Studies of an activity from endothelial cells that inhibits platelet aggregation, serotonin release, and clot retraction

THROYBOSIS RESEARCH Printed in the United

Vol. States

j, pp. 747-7j7, Pergamon Press,

1974 Inc.

STUDIES OF AN ACTIVITY FROH ENDGTHELIAL CELLS THAT INHIBITS PlATELET AGGREGATION, SERCYIONINRELEASE, AND CLOT RETIUCTION

S. R. Saba and R. G. Mason Department of Pathology, University of North Carolina Chapel Hill, North Carolina 27514

(Received

4.11.1974.

Accepted

by Editor

M.I.

Barnhart)

ABSTRACT present in endothelial cells of human umbilical vein An activity origin has been found to inhibit aggregation, serotonin release, and clot retraction. This activity appears to reside in molecules of small size that can be released from in situ or isolated endothelial cells by freeze-thaw treatment or by exposure of cells to ADP, collagen, epinephrine, or thrombin. Relative amounts of inhibitory activity released from endothelial cells vary with the specific plate let aggregating agent used as release inducer and are influenced by use of in situ or isolated cells. Varying the concentration of aggre gating agent varied proportionally the amount of inhibitory activity released from in situ but not from isolated endothelial cells.

INTRODUCTION An activity of endothelial cell origin was described recently (1) that inhibited aggregation of platelets.

This activity could be released from iso-

lated endothelial cells by disruption of the cells or by treatment of cells with certain platelet aggregating agents.

A broad spectrum of action of the

inhibitory activity of endothelial cell origin was shown in that it prevented aggregation of platelets induced by adenosine diphosphate (ADP), collagen, epinephrine, or thrombin.

This inhibitory activity was shown to reside in a

small, heat labile molecule (9OoC, 10 min.). Further characterization of the inhibitory activity of endothelial cell origin has been undertaken.

Semiquantitative studies of the release of the

inhibitory activity both from endothelial cells in situ and from isolated 747

738

INHIBITOR

endothelial

cells

conducted.

The inhibitor

gation

but

to

in

FROM ENDOTHELIAL

response

prevent

to

has

various

been

release

of

found

Vol.j,No.6

CELLS

platelet

aggregating

not

to

serotonin

only

fran

agents

prevent

platelets

have

platelet

and to

been

aggre-

inhibit

clot

retraction.

MATERIALS AND METHODS Endothelial

Cells

and

Human umbilical tals

modified fic

cords

and brought

The umbilical

vein

platelet

lumen of bation

to

(!$g*+-

the in

37’C

were

in

did

obtained

a segment

Ca*+- free)

vein

preparations. from

laboratory.withfn

aggregating

the

at

Inhibitor

this

cord

and

increase

(MTS).

yield.

at

delivery.

and rinsed of

with

the

was placed

22OC for

speci-

in

the

60 minutes.

The incubated

solution

of

endothelial

cells,

was then

supernatant

was used as the inhibitor preparation in studies of platelet aggre-

ment

A control of

the

consisted

same umbilical

of cord

15OOg for

an identical vein

from

activity

Incu-

which

at

inhibitory

of

agent,

centrifuged

the

time

hospi-

aggregating

gation.

now contained

the

A solution

investigation

incubated

inhibitor

of

collaborating

was cannulated

solution

in MTS under

segment

nearby

two hours

each

Tyrode’s agent

not

of

six

15 minutes

preparation which

the

from

aggregating

released at

from

220C;

a different agent

This

control

cal

were

used after intervals greater than 2 hours post delivery or

cords

negligable

inhibitor

activity

unless

seg-

was

omitted.

contained

the

umbili-

unless traumatized cords were used in which cases detectable amounts of inhibitor were present in these controls. Endothelial cells were isolated from human umbilical cord vein by treatment of the in situ cells with 0.22 collagenase (collagenase type IV, Worthington Biochemical Corporation, Freehold, New Jersey) in MTS as previously reported (1).

Isolated endothelial cells were washed two times in FITSand fin-

ally resuspended

in &fTS at

a final

concentration

of

approximately 1000 cells

Vol.j,No.6

per

IXHIBITOR

axa3.

sion

Isolated

test.

