Rise of factor VIII after exercise and adrenaline infusion, measured by immunological and biological techniques

THROMBOSIS Printed

RISE

OF

RESEARCH in the United

FACTOR

MEASURED

BY

VIII

AFTER

EXERCISE

IMMUNOLOGICAL

C.R.M.

Prentice,

University

Castle

(Received

Forbes

Royal

Glasgow

antigen

1972 Inc.

INFUSION,

TECHNIQUES

M.

Smith,

Infirmary,

G4 OSF.

by

has been made

as measured

activity, VIII-related

and Sandra

Accepted

23.8.1972.

ADRENALINE

BIOLOGICAL

of Medicine,

Street,

A comparison

AND

AND

C.D.

Department 86

ABSTRACT

Vol. 1, pp. 493-506, Pergamon Press,

States

Editor

B.

of biological

by coagulation by the Laurel1

Blombgck)

factor

assays,

VIII

and factor

electrophoresis

Factor VIII-related antigen is present in all technique. normal and haemophilic individuals but not in those with severe

von Willebrand’s

adrenaline fall

infusion

of factor these

VIII are

techniques;

exercise

pattern

of rise

by both biological

it is tentatively

circumstances,

rclcascd

Following

is a similar

VIII as measured

immunological under

disease.

there

additional

or and

and

suggested

amounts

that,

of factor

into the circulation.

INTRODUCTION Factor activity

VIII

(antihaemophilic

in both physiological

exercise

or adrenaline

activity

2) which

(1,

and in diseases disease

(7,

is seen.

such

administration can be blocked as the nephrotic

the basic

(9,

syndrome lo),

mechanisms rise

rise

adrenaline

administration

may

be due either

or to the release

of factor

inhibitors coronary

6),

sustained

controlling

of factor

493

(5,

a similar

The

the circulation

is a rapid

changes

Following

by beta-adrenergic

are unknown.

within

marked

situations.

there

VIII levels

factor

undergoes

and pathological

8) and thyrotoxicosis However,

globulin)

VIII after

plasma

of stored

(3,

4),

artery

elevation factor

exercise

to activation

VIII

or

of the clotting or newly

syn-

in

FACTOR

494

thesised

material.

technique was

Rizza

found

with

increased

Zimmerman

estimations

provided

antiserum

means

concentration

of factor

adrenaline

using

antigenic adapted

VIII

activity

of factor

technique

of factor

VIII

of the clotting

the Laurel1

VIII

This

activity

following

can be compared

an inhibitor-neutralisation

in rabbits.

the biological

Vol.l,No.5

concentration

concentration

prepared

by which

(11)

EXERCTSE

post-exercise

et al (12)

of the antigenic

monospecific

AFTER

and Eipe

that the increased

correlated

factor.

VIII

(13)

by use

for

of a

technique

has

and the immunological

exercise

and the

administration

of

simultaneously.

METHODS The

method

essentially Three

that

hundred

volunteers,

ml

of blood

30 minutes

glycol

between

400-1,000

to provide

Fine

units

Chemicals

collected

in 4 ml. aliquots

containing about

VIII

Freund’s

was

same

two

weeks

with

prepared

adjuvant

procedure the

Freund’s

solution

VIII

was

after

Blood

this

The

mixture,

weeks,

taken

was

the void tubes

to provide Antiserum

ml.

solution

gel

Those

pooled

per

4B

eluate after

with

as the

into

rabbits.

and after without

7,

8 and 9 days

and absorbed

as described

to

2 ml

as well

, intramuscularly two

for

activity.

were

of this

and poly-

column

VIII

VIII

factor

containing

immediately

intravenously

was

Crude

ethanol

pH 7.4.

factor

one unit factor

preparation

and centrifuged

to a Sepharose

Sweden)

filled

2 ml

normal.

plasma.

applied

was

modifications.

citrate

with

concentration

injected

minor

concentrate,

0. 15 M,

for

repeated

was

adjuvant.

poor

and injecting

VIII

was

platelet

Uppsala,

by mixing

of the factor

sodium

VIII

VIII

from

of 3.8%

and the tubes

factor

a few

by venesection

which

buffer,

e containing

complete

remainder ‘The

eluat

ml,

immediately

the highest

10 ml

factor

assayed

with

of factor

Inc.,

saline

of factor

by precipita%ion

8 ml per

in barbitone

were

taken

prepared

filtration

volumes

was

to provide

were

e’hylene

et al (12)

to l/ 10th volume

concentrates

(Pharmacia

and purification

of Zimmerman

added

at 2000 G for VIII

of preparation

a further

prior after

mixing the last

injection. The When

tested

antiserum

was

treated

by immuno-diffusion,

it gave

a single

precipitating

previously line

(12). against

Vol.

