thrombocytopenic purpura Willebrand factor ...

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1985 65: 1232-1236

Effects of fresh-frozen plasma and its cryosupernatant fraction on von Willebrand factor multimeric forms in chronic relapsing thrombotic thrombocytopenic purpura JL Moake, JJ Byrnes, JH Troll, CK Rudy, SL Hong, MJ Weinstein and NM Colannino

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Effects von

of Fresh-Frozen Willebrand Factor Thrombotic By

Remission

plasma

relapsing

thrombotic

contain thelial

to

of some von

those

frozen

plasma

(FFP)

in the

in

patients

with

patients

with

Three

remission

have

their

plasma.

with

FFP.

three

plasma

Two one

immediately

vWF

multimers

plasma

of

two

of

after

three (on

one

occasion). was

URING

the

immediately patient

in patients

patient

transiently

plasma

cular This

human During decrease

platelet may be

chronic

and attach

with

intravas-

thrombocytopenia. preferentially

to

platelets in vivo in the presence of a putative inciting cofactor released in association with inflammation or tissue necrosis. The unusually large vWF multimers then return to patient plasma as recovery occurs. The infusion of normal fresh-frozen plasma (FFP), often

in association

From

the

University

Boston

School

School;

the

Thorndike College

institutes

No.

of Health, June

Address tology.

reprint

Veterans

from

the

HL22355

and

HL13262

Bethesda,

Md.

Hospital-Mail TX & Stratton.

0006-497//85/6505-0027$03.00/0

1232

City University.

accepted

Houston,

Research

Rice

to Dr Joel

Methodist

by Grune

funds

77030. Inc.

Centers;

Nov

in

Boston

of Miami

Boston and

1 1. 1984;

The

Medical

Hematology Tex;

is effective

University

requests

Fannin,

© I 985

VA The

Laboratories, Waco,

by research

Submitted

Miami

Medicine;

Memorial

grants

plasmapheresis,

B. Castle

of Medicine, by

6565

and of

William

Supported and

with

Medical Laboratory.

Hospital;

Baylor

Houston. Administration

from

the

National

14. 1984.

L. Moake, Station

Medical 902.

Main

and

plasma

in a third

patient

who

HemaBldg.

was

multimers

to smaller results

fraction the

vWF

indicate of normal

metabolism

this

in partial

an

plasma

large in

& Stratton.

or preventing

harmful. The

have transfusion

contains plasma,

pernatant been found prophylaxis

in vivo chronic

in controland

relapsing

to

of normal

relapsing

normal platelets,3 of factor IX on be

ineffective

or

cryoprecipitate,

which

multimers present in normal Transfusion of the cryosu-

fraction of normal plasma, however, to be at least as useful as normal plasma against, or therapy for, TIP relapses

patients with chronic relapsing TTP.46 tant contains residual amounts of the multimers present in normal plasma. In this

report,

cryosupernatant on the plasma with chronic

we describe

remission. in order

between multimer

therapy forms.

Cryosupernasmallest vWF

effects

of FFP

and

The patients were studied during to allow a correlation to be made and any changes in plasma vWF It is not possible to make this correla-

tion during relapses relapses the unusually

in chronic TTP, because during large vWF multimer forms (and

even the in circulation)

in association

the

has for in

infusion, as well as plasma exchange, vWF multimer patterns in three patients relapsing TTP who were in complete or

partial remission

sometimes normally

phase.

multimers.

in chronic

reported

the largest vWF is also ineffective.46

fluid

Inc.

relapses

been

FFP large

cryosuperna-

vWF

TTP.2 In contrast, transfusions of albumin,4 -y globulin,5 or preparations fibronectin5

in the

some

in

abnormality Neither

in the

is involved

is defective

the

TTP

unusually

in vitro

of unusually

process

the

remission.

activity

its

disap-

from

relapsing

converted

forms

that

and

in vivo

multimers

reversed

fluids

or cryosu-

FFP,

rapid

chronic

directly

these

in

the

exchange

remisremoved

FFP

activity

vWF

with

cryosupernatant

controlling

purpura multimers similar to

endothelial cells in relapses, the unusuon disappear from

in association

agglutination because they

remission.

ling

large

patients

when

normal

an

plasma

patient

medium

cells, either

that

her

Unusually

from

culture

promoted

unusually

two

TTP patients. 0 1985 by Grune

infusions.

with

conclude

of

the

to

thereafter.

disappear

with

fraction.

of

returned

endothelial

in vitro

We

not

on

L. Hong,

persisted

or from

human

cryosupernatant plasma

did

samples,

incubated

that

plasma received

the

and

normal

tant

(on

who

after

multimers

were

These

trans-

after

relapsing thrombotic thrombocytopenic (TTP),’ von Willebrand factor (vWF) larger than those in normal plasma, and those produced by normal culture, have been found. ally large vWF multimens

patient

cryosupernatant

vWF hours

from

nor

large

from

following

or

on before

Unusually

serially

REMISSION

once

studied

disappeared

The

studied

in

Suchen

to 24

multimers

pearance

during

Fraction Relapsing

large

pernatant.

of

present

was

1 1/2 hours and

plasma

cryosupernatant

occasions)

occasions).