This

aggregating ture not

cells cell

agent

in )ITS and

of

agent

platelet

endothelial the

negligable

in

A control

platelet

inhibitor

vein

platelet

before

the

at

centrifuged

mix-

37Oc did

endothelial

preparation

consisted

treated

was omitted.

cells is

from

isolated

serotonin

preparation

of

of

in

isolated

identically

This

control

endothelial

cells

with contained

release

was the

supernatant

in HTS centrifuged referred

to

and

as

at

clot

contraction.

frau

three

15OOg for

freeze-thaw

(,5OO/nm13)

times

frozen

15 minutes

inhibitor

at

preparation.

KTS.

or

other

Aggregation

inhibitor

preparation,

preparation,

and 3)

consisted

(1)

of

at

1,000

2)

agent

was used

from

rpm.

to

endothelial

at

least out

with

platelets/mm3) from

in

consisted

MTS substituted

which

agent.

aggregation of

for

was added

aggregating

cells

days

the

of

inhibitor

was prepared

donors

prior

a Chrono-log

MTS substituted

induce

normal

seven

Controls

PRP to

ml of

(500,000

obtained

carried

with

ml of

and 0.1

release

blood

mixtures

mixtures 0.2

(PRP)

for

were

ml KrS,

aggregating

from

medication

studies

PRP stirred

0.1

plasma

previously

37OC with

induce

as the

cord

agent

the

of

incubation

preparation

same umbilical

platelet-rich

aspirin

ture.

ation,

A control

22OC; of

also

volume

exclu-

Aggregation.

as described

Tests

was used

supernataat

Citrated

taken

mixture

B dye

15 minutes

for at

739

erythrosin

an equal

22’~

The supernatant

preparation

of

consisted

Platelet

at

15 minutes

aggregating

endothelial

This

by the

with

yield.

inhibitor

inhibitor

and thawed

incubated

CELLS

activity.

studies

second

95% viable

was mixed

15OOg for

the

the

inhibitory

was used

22Oc.

from

that

A second

80 to

aggregation.

cells

exception

This

at

inhibitor

cell-aggregating studies

were

suspension

was centrifuged increase

FROM ENDOTHELIXL

to

venipunc-

aggregometer

of:

1)

the

for

the

ml of

In each platelets activity

at

control

inhibitor

aggregating

0.1

who had not

agents.

inhibitor case,

the

prepar same

as was used tested

with

to

750

INHIBITOR

these platelets.

FRO!4 ENDOTHELIAL

CELLS

Degree of inhibition of platelet aggregation was determined

by measurement of change in % transmission (ZT) of the aggregometer and by determination of change in the total area which lay under aggregation curves; the areas under curves were cut out and weighed on an analytical balance. Amounts of aggregating agents required to produce a 75% change in %T varied with individual plasmas; ranges of concentrations are given for each aggregation-inducing agent in footnotes to Tables I and II. Release. One tenth ml of serotonin solution (9.1 X 10m6 H, specific activity of 55 m Ci/m mole of Cl4 labeled serotonin creatinine sulfate, Amersham Searle, Chicago, Illinois) was incubated with 0.9 ml PRP for 30 minutes at 37OC.

Two

tenths ml of freeze-thaw inhibitor preparation in MTS was added to 0.2 ml of labeled PRP and incubated for LO minutes before 0.1 ml of aggregating agent was added with stirring for 10 minutes.

The final mixture was centrifuged for

20 minutes at 1500g at 22OC, and the supernatant solution was counted in a Nuclear Chicago Scintillation counter with PBD in toluene as fluor. consisted of:

Controls

1) the control inhibitor preparation, 2) mixtures with MTS sub-

stituted for inhibitor preparation, and 3) mixtures with WTS substituted for aggregating agent. Clot Retraction. To 0.2 ml of PRP was added 0.2 ml of freeze-thaw inhibitor preparation in MTS.

The mixture was incubated at 37OC for one minute before 0.1 ml of

thrombin (2 units/ml) was added.

The final mixture was incubated for 90 min-

utes at 37OC before the degree of clot retraction was quantitated by gravimetric determination of the amount of extruded serum.

The control consisted

of a mixture with MTS substituted for inhibitor preparation. Reagents. ADP and epinephrine were obtained from Sigma Chemical Co., St. Louis,

INHIBITOR

Vol.5,No.6

Missouri and

FROM ENDOTHELIAL

prepared fresh in KTS each day.

Achilles tendons by the method of Hovig (2). this

Collagen was

751

prepared from h-n

One to two and

stock collagen preparation were made vith MTS.

was of bovine origin and was

CELLS

1:4

dilutions

of

Thrombin, 300 units/ml,

highly purified; this was a gift from Dr. R. L.

Lundbled, Dental Research Center, Dnivtrsity of North Carolina, Chapel Hill, North Carolina.

