Interaction of lentil lectin with human platelets. Evidence against glycoprotein II as aggregation mediator

Vol.

92, No.

January

BIOCHEMICAL

2, 1980

29,

AND

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Pages 499-504

1980

INTERACTION OF LENTIL LECTIN WITH HUMAN PLATELETS. EVIDENCE AGAINST GLYCOPROTEIN II AS AGGREGATION MEDIATOR Pankaj

Ganguly

and Nancy G. Fossett

From the Department of Hematology, St. Jude Children's Research Hospital and the Department of Biochemistry, University of Tennessee, Memphis, Tennessee 38101

Received

October

11,

1979 SUMMARY

Human platelets bind on an average of 5 x 10' molecules of lentil lectin/cell with an apparent dissociation constant of 3 x lob7 M. The lectin binds mainly to surface glycoprotein II with an apparent molecular weight of Lentil lectin neither caused aggregation nor did it inhibit platelet 125,000. It had no influence on the binding of thrombin aggregation by other agents. or on thrombin-induced clot retraction. The hypothesis that to platelets glycoprotein II mediates platelet aggregation needs reevaluation.

Platelets

are

known

bosis.

When

logical

and biochemical

Recent

are

their

known

is,

as useful

of in

probes

glycoproteins. small

we have show

to bind

that

aggregation

these

to

allowing

lentil nor

leading

properties,

the lectin does

it

the

analogous

plug.

might

of

specific

interaction

of

interaction

of

lentil

of

that

of

with

platelets

by other

(2).

sugars

with

reaction

their

glyco-

and serves cell

surface

combining

sites

In this

study,

human platelets. but

it

1).

and lead

specific

lectins

reaction

lectin to

aggregation

is

in

by

and glyco-

with

relationship

type

tightly

of cells

polysaccharides

be inhibited

lectins

be mediated (reviewed

of a variety

structure-function

a more

affect

of a hemostatic

to antigen-antibody

advantage

binds

morpho-

process

precipitate may

of

glycoproteins

receptors

agglutination,

explore

for

the

and throm-

series

formation

of this

particularly

also

in hemostasis

a complicated

to the

aspects

some

many ways,

role

undergo

to surface

cell

An added

explored

an important

Lectins

like

Because

proteins

are

that

components,

which,

(2).

changes

agglutination.

proteins

play

platelets

suggest

surface

Lectins to

stimulated,

studies

platelet

to

neither

We causes

agents, 0006-291X/80/020499-06$01.00/0

499

Copyright t-1 1980 by Academic Press, Inc. All rights of reproducrron in any form reserved.

Vol.

92, No.

2, 1980

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MATERIAL AND METHODS Blood was collected from healthy volunteers in plastic bags or syringes utilizing 0.1 vol of 3.8% sodium citrate as anticoagulant. The red cells were removed by differential centrifugation and the platelet-rich plasma (PRP) was collected. If necessary, platelets were washed as described and resuspended in phosphate-buffered saline (PBS) (3). Lentil lectin was purchased from Sigma Chemical Co., St. Louis, Missouri. It contained both lentil A and lentil B which are known to be identical in terms of erythrocyte binding and mitogenicity for lymphocytes (4). The lectin was labeled with 1251 with chloramine T. The specific radioactivity of the lectin was in the range of 3.0 to 8.0 x 10' cpm/pg. The binding of the lectin to platelets was determined at least in duplicates by Millipore filtration (5). Different amounts of the labeled lectin was incubated with a constant number of washed platelets for 15 min at room temperature in a total volume of 1 ml. The samples were then filtered under reduced pressure and the filters washed with 4 ml of PBS. Nonspecific binding was determined by filtering equal amounts of lectin in the absence of platelets. The filters were finally counted in a Packard autogamma spectrometer. Platelet aggregation was measured in a dual channel aggregometer (Payton, Buffalo, New York) (6). The experimental sample was analyzed in one channel while a control was run in the other. The amounts of stimulants shown in Table I are final concentrations in a total volume of 0.5 ml. The procedures for gel electrophoresis and lectin staining of the separated platelet components have been described (5). After electrophoresis, the gel lane was fixed and washed repeatedly until the pH of the washing buffer (PBS) was 7.0. The gel was then incubated overnight with the labeled lectin containing 0.5% albumin. Excess, unbound lectin was removed by repeated washing of the gel with PBS until the radioactivity in the washing fluid reached a constant, low value. The gels were dried under vacuum and left in contact with Kodak x-ray films in the cold. The films were developed after l-3 days. Other details are provided in figure legends. RESULTS AND DISCUSSION The binding Fig.