TTP

transfused

exchange.

1/2 to

two

were

patient or

vWF

sion

form

multimers

received

third plasma

within (on

cryosupernatant

D

two the

decreased

FFP

exchange

the

relapsing

patients

large

of TTP

relapsing

ten

mul-

fresh-

or prevention

vWF

within

endo-

(vWF) human

as normal

chronic

chronic large

the

and

samples

fusion

of

of

occasions,

and

treatment

unusually

(TTP)

cryosupernatant

effective

the

chronic

normal

of the

is as

Unusually

with purpura

factor

by

infusion

of

TTP.

normal

The

fraction episodes

patients

Willebrand

produced

in culture.

cells

John J. Byrnes, Joseph H. Troll, Christine K. Rudy, Mark J. Weinstein, and Noreen M. Colannino

thrombocytopenic large

similar

Moake,

L.

samples

unusually

timers

Joel

Plasma and Its Cryosupernatant Multimeric Forms in Chronic Thrombocytopenic Purpura

with

largest vWF multimers disappear from patient the

in vivo

that is the pathophysiologic organ ischemia.

Blood,

Vol 65, No

platelet

basis

5 (May),

found plasma

agglutination

for

the

1985:

multifocal

pp

1232-1236


EFFECTS

OF

CRYOSUPERNATANT

ON

vWF

IN

UP

1233

PATIENTS

hours

in 0.15

changes),

Patient

1

For

five years,

either

FFP

TTP

this

relapses.

She

et al.’

counts,

is described

were

to

335,000/ML)

U/dL),

present

she

as part

of her

study

and

Patient

gave

unusually

(vWF

antigen

with

2 units

mL)

of normal

prophylactic

large

of either

She

consent

on the

This

was

program

understood

the

to

at the

purpose

of

for it to proceed.

This

34-year-old She

woman

was

is patient

once

has

B in Moake

intermittently

et al.’

she gave

permission

then

30 minutes

after,

she

was

in

vWF

forms

She

for blood

were

found

hen

On

infusion. and

plasma

(vWF

of FFP

This

one

of those

At

before, the

time,

unusually antigen

21-year-old

recovered unusually

large

vWF

she

level,

the

relapsing

occasion,

when

(platelets had

had

383

reappeared

143

patient

TTP.

She

had

partially

she

I l0,000/zL),

a 3-L

antigen,

from

chronic

one

multimens

vWF

obtained

On

relapse

U/dL),

(postexchange

had

et al.’

a TTP

168

were the

woman

from

antigen,

human

vWF and

in hen plasma

exchange

U/dL).

before,

and

with

180

then

is

blood

7.4. samples,

IRMA,

were

were

applied

gels

were

soaked

mol/L

NaCI

lgG

changes

acetic

acid

for

hours

and

placed

procedure.

and

The

preparation

plasma

of

(PPP)

from

described.’

vWF

quantified

by

high

Colloids

antigen

was

(IRMA)’ rate

also using

Chemical

(100%)

rabbit

of the

Agarose

mol/L

Patient

and

same

vWF two

2#{188} to out

2/2

and

containing ing)

for

(Dako

and

NY)

plasma

mented

and

contains

100

assay

Cryosupemnatant lated was

method

of Gralnick backing

sodium

pooled levels

They different

were

origin

hrs at 100 electrophoresed rabbit 16 hours

in the

samples

in the

same

0.5%

second vWF

and

-35

into

0.5%

dimension

then

for cut

agarose

(Calbiochem-Behrwere

then

washed

to

in an oven

at 56 ‘C for six Rochester,

screens

(E.

NY)

I. duPont

Wilmington,

de

Del)

for

veins

in gelatin

dishes

containing (at

anti-human

isolated

does

for

not

by

Laboratories,

medium

56 ‘C,

vWF

were

(Difco

199

supple-

30 minutes)

react

with

fetal fetal

calf

was and

prepared

for transfusion from

three

from

single

normal blood

donors

units in

was

different

in acid-citrate-dextrose. made

from

anticoagulated made

from

normal

obtained

and

from

anticoagulated

from pooled

donated

single

donors,

Cryosupennatant units

of blood

by

acid-citrate-dextrose. units and for

the PPP

of fresh

anticoagu-

in vitro

citnated

PPP

prepared

from

studies of four fresh

in hepanin.