RESULTS Results

of

tests for the presence of inhibitory activity recovered by

incubation of endothtlial cells in situ with aggregating agents are given in Table I.

In studies with endothelial cells in situ, it was found that throm-

TABLE I Effects on Platelet Aggregation of an Agent Released from Endothtlial Cells In Situ in Response to ADP, Collagen, Epinephrint, or Thrombin

X decrease in platelet aggregation* in presence of inhibitory agent (P n * S.D.) y area under curve By%T

Aggregating agent used to induce release of inhibitor from endothtlial cells

Number Concentration of experiments

ADP

10-5 H

36.3 + 12.8

32.3 + 11.4

Collagen

1:2 dilution

11.7 +

9.2

18.2 +

6.0

1:4 dilution

7.4 +

5.1

14.7 +

8.6

Epintphrine

Thrombin

10-5 M

14.0 +, 3.3

12.1 +_ 0.2

10'6 H

11.3 +_ 0.6

8.0 +_ 1.7

1 unit/ml

75.6 + 12.6

54.0 2 19.8

0.5 unit/ml

56.7 +_ 18.5

34.2 +_ 12.1

12.4 f 2.3 13.7 f 1.5 J 0.25 unit/ml _ _ . r . ~. * Ranges of final concentrations f agents used to 1Muce aggrtgatlon or platelets were: ADP, loss to 2X 14 8n ; collagen, 1:4 dilution; tpintphrine, 1 to 2 x 10-s H; and thrombin, 0.1 to 0.2 units/ml.

INHIBITOR

752

bin released released tion

the most inhibitory

progressively

release

phr iae,

or thrombin.

reverse

the action

Results

agent of

while

amounts of

incubated

inhibitory

Addition

CELLS

ADP, epinephrine, Decreasing

inhibitor. with

endothelial

activity

of calcium

Vo1.5,No.6

in tests

the concentra-

cells with

to the reaction

and collagen

in situ

collagen,

mixtures

resulted epine-

did not

of the inhibitor

of tests

endothelial

activity

lesser

of the aggregating

in a decreased

lated

FROM ENDOTHELIAL

with

cells

inhibitory

activity

with aggregating

obtained

agents

by incubation

are shown in Table

of

iso-

II.

TABLE II Effects Endothelial

on Platelet Aggregation of an Agent Released from Isolated Cells in Response to ADP, Collagen, Epinephrine, or Thrombin

Aggregating agent used to induce release of inhibitor frcm endothelial cells

Concentration

ADP

5 x 10’5

Number

of !xper iment d

Z decrease in platelet aggregation* in presence of inhibitory agent (I !an + S.D.) By area under curve By7,T 5.6

18.9 +

4.9

10-5 M

30.4 + 15.6

23.5 +

4.5

1:2 dilution

40.7

+ 18.4

39.2 f 20.7

1:8 dilution

48.5

+, 18.6

42.7

Epinephr ine

5 x 10’7

21.0

+

7.3

23.8 +

5.4

Thrombin

0.5

5.8 +_ 3.7

11.0 +

0.9

Collagen

*

M

units/ml

0.25 units/ml 8.1 of agents used Ranges of fina concentration platelets were: ADP, 2 X loto 2 x lo-6 M; epinephrine, 10’5 to 2 X 10-6 M; thrombin, 0.2

Collagen

was found to release

aggregating Lesser

agents

amounts of

thrombin.

In teats

the

were incubated inhibitory with

+

26.0

n

largest with

activity

isolated

3.8 f 4.4 1 5.7 f to induce aggregation of collagen, 1:4 dilution; to 0.44 units/ml.

amounts of

isolated

inhibitory

endothelfal

were released

endothelial

2 13.3

cells,

cells

activity in hTS.

by ADP, epinephrine, decreasing

when

and

the concen-

I?i-HIBITOR FROM

tration

of ADP or collagen

amounts of induced tained

inhibitory

by each of

activity. the agents

from isolated

Effects platelets

is

of

slightly

shovn in Table

of

CELLS

did

not result

Similar

inhibition

tested

endothelial

the presence

ENDOTHELIAL

could

cells

in release

of

of platelet

be demonstrated

smaller aggregation

with

inhibitor

ob-

by freeze-thaw.

inhibitor

III.

