THROMBOSIS RESEARCH Printed in the United

COMMUNICATION

BRIEF PLATELET

O.PONARI,

FUNCTION

E.CIVARDI,

1975 vol. 6, pp. 443-450, Pergamon Press, Inc.

States

FOLLOWING

A.MEGHA,M.PINI,

VENOUS OCCLUSION

R.POTI'

and A.G.DETTORI

Division, Centre for Haemostatic Diseases. Riuniti di Parma . Parma, Italy .

5th Medical

IN MAN.

.

Ospedali

(Received 5.8.19'74; in revised form 19.12.19’74. Accepted by Editor I.M. Nitsson. Received by Executive Editorial Office 17.3.1975) ABSTRACT

-

The possibility that platelets interfere in changes evoked by venous stasis in the hemostatic system of local blood was investigated in man with the tourniquet test. A significant rise in PF4 (anti-heparin) activity of PPP and an inconstant increase in PF3 availability were the only changes seen in samples after stasis , while platelet number, adhesiveness, ADP-aggregation, and platelet fibrinolytic components ( antiplasmin and proactivator activities) remained unchanged .

A transient been reported

hyperfibrinolytic

in local blood during

by tourniquet

test

The possibility platelets

venous occlusion

that quantitative

has received

work has been limited results

to be unchanged

increase

obtained

in man

changes

in

in the chain of events elicited

by

Platelet

a reduction;

in platelet

research and has led

number

been found

some

behaviour 443

Previous

count and adhesiveness

adhesiveness,

(5,2) and peculiar

functional

little attention.

to platelet .

and/or

after venous occlusion

et al. (6) reported variations

venous stasis

state has

(1,3,15-19).

can also play a r81e

to equivocal

and hypercoagulable

has generally

(2-5) whereas authors

others

was

(3,6)

found a

only HLADOVEC observed

no

Significant

seen by NERI SERNERI et

PLATELET

444

al,

with an in vivo A detailed

method

analysis

after venous stasis

AND VENOUS

fasting

of the functional

value between

were carried

systolic

citrate

out on ten subjects,

(7)

level, unless (300,000 per platelet

otherwise ~1)

aggregation

in centimeters

of

recorder

platelet

kaolin)

of

of the

mixture

to BRECHER

(PRP) with

. with the method

of the

out with the method Milano,

induced by ADP (Biochemia,

Italy). was

to a Philips

crude O.D. changes

according

of

Milano)

(Mod. 169) connected time,

value

after a few days in

kit, &fascia e Brunelli,

. Reaction

The platelet

to a standard

rich plasma

was carried

final concentration

to O'BRIEN

of ADP (1.25&g/ml)

and

et al. (10) and given

.

factor

SPAET and

according

of tests were performed.

slope of the curve were calculated standard

count

to glass was measured

by an EEL Aggregemeter

Platelet

a

at an intermediate

the completion

stated, was adjusted

and adhesiveness

direct writing

using

applying

of blood + 1 volume

before

stasis was repeated

(9) ("Adeplat"

followed

by

. Two venous blood

(PPP) from the same sample

adhesiveness

same subjects

Platelet

(9 volumes

a platelet

by diluting

I (8). Venous

SALZMAN

pressure

immediately

and a series

poor plasma

Platelet

obtained

.

On these blood samples and CRONKITE

was

of both sexes,

from the same arm, the first under basal

and the second

stasis period

study.

METHODS

and diastolic

on

3.8% sodium citrate)

HELLEM

AND

of platelets

in the present

cuff to the arm for ten minutes

samples were taken

conditions

behaviour

in man has been performed

and at rest. Venous stasis

sphygmomanometer

Vo1.6,No.5

(4).

MATERIAfS The experiments

OCCLUSION

3 (PF3) availability

CINTRON

(II). Sub-samples

was determined

by the method

of the incubation

were made after the first and every five minutes

clotted with Russell

viper venom + calcium

(Russell's

viper

+

(PRP

and

venom

PLATELET

Vo1.6,No.5

Reagent,

Warner

In vivo heparin

release

poor

extract 1 %

plasma

minutes

at 37"

solution

(Warner

For

the

activator times

assays

per

ml

was

0.1

ml

0.1

20 u/ml);

Lambert,

(Topostasin, after

(0.1

ml

Roche,

clotting

ml),

then

solution Roche;

assayed (Warner

10 NIH

u/ml)

and

was

solution

as test

Plain,

then

incubated

Lederle,

lytic

clotted

in 0.025

Wayne,

activity

M calcium

0.1

ml

U.S.A.;

solution

time was

test

5,000

of thrombin

recorddetermin

material with u/ml,

table

are

reported

1; statistical

as mean

analysis

values

was

(+ standard

performed

with

(Topostasin,

.

deviation) the

0.1

% fibrinogen

RESULTS Results

:

( Fibrinogen

of a 0.6

chloride

(13)

minutes

minutes

U.S.A.); ml

three

Detroit,

lysis

(14):

for ten

on 0.3

with

et al.

activity

GROSS

pro-

Antiplasmin

of thrombin

and

.

(100,000

solution

added

and

washed

C for ten

ml

,

and thawing;

Parke-Davis,

0.1

five

Roche

and

medium

proactivator

HOLEMANS

of

recorded

freezing

at 37"

and

U.S.A.),

were

of MORIAU

fibrinogen

were

was

to

method

U.S.A.)

of a

solution

time

material.