1,No.

normal prior

FACTOR

5

and haemophilic

(factor

or plasma

but there from

VIII concentration

antiserum

was

minutes

mixed

AFTER

as well

plasma,

to gel filtration,

fibrinogen

VIII

was

as the factor

with severe

Additionally,

with four

volumes

at 37OC it ncutraliscd

over

VIII concentrate

no cross-reaction

a patient 1%).

495

EXERCISE

with purified

von Willebrand’s

disease

when one volume

of undilute

plasma

95% of the factor

for

of undilute sixty

VIII activity

of the

plasma. Immunoelectrophoresis using

a Shandon

powcr

electrophoresis

Camberley,

Instruments,

Surrey)

the test

and standard

0. 1 ml,

0.01

M barbitone

solution

wcrc

used

ing 2 x 3 inches

for

three

The

were

saline

and stained

estimations

as 2501.

Accordingly

VIII antigen The wells of factor was

in whole

were

period

filled

using

40 amps,

by Breckenridge

slides

measur-

containing

0.03

was

carried

and 250 volts

out

per

distilled

water

for four hours,

quantities

was

were

per

of plasma

of factor

VIII-related

180 volts

VIII assays

in

of this

then immersed

slide. in dried

prior

for

differed

by as much

estimation

of factor

concentration plasma,

slide.

the height carried

and Ratnoff

by ethanol.

and only

with the agarose. without

for immuno-

VIII are precipitated.

antigen

devised

temperature,

Factor described

current

10 microlitres

did not increase

dissolved

at 4’C,

without

of time

were

hours

was mixed

out at room

2. 5 ml of

B.

a technique

with

and D. C.

from

Glass

and electrophoresis constant

of factor

plasma,

made

agarose

in the use of Cohn fractions

VIII antiserum

carried

hours

ml 0.9%

hours,

platen

and 10 microlitres

with 6.5

is that variable

Duplicate

was

in diamctcr.

1% azo-carmine

One problem

cooling

3 mm

30 amps,

technique

(Shandon Southern

the precipitates

pH 8.4

VIII antiserum

forty-eight

with

electrophoresis

buffer,

kept for forty-eight

for

U77

with water

samples;

covered

ho-drs using

out by the Laurel1

I-O prccipit@

to fill wells

were

factor

slides

0.9%

plasma

carried

apparatus

Cohn fraction

supply.

ml undilute

was

water

Electrophoresis cooling,

Electrophoresis of the precipitation out by the one-stage

(14).

15 microlitres

for three for

a longer

peaks. technique

as

FACTOR

496

VIII

AFTER

EXERCISE

Vol.l,No.5

RESULTS In fig. normal for

1 is seen

pooled

plasma

measurement

of the precipitate

factor

VIII,

a straight-line

filled

with

tion

plasma

VIII

less

was

in three

than

Willebrand concentration

severe fifth

was

wells von

contained Willebrand

of Cohn from plasma,

left

The

plasma

fraction to right: haemophilic

I-O

fourth

wells

When of

well

(factor

contained

which

was

was

disease

VIII

concentra-

in the von a normal

biologically

inactive.

1

for

factor

normal plasma

the

contained

material

plasma

antigen

samples.

von Willebrand’s

no cross-reacting

VIII-related

standards

on a semi-logarithmic

and sixth

of haemophilic

wells,

the concentration

factor,

with

but the haemophilic

of factor

against

obtained.

three

to provide of test

was

FIG.

Electrophoresis

dilutions

plotted

The

2%).

There

1%).

plasma,

was

a patient

1 : 2 dilutions

in the first

concentration

relationship from

in which,

by the dilution

concentration

and

used

peaks

as determined

scale

undilute

plate

of the antigenic

height

(factor

a Laurcll

VIII

plasma

estimation. (undilute,

(undilute,

1 : 2).

The 1 : 2,

1 : 4).

Vol.l,No.5

FACTOR

VIII

AFTER

EXERCISE

497

Exercise Five possible,

healthy

and blood

and after

samples

exercise

immunological VIII

activity

rise

of factor

measured

ran were

and measured

after

exercise

accompanied

taken

been

factor

plotted

VIII

assajr

before

and the of facdor

concentration, the same

in biological

in antigenic

estimation

as

the percentage

to approximately

that the increase

as fast

increase

against

factor

a mile

VIII

the one-stage

The

rose

by nn incrcasc

of half

2, the perccntagc

antigen.

techniques,

for

by both

has

VIII-related

indicating

a distance

In fig.

technique.

by both

exercise was

volunteers

activity

extent

of factor

concentration

as after VIII

of the clotting

factor.