RESULTS Unusually

the

before

gel slab were

antibodies V. Gels

agarose

in two

glycenol/l0%

8.6.

dimension

gel lanes

in 2%

Dept.

8.0,

‘25l-anti-human

(Kodak,

umbilical

grown

heat-inactivated

plasma

collected

human

to

buffer first

human

was

pH

adjusted

running

pH

HGT

buffer

acetate,

were

first-dimensional

in the

SeaKem The

sodium

electrophoresed

anti-human at 10 mA

0.5%

barbital-acetate

wells

V. The

using

by a modifica-

dimensions.

mol/L

plasma

with

al7

both

barbital/0.080

normal antigen

et for

done

(Cohn

Louis),

for 48 hours

film

Products

and

individuals

different was

20%

Fresh-frozen

U/dL

albumin St

vWF).

normal

rabbit

from

(Rabbit

vWF

PPP

dried

of deionized bonate/0.I50

rabbit then

at I 00

isopnopanol.

serum

intensifying

Photo

35-mm

serum.9

Accu-

Plus

Inc.

cells

with

serum

rabbit

Calif).

Antibodies,

Westbuny,

pooled

Detroit)-coated

backing

immunoradiometnic

vWF

immunoelectrophonesis

electrophoresis. from

GelBond

La iolla,

been

calf

were

XAR-Omat

treatment

were

SeaKem

and

buffer.

hours

sodium

with

NaCI, gels

running four

Co,

pH 20 L

hours at -70#{176}C.

collagenase

antigen.

on GelBond

0.048

0.5% Me)

Corp.

Corp.

Normal

on

Rockland,

by solid-phase

Scientific

plasma

using

Corp,

has

normal

Aganose

anti-human

vWF.

vWF

FMC

quantified

Two-dimensional tion

(HGT)

platelet-poor

samples

and

(Calbiochem-Behring

and

‘25l-anti-human

blood

in patient

Co.

23 ‘C

SDS,

changes

in borate/NaCI,

The

with

two

mol/L

Chemical

at

mol/L

Lightning and

in

in 0.02 and

acid/lO%

bovine

Sigma

18 hours

hour.

‘/2

Endothelial

pooled

electroimmunoassay5

Div.

vWF

normal

venous

levels

temperature

anti-human

and

citrated

antigen Laurell

gelling

(Marine

patient

free,

of 0.15

Chronex

24 to48

METHODS

AND

for

hour

in 0.036

(1 x l0 cpm/mL)

Nemours MATERIALS

one

of

at 50 V and

I mg/mL

acid

three

after,

for

a

hour

and

plasma

I 5 minutes,

by 10% acetic

30 minutes

containing

vWF

samples

layer

V. fatty

(vWF

immediately

under

in the gels

pooled

urea/2%

gel

washed

incubated

mol/L

the

for

and

EDTA/8

for

fixed

normal

of

at 60 ‘C

was

mol/L

EDTA/0.l%

and

by dilution

incubated

V, protein

I .5 x

0.04

mmol/L

the

by electnoimmunoassay

15 U/dL

wells

1% of

gel slabs,

buffer,

in patient

quantified

for one

sulfate ‘251-antiusing

(1%)

running

were to

water,

the levels

mmol/L

dodecyl

in a modification

acetate/2

antigen to

electrophoresis

normal sample

rabbit

autonadiognaphy Aganose

in

sodium

vWF

Following

and

of the origin

with

system

Zimmerman.S

previously

samples

and

reproducible.

by sodium

by

formed

TRIS-HCI/2 The

patient

of patient

overlaid

analyzed

adjusted

the

same

the expeni-

of

always

matching

separated

buffer

were

The

plasma

fraction

FFP

and

mol/L

pH

8.0.

were

and

mm,

Under

gels

exact

in

photographed.

a continuous

when

normal

Venous

and

IgG,

and

for the

positions were

electrophonesis,

of Ruggeni

x

mol/L

large

and

acid,

samples

chamber.

individual

multimers gel

aganose

The

A in Moake

together, vWF

plasma

relative

after

(two

Feinbiochemiacetic

concurrently

patterns

Blue-stained

R, Serva

normal

the

superimposed

taped

Plasma

SDS,

3

patient

were

Coomassie

with

acid.

performed

vWF

water

stained

Blau

electrophonesis

plasma

Coomassie

wells,

80

U/dL).

