Effect of exercise on F VIII-complex: Proportional increase of ristocetin cofactor (von Willebrand factor) and F VIII-AGN, but disproportional increase of F VIII-AHF

Vol. 10, pp. 163-168, 1977 Pergamon Press

THROMBOSIS RESEARCH

BRIEF

EFFECT OF EXERCISE

COMMUNICATION

ON F VIII-COMPLEX:

PROPORTIONAL

INCREASE OF RISTCCETIN

COFACTOR (VON WI LLEBRAND FACTOR 1 AND F- V I I t -AGN, DISPROPORTIONAL

Division of University

Haemostasis Hospital

BUT

INCREASE OF F VIII-AHF.

Jeanne St i bbe and Thrombosis Research, Department of Hematoloov, Rotterdam-Dykzigt, Rotterdam, The Netherlands.

(Received 8.9.1976; in revised form 19.11.1976. Accepted by Editor K.M. Brinkhous)

INTRODUCTION -__-Both Factor VIII procoagulant activity (F VIII-AHF) (1) and Factor Vlllrelated antigen (F VI I I-AGN) (Z-4) are known to increase after exercise. We showed that in platelet rich plasma (PRP) obtained from healthy volunteers after exercise, platelet aggregation could be induced with lower ristocetin concentrations than in PRP from the same individuals obtained before exercise (5). The results were interpreted as an increase of the ristocetin cofactor. We subsequently showed that plasma proteins such as fibrinogen and albumin bind ristocetin, which is then not available for the interaction between the ristocetin cofactor and platelets (6). The effect of exercise on ristocetin-induced platelet aggreqation in PRP, therefore, might reflect either an increase in ristocet’in cofactor or a decrease In plasma proteins that bind ristocetin, or both. We report here that in 10 healthy volunteers and in one patient with von Willebrand’s disease the rlstocetin cofactor increased after exercise. The ristocetin cofactor was assayed quantitatively using formalin-fixed platelets which react specifically with ristocetin and the ristocetin cofactor or bovine Factor VIII without secondary release and aggregation (7) as has been pointed out first for paraformaldehyde-fixed platelets (8). accounts for the effects of plasma proteins on ristocetinThe assay used, induced agglutination (6). In these 10 healthy volunteers F VIII-AGN increased to the same extent the ristocetin cofactor; F VIII-AHF, however, increased significantly The patient with von Willehrand’s disease also showed a disproportional and high increase of F VI I I-AHF after exercise.

MATERIALS -__l--From

10 healthy

volunteers

AND METHODS

(6 males

and

143

4 females),

ranginp

in

age

as more.

164

EFFECT:EXERCISE ON vWF & F.VIII

Vol.l.0,No.l

from 19 - 38 years (mean 30 years), and 1 male patient, 23 years old, with von Willebrand’s disease, blood was collected Immediately before and 5 - 10 min. after exercise. The exercise comprised of running up and down a staircase for 3 min. as fast as possible. Two females exercised for only 2 min. because they were exhausted.

of

Blood (5 ml) 0.55 M sodium

was collected citrate.

into

polystyrene

tubes

containing

0.1

ml

Platelet-poor plasma (PPP) was prepared by centrifuging the blood for 10 min. at 1,000 x g at room temperature and subsequent centrifugatlon of the sugernatant plasma for 20 min. at 20,000 x g at 4’C. The PPP was stored at -70 C and used within 2 weeks for assay of ristocetln cofactor, F VIII-AGN, F VIII-AHF and fibrinogen. The samples were thawed only once and were then tested immediately. Pooled normal PPP, used as standard in the assays of ristocetin cofactor, F VI I I-AGN and F VI I I-AHF, was part of the pool used routinely for these assays in our laboratory, The pool is obtained from the blood. from 30 healthy male volunteers and stored at -7O’C for several months. Each newly prepared pool is tested against the former one. Following this procedure over the years, it is our experience that F VIII-AHF and F VIII-AGN are stable during the storage period. Although our experience with the assay of the ristocetin cofactor is relatively short, the impression is that no important changes in the concentration of the ri stoceti n cofactor occurs during storage at -7O’C for several During the experimental period two different batches of pooled months. normal PPP were used. For all plasma samples obtained from one Individual one and the same batch was used as standard for all three assays. Tris-saline buffer was Tris-saline-albumin buffer (Sigma them. St. Louis).