400 -

300 INCREASE IN FACTOR VIII ACTIVITY - PER CENT 200 -

I 100

??

I 200

I 300

1 400 RELATED

INCREASE IN FACTOR VIII ANTIGEN PER CENT FIG.

Comparison antigen

between

immediately

of pre-exercise 0Cra~J”n.s.

levels.

rise

of factor

after

cxcrcisc, Two

VIII

2

activity

cxprcsscd

of the five

and factor-VIII-related as pcrccntage

individuals

were

increase

tested

on two

FACTOR VIII

498

In two healthy factor

VIII activity

techniques, exercise

antigenic

plate

samples

whole

for

VIII-related

a period

over

The pattern

measurement antigen

rise

was

tested

tested

of subseq-lent and fall

in fig.

of factor

VIII,

that all the

of pre

4.

is shown

exercise

and post-

A similar in fig.

dilutions

samples.

following

after

A representative

fractions

in three

peak of

hours

suggesting

activity.

is seen

were

and fall

similar

precipitated

compared

samples

sample

was

1,No.

and immunological

of twenty-four

of rise

biological

ethanol

were

plasma

the pre-exercise itandard

taken

possessed

in which

of the post-exercise

by both coagulation

by the two tcchniqucs,

material

exercise

the decline

Vol.

EXERCISE

measured,

3 a and b).

as measured

which

was

in samples (fig.

Laurel1

volunteers

AFTER

5.

plate In this

to provide The pattern

in case

a of factor

is seen.

400-

V+-iO

-

Factor pm-

related

-----

Factor m

activity

2 TIME

AFTER

FIG.

4

6

EXERCISE

3 aandb

Comparison of factor VIII activity and factor following exercise in two normal people.

VIII-related

antigen

antigen

12 hours

5

Vol.

I-No.

FACTOR VIII

5

499

AFTER EXERCISE

I i ‘

FIG. ---

Factor

VIII-related

samples;

antigen

the wells,

samples

taken

before

12 and 24 hours

from

before

exercise,

Factor

dilutions,

Cohn fractions

1 : 2 dilutions

post-exercise,

1,

antigen

before

the pre-exercise

2,

4,

immediate

post-exercise,

5

and after sample, 2,

4,

exercise, undilute,

plasma

I-0

of

post-exercise.

VIII-related contain

exercise,

contained

immediately

FIG.

the wells

4

and after

left to right,

.

samples;

1 : 2 and 1 : 4

6 and 10 ho-drs post-exercise.

6,

FACTOR VIII

500

-The effect 01 factor

of adrenaline

VIII was

stuclicd

adrenaline

infusion

by Ingram

and Vaughan

of the infusion Again,

as seen

measured almost

in

AFTER

on the activity volunteer

one

in a dosage Jones

and antigenic

who rcccivcd

of 10 pg per minute Blood

(3).

as well

as at intervals

in fig.

6, the pattern

by both the coagulation

was

over

Vol.l,No.5

EXERCISE

for

taken

concentration

an intravenous 15 minutes

before

and at the end

the next twenty-four

of factor

VIII rise

and immunological

as used

hours,

and fall

as

methods,

was

identical.

300 h

-

Factor Sm- related antigen

----

Factor Sm activity

I

I

1

2

4

6

TIME AFTER

II II

1

12 hours

ADRENALINE

. FIG.

Factor VIII activity following adrenaline

and factor infusion.

VIII-related

6

antigen

co;lcentration

FACTOR

Vol.l,No.5

VIII

AFTER

501

EXERCISE

DISCUSSION By use estimation bctwcen

of both biological

of factor

VIII it has been possible

the antigenic

Zimmerman antigen

concentration

in both normal

people

with von Willebrand’s

haemophiliacs

contain

similar

Zimmerman it may

to normal

et al (15)

bc possible

significantly

We have

activity

of factor

in normal seen

suggest

Willebrand’s unstable

human

factor

from

that either disease

there

indicated

as carriers

prepared

a precipitating In contrast,

factor

against

of factor

against

a component was

disease.

This

VIII synthesis

of such

identity

USC

VIII.

no such reaction

with von Willebrand’s

that it has no antigenic

that

the biological

line

occurs

that

possess

in rabbits

VIII both neutralises

or that synthesis

suggest

by the combined

VIII than biological

is failure

but

inactive.

(16) have

techniques,

patients

patients,

would

ol hacmophilia

plasma.

factor.