1.

of

The

observed

in

1251-labeled

binding studies

concentration

of lectin

the

Analysis

there

are

about

dissociation values

10m7

of

platelets lentil

lectin

5 x lo5

8 x lo5

(5).

of sites/cell

for Thus,

with

added

of

of

double

break

reciprocal

4 x 10e7 M.

plots

both

fresh

of

500

the lectin

These

data

compare

lentil

dissociation lectin platelets

are

in

those

platelets

or

increasing

30 to 40 pg/ml

data

indicated

of that

with

an apparent

favorably

with

constant to

shown to

with

at about

lentil

and fixed

parameters

to

increased

of

is

similar

for

and an apparent binding

kinetics

bound

a distinct

sites/platelet

platelets

phytohemagglutinins

lectin

with

to fresh

saturation

binding

the

similar

lectin

showed

amount

of

constant

M reported

the

The

(4).

lectin.

curve on

erythrocytes

lentil

the

of 3.2

formaldehyde-fixed capable

of binding

x

Vol.

92, No.

2. 1980

BIOCHEMICAL

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-0.04

20

I

30

40

RESEARCH

0.04 0.08

00

IILECTIN

v I I I IO

BIOPHYSICAL

COMMUNICATIONS

0.12 0.16

ADDED pg-’

I

I

I

I

I

I

50

60

70

80

90

100

LECTIN

ADDED

pg/ml

Fig. 1. Binding of lentil lectin to washed platelets in phosphate-buffered saline. Platelets (2.5 x lo*) were incubated with different amounts of ['251]lentil lectin for 15 min at room temperature in a total volume of 1 ml. The amount of lectin bound to platelets was determined by millipore filtration. The insert shows a double reciprocal plot of the binding data for the and the apparent dissociation determination of the number of binding sites constant. Platelet analyzed of

membranes

by SDS gel

bands

ranging

I 1251]lentil weight 2a).

in

Platelets at

aggregated

peaks

to other in

nor

these

concentrations

the

were

the the

with

The

lectin

at

it

affect

platelet

were

also

mainly

with

molecular

observed

(Fig.

a prominent

previous

peak

reports

based

to platelet

GP-II

8). different

amounts

aggregation

In contrast,

of

concentrations

wheat

were

repeated

same as with

PRP.

Furthermore,

with

lentil neither

by thrombin germ agglutinin

of platelets.

experiments

501

Staining

showed

saturating

50% to 80% aggregation

a number

an apparent

confirm binds

(7,

incubated

with

and

revealed

25,000.

again

lectin

I).

to

bands

data

components

or ADP (Table

glycoproteins, results

did

caused

200,000

faint

centrifugation

protein

component

These

lentil

were

platelets

germ agglutinin

contains

plasma

other

2b).

surface

10 min.

from

for

of autoradiographs

that

37O for

wheat

of

(Fig.

chromatography

and more weakly

lectin

scanning

gradient

Staining

a prominent

A number

smaller

by density

weight

showed

125,000.

on affinity

and

molecular

Densitometric

and other

isolated

electrophoresis.

lectin

of

were

Since washed lentil

or at

plasma

platelets lectin

did

Vol.

92, No.

BIOCHEMICAL

2, 1980

AND

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Specificity of binding of lentil lectin to platelets. Isolated Fig. 2. platelet membranes were solubilized and the components separated by SDS gel electrophoresis. Gel lanes were stained with [1251]lentil lectin and auto(a) Autoradiographs of two different radiographs were developed after 24 hrs. gels are shown. The most prominent band (arrow) has an apparent molecular weight of 125,000. (b) Densitometer scan of an autoradiograph which showed one prominent peak and other smaller peaks.

TABLE

Lectin

I.

Effect

of

Lentil

Aggregation

Adenosine Adenosine Adenosine

0 30 50

Wheat Wheat

0

50 "Average

of

block binding binds

of to

four

determinations

clot

retraction

labeled platelets

germ germ

80 mU 80 mU 80 mU

thrombin but

lo-' M lOI M 10 ' M

50 55 55

agglutinin agglutinin

40 pg 40 I-18

50 50

PRP to

has

70 70 70

diphosphate diphosphate diphosphate

of

no

%-k

0 0

Thrombin Thrombin Thrombin

0

30 50

lectin

Aggregation

0 0

50 100

the

on Platelet

Stimulant

Pg

neither

Lectin

by

platelets. influence

retraction.