753

on the release

ADP, collagen,

of

serotonin

epinephrine,

from

or thrombin

was

TABLE III Effects of the Inhibitory Serotonin from Platelets

Agent used to induce release of serotonin from platelets

Activity of Endothelial Induced by ADP, Collagen,

2 x lo-4

1

of exper intents

X inhibition of release of serote nin by inhibitor obtained from endothelial cells by freeze-thaw

X release of serotonin

Number

Concentratioc

Cell Origin on Release of Epinephrine, or Thrombin

with inhibitor

vithout inhibitor

M

50.6 5

6.6

9.1

+

9.7

82.0

1 x 10’4 M

39.7 +,

9.3

5.7 +

5.6

85.6

Collagen

1:l

66.3 + 10.1

14.6 +_ 9.1

78.0

Ep inephr ine

2 x 10’4 H

64.0 +_

7.9

0.3 +_ 0.3

99.5

1 x 10’4 M

61.8 f.

3.4

0.3 +,

0.3

99.5

1 unit/ml

86.5 +

3.9

38.8 f

13.0

55.1

0.5

77.2 f

9.5

5.1 f

6.7

93.3

ADP

Thraubin

unit/ml

used to induce

release

from endothelial

cells

percent dothellal release

serotonin cell

of

by freeze-thaw

release

origin.

serotonin

treatment.

in the absence

Results

and presence

agents

tested,

also

induced

was obtained expressed

the inhibitor inducer

of

appreciable

as of en-

serotonin release.

the amount of inhibitory activity

was sufficient to prevent 55.0 to 99.5% of the release platelets.

are

of

Thrombin was the most efficient

but ADP, epinephr ine , and collagen

With the release-inducing

Inhibitor

from platelets.

of

eerotonin

from

INHIBITORS

754

The inhibitor

of endothelial

prevention

of clot

endothelial

cells

retraction.

cell

by 58%.

centration,

500/&)

Addition

of

isolated

of clot

CELLS

was also of

the

when added

to recalcified

60X inhibition

origin

Preparations

by freeze-thawing,

retraction

mately

FROM ENDCTHELIAL

intact

titrated

Vo1.5,No.6

an efficient

inhibitor

agent

released

for

from

to titrated

PRP, reduced

clot

endothelial

cells

con-

PRP likewise

(final

resulted

in approxi-

retraction.

DISCUSSION The inhibitory potent This

agent inhibitory

cells

the

for

is

intact

of

labile

plate let

this

cells

is

tory

a broad

is

or of cell

demonstrated

inhibitor

which

activity

fragments. molecule

easily

when

contain

ADP, pre-

and the release

epinephrine,

of platelet

to the presence

of

can be

not only

retraction

by ADP, collagen,

in response

small

mixtures

clot

by exposure

The inhibitor

cells

inhibitory

prevents

spectrum

cells

cells

to be a relatively activity

and retraction.

or thrombin.

function of

and is

commonly used

agents. activity

from isolated cells

indicates

could

the cells

can be recovered

endothelial

of endothelial

activity

cubating

induced

from endothelial

endothelial treatment

endothelial

This

but also

release,

agents.

added to reaction

or thrambin.

from platelets

The inhibitory or

are

aggregation

activity

aggregating

intact

has been shown to be a

from endothelial

aggregating

Inhibitory

(1).

epinephrine,

released

situ

can be released

of

cells

aggregation,

has been shown previously

endothelial

serotonin

Thus,

of platelet

in the absence

heat

collagen, vents

prevention activity

activity

which

from endothelial

to conmonly used platelet

demonstrated This

activity

that

cells

be recovered with

cells. the

with

either

Recovery

from endothelial of

inhibitor

inhibitory

activity

collagenase.

(b

from endothelial

MTS in the absence

is

rare

cells

of aggregating

cells

from in situ

not generated

occasions,

in situ agents.

in

by

inhibi-

simply

by in-

In each

INHIBITOR

Vol.j,N0.6

case in which release sence

of

FROM ENDOTBELIAL

inhibitor

of added aggregating

signs

of

thrombin

of

cells.

lated

endothelial

release.

the process

might

render

thrwbin.

relatively

in situ

high since along

in these

the cord

work is

the differential

with

of

isolating

normally

frees

surface.

in situ of

The ability release

antithrombotic induced

of

the

likely

cord

showed obvious

this

are

cells

inducer

of

endothelial

is cells with

but

a possible

inhibitor in tests are

vein. less

such sensi-

not known.