Patient

Dried

and

always

in deionized

‘C,

methanol/lO% acetic

described,

TRIS-HC1/0.02

unit

to be taken

of the

normal

hours

0.1%

45%

patient

in the same

control

method

since

deficiency”

relapses.

308,000/.tL)

in

of TTP

a single

samples

completion

(platelets

episodes

T1’P

of

conditions

plasma

G plus in

was

mental

four

methanol/7%

(SDS)-agarose

“thrombopoitin receives

against

and

remission

periodic

to have

as prophylaxis

occasions,

had

believed

periods

for

at 37 to 45

Blau FRG)

dimension

time

vWF

(0.1%

in 25%

second

122

FFP

plasma.

Blue

Electnophonesis

C

(platelet

NaCI,

in an oven

Heidelberg,

destained

patient

levels,

transfusion

Center.

informed

and

remission

when

of

to prevent

2

infancy. and

and

Medical

transfusion weeks

in clinical

plasma

(-.400

regular

of Miami

required to four

5 in Bynnes2

was

transfused

fraction

University the

patient

in hen

was

has

three

as patient

the

cryosupernatant done

woman every

When

190,000

multimers 235

26-year-old

or cryosupennatant

Moake

in

Bnillant ca,

mol/L

dried

for 18

plasma longer tive

of two detected, amounts,

large

in citrated

blood obtained of either normal or FFP

vWF

multimers

in the

chronic relapsing TTP or were considerably

in patients

PPP

30 to 90 minutes cryosupennatant I and

2 (Fig

samples

remission

patients reduced

were no in rela-

prepared

from

after the in patient

transfusion 1 (Fig 1),

2). These

changes

in


MOAKE

1234

NP

EC

Pre

t5i

ET

AL

24h

Pre

__ ____

(.

...

.

+

n i

1.5 h Fig 1 . Following the transfusion of normal cryosupernatant (-400 mi), venous blood samples were obtained from patient 1 at 1 .5, 12. and 24 hours (1 .5h. 12h. and 24h). Analysis of patient plasma samples was by two-dimensional immunoelectrophoresis in 0.5% agarose (A). and by SDS-agarose (1 %) electrophoresis and autoradiography (B). (A) n. normal platelet-poor pooled plasma; p. patient 1 plasma. (B) NP. normal pooled plasma; EC. normal

.,.rt

12h

es

A

..

i-n 24h

circulating transfusion

vWF forms were of 2 units of FFP

cryosupernatant (on two 1 . Similar changes were the transfusion hours later the

W.

of I unit unusually

observed (on one

B

4.

following occasion)

the or of

of three occasions) in patient also seen immediately after of FFP in patient 2. Ten to 24 large vWF patterns were the

same as the pretransfusion patterns in patient I , and remained unchanged thereafter as long as the patient was in remission. Platelet counts did not decrease, plasma vWF antigen levels did not change detectably, and the clinical status of patients 1 and 2 was not altered following the transfusion of cryosupennatant or FFP. These results indicate that the disappearance of the unusually large vWF multimers was not the result of their attaching to platelets and Unusually

causing agglutination large vWF multimens

the plasma of plasma exchange during

a period

patient (Fig when

in vivo. not

were

present

3 immediately following 3). This procedure was her

platelet

count

was

in 3-L done

progres-

sively normal.

endothelial

cell

increasing and Disappearance

culture

her

medium.

clincal status of the unusually

had become large vWF

forms in patient 3 could have been the result both their physical removal and their conversion in vivo somewhat smaller multimens (as described below). Unusually large vWF multimers did in vitro from remission plasma samples when they were incubated with normal tant or normal obtained when were incubated vWF multimens

of to

not disappear of patient cryosuperna-

plasma (Fig 4). Similar results were remission plasma samples of patient 3 with normal plasma. Unusually large also did not disappear in vitro from the

medium removed from cultured human cells when this medium was incubated phase with normal cryosupernatant (Fig vitro results were not affected by the

endothelial in the fluid 5). These in presence or

absence ofcalcium (12.5 mmol/L), additional hepanin (14.3 U/mL) or proteolytic inhibitors (2 U/mL hirudin

+

100

fluorophosphate

U/mL

aprotinin (DFP)

+ +

2 mmol/L

1 zmol/L

diisopropyl-

p-amidinophenyl

Pre

+

Fig patient normal ma.