0.01 M Tris, 0.15 M NaCl, pti 7.4 contained 40 mg/ml of bovine serum

albumin

Assay of ristocetin cofactor was performed according to Macfarlane (9) as modified by Stibbe and Kirby (6) : 0.35 ml of prewarmed et al. formalin-treated washed norm81 platelets suspended in tris-saline buffer (final platelet count 3 x 10 /ml) were incubated for 1 min. with 20 ul rlstocetln (final concentration 1.0 mg/ml). Agglutination was then induced by 0.05 ml PPP or dilutions of PPP In tris-saline-albumin buffer. Agglutination was performed at 37’C in a Payton aggregometer, with a stirring speed of 1,100 RPM. The ristocetin concentrations in this assay is chosen such that it is high enough to overcome inhibitory binding of ristocetin to plasma protelns (fibrinogen, albumin) and low enough to In no instance precipitate formation prevent fibrinogen precipitation. Two samples with both high total serum proteins (89 and 76 g/l) occurred. and high albumin concentrations (64 and 63 g/l) were tested on inhibiting binding of ristocetln by performing the assay with higher ristocetin concentrations,(l.Z and 1.4 mg/ml).As this did not result in a higher agglutination response, inhibitory binding could be excluded in these samples (6) and thus in all samples assayed as they had lower protein concentrations. Rlstocetin (H. Lundbeck and Co., Copenhagen, batch 7) was dissolved in-saline-buffer and stored at -26’C. F VIII-AGN the Laurel1 human Factor F VIII-AHF

was measured technique VIII.

in

PPP by electroimmuno

(10)

using

rabbit

was assaved

usinn

a one

antiserum

stage

assay

according

(Behringwerke)

procedure

according

to against

to

EFFECT:EXERCISEON vWF & F.VIII

Vol.10,No.l

Veltkamp

et

al.

(11)

Fibrinoqen

as modified

was measured

by Veltkamp

according

to

and

Clauss

165

Van Tilburg

(12).

(13).

Total serum protein was measured by the bluret method according to the -_.-Albumln and olobullns were measured by WA-12-60 procedure of Technicon. veronal buffer pH 8.6) stained with electrophoresis (cellulose acetate, Ponceau S and measured by densitometry (Dr. E.G. Blljenberg, Central University Hospital Rotterdam-Dykzigt). Clinical Chemical Laboratory, The

bleeding

Platelets

time were

was measured

counted

according

according

to

to

Brecher

Ivy

et

al.

and

Cronkite

(14). (15).

RlSlOCETlN

FIG.

Effect of exercise on 1n r i stncet cofactor. F VIII-AGN and F VI I I-AHF in 10 healthy volunteers. The thick I ine I n each‘ col umn 1nd icates the mean.

2.L

2.0_

IS_

i.

-

I .Q.

:B 0.L

1

/

RESULTS AND DISCUSSION

A.

Normal

1.

Increase The

lndivlduals. of

ristocetln

rlstocetln cofactor

cofactor increased

after

exercise, in

all

volunteers

(fig.

1).

The change In flbrlnogen concentration after exercise was neglible. The changes in concentrations of total serum protein and albumin were variable, showing both mild increases and mild decreases after exercise. As described under methods, these changes do not influence the rlstocetin cofactor assay. Only small changes in serum globulins occurred. 2.