VIII which is anti-

and Huehns

factor

of this

VIII-related

VIII but biologically

carriers

VIII and gives

plasma

molecule

factor

This

of factor

that antiserum

and haemophilic

against

would

antigcnic

confirmed

purified

form

and immunological

more

activity

and haemophilic

disease.

and Bennett

to dctcct

of the coagulation

partially

an altered

for

to study the relation

and the clotting

not in patients

genitally

techniques

(12) h ave shown that the factor

and Ratnoff

is present

and immunological

in von

an abnormal

or

with the normal

factor

VIII molecule. It is known that the biological exercise have

(1) or adrenaline

fo.und that,

activity

the rise

correlated

VIII reverted detailed

required,

therapy

studies

hours noted

which following

value

on the turnover of factor

decline

of factor

factor

of factor

VIII-related rise

we

VIII in this

VIII

in factor

six to ten hours.

Although

situation

are

would

seem

to be four

than the usual

value

of ten to

of haemophiliacs

coagulation

studies

in biological

exercise

faster

the transfusion using

within

after

and adrenaline

post-exercise

VIII after

is significantly

by observers

exercise

the imtnediate

VIII increases

In the present

with the concentration

to the pre-exercise

the half-life

or rive hours, twelve

and subsequent

closely

of factor (2).

both strenuous

By both methods

antigen.

more

administration

following

administration,

activity

assa;rs

with replacement (17,

18).

W hethc r

FACTOR

502 the

increased

exercise, isms

turnover

or

Our exercise

show

adrenaline

and antigenically,

Similarly

techniques

no differences

in patients

with

with

infusion

is

between

(20)

proportion

This

the rabbit

haemophilic

patients

VIII

(12,

23).

VIII

antigen

that these study factor

patients

antigenic

patients

antigen

Rizza

VIII

is the

same

and Eipe of factor The

even

(unpublished (9) h ave VIII present

VIII

concentration

of additional

factor

VIII

from

or to activation

of factor

circulation

is not yet

view

single

of the

VIII,

known.

precipitation

these

outside

by monomer

It is more arcs

greater

after

stores

possibly

we have

seen

a

in rabbits

absent

found

factor

In a that

than that

seen

is

in the

likely after

a rise

using

by use

with

factor

(16) using

not excluded.

that there

study,

in antigenic

in

but the possibility

exercise

is in agreement

technique,

antigen

in

results).

noted

after

with

was

inhibitors or

VIII

in

in 18 of 20

patients

disease

antigenic

prepared

VIII,

that

prepared

and Huehns

to factor

factor

inhibitors

technique.

immunological

release

with

concentration

neutralisation

rise

had inhibitors

of

cross-reacting

factor

to be present

that the two

found

to fewer

an antibody

had von Willebrand’s

without

Recently

with

present

inhibition

antibody

with

VIII

out by Hoyer

They

localised

Bennett

material

suggested

have

VIII-related

haemophiliacs

the

They

might

of seven

technique

carried

material

than the

resting

individuals.

inhibitors.

cross-reacts

under

measured

had antigenic

biologically

of factor

been

who

after

gel-filtration

levels

have

VIII

molecule

cross-reacting

.

using

is probably

22,

both

and in normal

antibody

coagulation-neutralisation VIII

found

VIII

factor

antibody

rabbits

haemophiliacs

after

mechan-

produced

identical,

et al (21)

of haemophiliacs

on the factor

factor

VIII

clearance

in the circulation

states

on factor

by acquired

the inhibitor.

found

Vol.l,No.5

of factor

VIII

the elevated

and Denson

determinants

all

normally et al (19)

studies

activity

since

VIII

factor

apparently

“hypercoagulable”

and Breckenridge

a small

additional

Penick

Immunological

VIII

instability

of the normal

that the

to factor

conditions.

factor

stimulation

EXERCISE

is not known.

studies or

AFTER

is due to increased

to increased

by exercise

VIII

an inhibitor

of a more

Whether

findings. exercise

direct

is due to

the normal

circulation

formation,

within

to be a release exercise

reaction

in the Laurel1

the in

Vol.l,No.5

FACTOR

technique

3)

(fig.

but

VJ 1’1. :i.S accolllpnrlietl cannot

be

by

completely

VIII

AFTER

the

possibility

tile

UlIcovel’ill~~

503

EXERCISE

that 01’

activation

l’Ul’i,llCJ’

of

;lJlLi(:eJl

factor i (!

s

i t.os

excluded.

ACKNOWLEDGEMENT

This to

C.

R.

work M.

was

supported

by

a grant

from

the

Wellcome

Trust

Prentice.

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