The

to collagen

is

preparation

attachment.

endothelial

from the Achilles of

iso-

of

some form of collagen

from their

the efficiency

was

with

cells cord

iso-

amounts

inducer

the collagenase

which anchor

thranbin

enhanced

cells tendon

used

However, may differ collagen

as a release in-

effect

of

thrombiu

in

collagen.

inhibitor

to prevent

serotonin from platelets properties.

in contact

At any rate,

may reflect

in tests

to collagen

endothelial

large

thrombin

hand,

alteration

and reactivity

Finally,

cells,

of

and with

released

as a release that

effect

in situ

them from umbilical

isolated

structures

subendothelial of

is

the endothelial

cells

endothelial

effective

of

these

used here.

the presence

nature of

one cell

thrombin

more sensitive

sensitivity cells

cells

was the most active

It

in composition

preparation

tion

in the ab-

the umbilical

or that

On the other

situ.

The exact

studies

considerably

also

occurred

be shown that

in situ,

moderately

the cells

the collagenase-sensitive

ducer

cells

endothelial

isolated

collagen

in

with

inducer.

was only cells

during

alteration

but’with

cells,

endothelial

interest,

the present

cells

as a release

Collagen

to

of

when tested

activity,

inactive

altered

could

postdelivery

With endothelial

nearly

tive

aspect

and collagen

inhibitory

with

it

755

trauma.

An interesting

lated

from eudothelial

agents,

used was more than two hours

CELLS

The inhibitor

by ADP, epinephrine,

not only

indicates prevents

or thrombin.

platelet

that the

this first

In addition,

aggregation agent

but

has potent

wave of aggregathe

inhibitor

756

IXHIBITOR

prevents

FROM ENDOTHELIAL

the second wave of platelet

the release

reaction

Diminution evidence

of

of clot

retraction spectrum

is not essential

inhibition

traction.

hence the

inhibition

of

Prevention inhibitor lation

of clot

of calcium

cells

inhibitor

is

take

up the

inhibitory

trum inhibitor

amounts (4). is

of

studied

is

platelet

Further

on platelets.

functions

agent

inhibitor for

of

not known.

Alternatively,

characterization

clot

re-

retraction

suggests

by

that

intracellular

the

reguIt

release

is

from

thrombostheain.

activity

within

synthesize

stimuli

the endothelial it.

in plasma

these

endothe-

and store

to appropriate

and concentrate

both of

release

to prevent

of

may be present of

retraction

of thrasbostheain.

cells

in response

aggregation

clot

such as the

inhibitory

from plasma

clot

and aggregation

be released

Platelet

inhibited

may influence

contraction

further

the platelet

reaction

may function

of the

is

Recently,

and contraction

Whether endothelial

activity

platelet

this

here

release,

the

activity

release

retraction,

location

not known.

agents

by prevent ion of

inhibitory

upon the occurrence

of Ca2+ needed

which can then

as aggregating

apparently

retraction.

reaotion.

that

The ultrastructural Lial

clot

ion concentration

sites

of

release

basic

to speculate

intracellular

effects

the platelet

inhibitor

influences

tempting

of

the platelet

by the

for

has been shown to be dependent

reaction (3);

aggregation,

Vo1.5,No.6

of platelets.

the broad

aggregation

CELLS

inhibitory

cell A broad

this such may spec-

in small activities

in progrese.

ACKNWLEWEMENTS

Supported in part by grants !%04-HL 46351, HL 13296, and M 14228 from the National Heart and Lung Institute, cootract P&43-68-977 with the Artificial Kidney-Chronic Uremia Program of the National Institute for Arthritis, Metabolism, and Digestive Diseases, and grant 938 from the Council for Tobacco Research--U.S.A., Inc.

Vo1.5,No.6

INHIBITOR

FROM ENDOTHELIAL

757

CELLS

RlwERENCES

S.R., ZUCKPX, W.H., and WbSCN, RX. Some properties isolated from hman umbilical cord vein. 3. m.

1.

S&A, cells

2.

HOVIG, T. Release of a platelet-aggregating phate) frop rabbit blood platelets induced Thromb. Diath. Hsemorrh. 2: 264, 1963.

3.

DE GARTANO,G., BUT'TECCHIA, D., and VKRMYIKN,J. Retraction of reptilaseclots in the presence of agents inducing or inhibiting the platelet adhesion-aggregation reaction. Thromb. m. 2: 71, 1973.

4.

MASON, R.C., SABA, S.R., of platelet aggregation.

and CHUANG,.H.Y.K. &I. J. m. 74:

of endothelial 6: 456, 1973.

substance (adenosine diphosby saline “extract” of tendons.

Naturally 91a, 1974.

occurring

inhibitors