502

thrombin

(Fig. These on

platelet

3) results

nor

did show

aggregation

it that

affect lentil or

clot

Vol.

92, No.

2, 1980

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Fig. 3. Lack of effect of lentil lectin on clot retraction of platelet-rich plasma by thrombin (0.4 U/ml). The first tube on the left is control without lectin, the next two tubes contained 30 ug/ml and 50 pg/ml of the lectin respectively.

Recent

studies

conditions

have

A

of

number

deficient is

in

are

normal

for

the

GP-II

gation

thesis

of

question

is

GP-II

patient

did

not

precipitation platelet

platelets

the

observed

tested

observation

by

change,

secretion)

glycoprotein

platelet

surface difficult

in

that

normal

a variety

antibodies

interpret

receptors (10).

induce

503

detected

weight

defects

responsible

may not

to the This with

hypoconcept

subagglu-

in a thrombasthenic (1).

may

120,000 and

aggre-

glycoprotein

treated

antibody

The

platelet

aggregation.

agents

stimuli

Monovalent

has led

platelets

of

the

Fc

this

platelet

are

affect

finding

(IgG)

molecular

not

as the

to

platelets.

that This

role

contains to

did

as well

retraction.

deficiencies

GP-IIIa)

(9).

of

show clot

are

Thrombasthenia

shape

thrombasthenic

that

8). count

suggesting

suggested

(1,

(1).

platelets

platelet

of an alloantibody

aggregate

or fibrinogen

(or

a vital

surface

are

in

glycoproteins

the

and do not

aggregation

studies

IgG

and GP-IIIa)

pathological

thrombasthenic

glycoprotein

to GP-III

plays

by the

GP-IIb which

different

surface

that

in

these

with

in their

(binding,

whether

agents

patients

shown

to aggregate

platelet

concentrations

intact

fail

by any of the in

(or

disorder

defects

tinating

that

has

of an antibody

was supported

defects

platelets

functional

that

specific

bleeding

they

involved

from

and GP-III

but

fragments

platelets

investigators

response

crucial

be

revealed

a congenital

initial

with

Double be

Heterologous in platelet

directed

(1).

results

antibody

It

to is

obtained antibodies aggregation

a

known with to (11,

Vol.

92, No.

2, 1980

It

12).

is

essential

Fab fragments release

of

reaction

lentil

lectin

lectin

at

did

it

alloantibody

when

tightly

physiological

GP-II

agents

by other plays

should

agents

a crucial

treated

aggregate

but

to

caused

or clot role

COMMUNICATIONS

with

will

in

study

GP-II.

platelet

retraction platelet

monovalent

undergo

of the present

primarily

neither

RESEARCH

platelets

results

platelets,

concentrations

aggregation

not

The

to

EIOPHYSICAL

normal will

stimulated.

saturating

that

AND

to show that

the

binds

affect

hypothesis

BIOCHEMICAL

normal

show that

However,

the

aggregation

nor

by thrombin.

The

aggregation

by all

be reevaluated. ACKNOWLEDGMENTS

This

study

was supported

by grants

HL 16720

and CA 21765

from NIH and by

ALSAC. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.

10. 11. 12.

Nurden, A. T., and Caen, J. P. (1979) Semin. Hematol. 16, 234-250. 177, 949-959. Sharon, N., and Lis, H. (1972) Science Ganguly, P. (1972) J. Biol. Chem. 247, 1809-1816. Majerus, P. W., and Brodie, G. N. (1972) J. Biol. Chem. 247, 4253-4257. Ganguly, P., Gould, N. L., and Sidhu, P. (1979) Biochim. Biophys. Acta 586, 574-583. Ganguly, P. (1977) Biochim. Biophys. Acta 498, 21-27. Clemetson, K. J., Pfueller, S. L., Luscher, E. F., and Jenkins, C.. S. P. (1977) Biochim. Biophys. Acta 464, 493-508. Phillips, D. R., and Agin, P. P. (1977) J. Clin. Invest. 60, 535-545. 28, 551-560. Kaplan, K. L., and Nachman, R. L. (1974) Br. J. Haematol. Moore, A., Ross, G. D., and Nachman, R. L. (1978) J. Clin. Invest. 62, 1053-1060. Clancey, R., and Firkin, B. (1973) Thromb. Res. 3, 375-381. Tollefsen, D. M., and Majerus, P. W. (1975) J. Clin. Invest. 55, 12591268.

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