Disproportional increase of F VIII-AHF. Flq. 1 shows that F VIII-AHF increases considerablv more after exercise than both the rlstocetln cofactor and F VIII-AGN. Thls’increase of F VlllAHF Is slgnlficantly more than both the rlstocetin cofactor and F VIII-AGN, whether the increase is expressed as U/ml (fig. 11, (p
166

EFFECT:EXERCISEON vWF & F.VIII

as % of

the

F VIII-AGN difference significant.

value before exercise (54, and F VIII-AHF respectively, in increase between rlstocetln

Vol.10,No.l

46 and 126% for rlstocetin ~~0.05, Newman-Keuls test), cofactor and F VIII-AGN

cofactor, The is not

The finding that F VIII-AHF rises more than F VIII-AGN in normal individuals after exercise Is not in agreement with former results in which proportional increases are reported (2-4). Bennett and Ratnoff (2) and Denson (3) measured the F VIII-AGN,in an ethanol precipitate of plasma and not in whole plasma. Their results are therefore not comparable to those reported here. Also, the exercising used by Bennett and Ratnoff (2) seems to be milder than we used: the mean increase in F VIII-AHF in their study in 10 volunteers was 0.28 U/ml as compared to 1.04 U/ml In our study. Prentice et al. (4) found In 5 healthy volunteers who ran a distance of half a mile as fast as possible,that F VIII-AHF and F VIII-AGN rose to approximately the same extent. The F VIII-AGN was measured In whole plasma, The difference between the results of Prentice et al. (4) and ours may represent individual differences or differences in methodology. In a number of carriers of hemophilia-A the plasma concentration of F VIII-AHF is lower than that of F VIII-AGN, although the F VIII-AHF may be well within the normal range, This discrepancy is used for the detection of these carriers. In view of the available reports on the combined effect of exercise on F VIII-AHF and F VIII-AGN (Z-4, this paper) a re-evaluation of the effect of mild exercise on these two parameters seems to be indicated. A carrier may escape detection if a disproportional increase of F VIII-AHF occurs. The female using the

volunteers pill or not.

In

this

study

were

The results represented here indicate in normal individuals, F VIII-AHF can AGN and the ristoceti n cofactor. B.

Von Wi I lebrand’s A decrease of

disease. bleeding

time

and

Effect

of -_-_I Exercise

platelet coqt (x 10 /I)

in

von

that vary

an

TABLE

not

asked

whether

under certain independently

increase

of

were

conditions of F Vlll-

F VIII-AHF

after

I Willebrand’ s Disease -_-_____* ristocetin cofactor U/ml

bleeding time

they

F VlllAHF U/ml

F VlllAGN

before

315

12 min

co.05

no neak

0.14

after

417

5 min

0.10

no peak

0.98

exercise on the

has been ristocetin

described cofactor

in and

von Willebrand’s F VIII-AGN is

disease unknown.

(161,

but

the

effect

Table I shows the effect of exercise on the bleeding time and the Factor VIII complex in one patient with a classical von Willebrand’s disease. The bleed/q time decreased with a concomitant small, but clearly distinguishable increase of the ristocetin cofactor. Whether this small

increase time is,

in at

I67

EFF'ECT:EXERCISE ON vWF & F.VIII

Vol.10,No.l

the

decrease

the

F VIII-AGN

assay.

A similar instantaneous Increase in F VIII-AHF without in F VI I I-AGN and ristocetin cofactor has been described the infusion of eplnephrine in von Willebrand patients

comparable recently (17).

increase after

Both before F VIII-AHF

ristocetin cofactor least, debatable. and after exercise was increased 7-fold

It is conceivable exercise in both mechanism.

is

responsible

for

no peak was seen after exercise.

that the disproportional normals and von Wlllebrand’s

In

in

bleeding

increase of F VIII-AHF after disease has a common

ACKNOWLEDGEMENT __The expert technical 1s acknow I edged. Cassa dinqen, for statistical qift from the Red Cross

assistance of Ria de Deugd, Hans van Daele and Els I thank Jacobus Zaalberg, Unilever Research, Vlaaranalysis of the results. Human platelets were a Rlood Bank (Head Dr.F.C.H.A. tithe), Rotterdam.

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3.

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9.

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zur

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human

of