/ml(feline platelets) n=5

396

in 2.5

BIOCHEMICAL

Vol. 99, No. 2,198l

saline

BIOPHYSICAL

RESEARCH

16:0-GP(4)

16:0-GP(l0)

16:0-GP(lO)

16:0-2:0-GPE(2)

saline

AND

16:0-GP(l0)

16:0-2:0-GPC('20)

16:0-GP(l0)

I&O-GP(4)

ADP

COMMUNICATIONS

ADP

c

I

Figure

Feline than

gation

did

methacin induced

I

6 Tachyphylaxis of human platelets treated with different phospholipids on subsequent addition of 16:0-GP. Numbers represent final concentrations in ug/ml of added phospholipids. The final concentration of ADP is 5 mM. Arrows indicate the times of additions of the phospholipids and ADP in

study

I mln

platelets

50 ~1 saline.

were

more sensitive

human platelets. not

involve

blocked

the

by arachidonic

AT=change

The effects

endogenous

transmission.

tested

of the phospholipids

on platelet

synthesis

aggregation but not

light

to the phospholipids

prostaglandin

irreversible acid,

in

because

of human and feline

by these

in this aggre-

5 mM indoplatelets

phospholipids

at any concentration

concentration

of 16:0-GP

tested. Human platelets centration lipid

inducing

effects

exposed

to 1-acyl-GP

observed

not

phospholipids

only

to the

reversible

was subsequently

phylactic

the

exposed

added,

aggregation

between

became desensitized

as reported

was observed were

threshold

with

all

the most various

by Schumacher the active

tachyphylactic. molecular

such as 16:0-2:0-GP,

species

16:0-2:0-GPM,

397

when this

et a1.(4).

phospholipids,

16:0-2:0-GPE,

phospho-

This

tachy-

and the platelets

Cross-tachyphylaxis of LPA but

or con-

also

was 16:0-GP

and

16:0-2:0-GPP,

Vol. 99, No. 2,198l

and 16:0-2:0-GPC, potentiated

platelets. phospholipids

respectively.

the

representative

BIOCHEMICAL

aggregation tracings

Feline also

of these

phospholipids

aggregation

by interaction

exposure

of human platelets of results

similar

with

to active

produced

to threshold

that the

Figure

properties

with

species

same binding

phospholipidr

by ADP.

effects.

together in both

COMMUNICATIONS

concentrations

tachyphylactic

phospholipids, suggests

RESEARCH

on the tachyphylactic

exposed

exhibited

among the active

BIOPHYSICAL

However,

platelets

phylaxis

AND

sites

the these

6 shows

of human of active

This structural compounds

on the platelet

cross-tachysimilarities cause surface.

ACKNOWLEDGEMENT We thank Miss Masako Kawakami and Miss Miyuki Nakata for technical This work was supported in part by a grant from the Japanese Ministry Science and Culture.

assistance. of Education,

REFERENCES 1. Vogt, W. (1960) Naunyn-Schmiedeberg's Arch. Exp. Pathol. Pharmakol. 240, 134-139 2. Vogt, W. (1963) Biochem. Pharmacol. 12, 415-420 3. Tokumura, A., Fukuzawa, K., Yamada, S. and Tsukatani, H. (1980) Arch. Int. Pharmacodyn. Ther. 245, 74-83 1. Schumacher, K.A., Classen, H.G. and SpZth, M. (1979) Thrombos. Hemostas. 42, 631-640 5. Gerrard, J.M., Kindom, S.E., Peterson, D.A., Peller, J., Krantz, K.E. and White, J.G. (1979) Am. J. Pathol. 96, 423-438 5. Tokumura, A., Fukuzawa, K. and Tsukatani, H. (1978) Lipids 7, 572-574 7. Tokumura, A., Fukuzawa, K., Akamatsu, Y., Yamada, S., Suzuki, T. and Tsukatani, H. (1978) Lipids 7, 468-472 E.J. (1967) Biochem. J. 102, 221-229 3. Long, C., OdaviC, R. and Sargent, R.N. and Hanahan, D.J. (1979) J. Biol. Chem. 254, ). Demopoulos, C.A., Pinckard, 9355-9358 1. Hanahan, D.J., Demopoulos, C.A., Liehr, J. and Pinckard, R.N. (1980) J. Biol. Chen 255, 5514-5516

398