I

2. Venous blood samples were obtained from 2 before (Pre) and after the transfusion of 1 unit of FFP. n. normal pooled plasma; p. patient 2 plas-

O.5h


EFFECTS

OF

CRYOSUPERNATANT

ON

vWF

IN

UP

1235

‘e.,

-

-

.

-

-;

Pre

-.

r,’

+

___1i

Fig

3. Venous blood samples were obtained from 3 before (Pre) and immediately after a 3-i plasma exchange (Post). n. normal pooled plasma; p. patient 3

patient

Post

plasma.

80% I-TIP NP

0

20% 8h

20%

%

+ NP24h

EC

0

r

CS-

-TTP

24

8h

CS

80%

20% ‘

0

NP

8.i

Fig

4. Patient remission plasma (TTP) was with normal platelet-poor plasma (NP) or with normal cryosupernatant (CS) in the ratios indicated. The mixtures were either sampled immediately (0 time) or were incubated at 37 ‘C for eight and 24 hours (8h and 24h) before sampling. EC. normal human endothelial cell culture mixed

:

4.

medium.

!ctCs NP

Samples of normal human cell culture medium (EC) were removed and mixed with equal volumes of normal human cryosupernatant. The mixtures were either sampled immediately (0 time) or incubated at 37 ‘C for one hour (lh). Some of the mixtures contained calcium (Ca) or calcium plus additional heparin (Ca + Hep). as indicated. and all in this experiment contained hirudin + aprotinin + DFP + p-APMSF). NP. normal pooled plasma; and CS. normal cryosupernatant.

I_____ 10

Ca ii1IO

r

Hsp;;I $1I

W1NPIO

c 11,110

Hsp.Cal Th”O

Ih1NP

CS

Fig 5. endothelial

II

up

#{149}

+


1236

MOAKE

methanesulfonyl ing the time

fluoride of incubation

(p-APMSF), to eight hours.

FFP TTP were

30

to 90

minutes

after

or cryosupernatant patients in remission, present in relatively

disappeared, multimers patient, venous

into two unusually reduced

exchange

was in partial samples were

transfusions

with

on different

The

during patient

relapsing possible

that the contains conversion,

TTP remission explanation for

large

to the in vivo

cryosupernatant an activity that of unusually

plasma. these observations

fraction converts, large

corollary of this explanation of unusually large vWF circulation of the TTP cryosupernatant must

of normal plasma or potentiates the vWF multimers to This are A

would be that the turnover multimers already in the

patient at the be rapid.

time

of plasma

in the

treatment

pernatant

on the

forms indicates transfused was

of relapses

metabolism

in chronic

or

of unusually

that the short-lived

short-lived effect yet uncharactenized

is

smaller forms only on endothelial cell surfaces. may occur as the unusually large multimers secreted from endothelial cells into the circulation.

multimers from TTP patient are capable of distinguishing

that

may TTP

because they contain an activity that promotes the breakdown or removal of unusually large vWF multimers before they can attach to agglutinating platelets. The rapid dissipation of the effect of FFP or cryosu-

studies, the conversion of unusually large vWF forms did not occur in the fluid phase when normal cryosupernatant was mixed with ( 1 ) the medium removed previously from endothelial cells in culture or (2) with chronic One

vWF

these two possibilities are not yet available. FFP and its cryosupernatant fraction

be useful

the next ten to 24 who was studied

In contrast

large Techniques

Normal

when after

unusually

unusually plasma. between

large vWF of a third

at the time, immediately

FFP.

occasions.

of

chronic relapsing large vWF forms amounts, or had

remission obtained

multimers reappeared in the plasma of the

serially

transfusion

from their plasma. Unusually were also absent from the plasma

who blood

vWF hours

the

AL

A second possible explanation for our results, which cannot be excluded, is that the transfusion of FFP or cryosupernatant somehow accelerates the removal of

or by extend-

DISCUSSION

Within

ET

may

active in the

be an substance

large

vWF

substance that patient studied.

intrinsic in FFP

was This

property of the and its cryosu-

pernatant fraction. Alternatively, an autoantibody in the blood of the chronic relapsing TTP patient may have inactivated within a few hours the effective substance in transfused normal FFP/cryosupernatant, or interfered with its attachment to endothelial cell surfaces. Although this is speculation, it is compatible with clinical observations that normal FFP often must be infused

frequently

for days relapsing

(with

or without

plasmapheresis)

relapses It is also

in some compatible

in order to control TTP patients.2’4

reports that (glucorticoids,

agents with immunosuppressive vincnistine,’#{176}

beneficial effects with the chronic

in the relapsing

)

treatment of form of TTP.

chronic with

properties may have

some

patients

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