(."Elase",

% bovine

from

times

incubated

0.2

(antiplasmin

same

the

to

incubation

clotting

in the

used

or platelet

(Topostasin,

components

five

using

of heparin after

u/ml);

added

following

Lambert)

ml

as anti-

PLains,

resuspended

extract)

for

added

PRP

were

( Varidase,

was

solution

. The plasminogen

derived

plasma

titrated

100 u/ml)

of platelet

streptokinase

was

subjected

a 0.3

Morris

a method ml

of

1

rich

, Morris

0.1

measured (12)

from

extract

of plasmin

of PPP

and

;

platelets

measured

DEUTSCH

Lambert

fibrinolptic

extracts

from

. was

4 (PF4)

of thrombin

u/ml)

and

of platelet

Warner

ml

saline,

fll),

platelet

activity

ed by

of

in buqfered

these

with

0.1

activities)

elements

0.1

C

ml

plasma,

Switzerland

; 20 NIH

Switzerland

0.1

445

U.S.A.)

of platelet

.

Roche,

Plains,

derived

instead

bovine

OCCLUSION

factor

material

of titrated

(Liquemin,

, Morris

in a system

as test

ml

VENOUS

of platelet

fibrinogen

0.2

ed

Lambert

activity

platelet

AND

Student's

in

446

“tl’

PLATELET'AND

test

vo1.6,No.5

OCCLUSION

.

Platelet adhesiveness stasis

VENOUS

count

(corrected

for haematocrit

(with both methods)

. The parameters

showed no significant

of platelet

variations

TABLE Variations

remained

in Platelet

variations)

unchanged

ADP-induced

and

after venous

aggregation

also

.

1

Function Tests Induced Stasis .

PLATELET COUNT (x103/+

in Man by Venous

BASAL VALUES (Mean 2S.D.)

AFTER STASIS

285291

286+86

n.s.

P

(Mean +S.D.)

ADHESIVENESS (% retention) - Hellem I Method - Salzman I1 ADP INDUCED AGGREGATION (cm) - Reaction Time

- Crude O.D.Changes - Slope

PF3 VAILABILITY (substrate Incubation time 1' 5' 10'

15" 20' 25' PF4 In Vivo

RELEASE

27.6426.25 38.3Oi8.08

n.s. n.s.

6.00 2 2.02 4.47 + 1.35 6.99 + 3.13

7.21 f 2.13 4.99 + 1.84 6.00 i 2.85

n*s. n.s.

times

in

64.90i23.22 46.80k14.86 33.2e11.24 25.90+ 9.18 23.40+ 8.30 23.502 8.30

n.s.

SeC)

56.50218.27 40.90+11.52 29.10+ 8.50 24.502 8.95 23.502 8.75 23.50+ 8.75

nasa n.6.

n@s. nos. noso n.6.

(substrate coagulation times in set) 68.10+13.90 52.10+12.01 43.02

Heparin, 1

U./ml

ANTIPLASMIN

(Lysis t.,min)

PROACTIVATOR

COagdatiOn

24.3625.70 36.6OLll.07

(Lysis t.,min)

1.33

8.05k 1.06

noso

10.75? 2.91

10.82+ 3.10

nDso

7.99+

PLATELET

An

increase

taken

after

in the

venous

particularly

minute),

while

because

outstanding

samples

basal

range

were

after

to

disappear

with

in single

much

venous

stasis

towards

in the

in the

with

to

the

5th end.

analysis

group.Nevertheless

.

cases

greater

in these

(1st

statistical

of values

demonstrated

was

obtained

in vivo

in platelet respect

to

release

poor

of

plasma

samples

in

. were

differences

components,

tended

were

in samples

of incubation

stages

observed

activity

taken

conditions

No

were

demonstrated

times

significant

wide

variations

: 'anti-heparin

from

not

of the

changes

Significant PF4

difference

were

was

: shorter first

447

OCCLUSION

availability

in the

this

differences

probably

PF3

VENOUS

occlusion

samples,

Mean

AND

i.e.

observed

antiplasmin

in the

and

platelet

fibrinolytic

.

proactivator

DISCUSSION From only

reported

marginally

homeostasis

were number

observations

with

to

greater

the

venous studies

more

stasis . In

it would changes

appear

induced

that

by

seen

in only

was

some

The

finding

sequestration PF3

by

availability,

general

trend

. This

has

fact,

venous

evidenced

causes

changes

indices,

shortening

of activated

cephalin

time

(15)

especially

an evident

activity

(1,3,15,16).

The

only

were

those

poor

plasma

changes

which

reached

regarding

PF4

activity,

obtained

after

stasis.

was

inconstant,

elastographic

and

with

hypercoagulability

previously

occlusion

(6)

a level

PTT rise

a constant

This

finding

most

supported.

fits

in well

found

after

and

in thrombo (1) or in Factor

rise

suggests

previour

number

not

in our

of statistical

with

blood.

while

in platelet

al.

although

towards

been

parameters,

of a reduction et

on the

in local

is in accordance

HLADOVEC

participate

occlusion

system

functional

. This

unchanged

platelets

venous

coagulation/fibrinolysis

(2-5).

attributed The

in the

of the

Variations platelet

results

of

other plasma

VIII

significance in platelet that

some

448

PLATELET

platelets during was

in local

the

period

proposed

activity"

by

In any reaction number

(20)

was,

Finally,

system, since

ADP

after

our

fibrinolytic

wish

extracts

to

the

components showed

undergoing

be

great

the

since

substantially

release

platelet

. Moreover

unmodified

support-

activation after

the

both

unchanged

technical

hypothesis

of the

venous

(antiplasmin

no variations

the

intravascular

(21).

not

were

not

demonstrated

aknowledge

could

"PF4-like

in

.

did

with

induced

reaction

interpretation

of plasma

of platelets

aggregation

findings

was

rise

pathology

mentioned,

stasis

interfere which

platelet

We

already

and

taken

stasis

release

. A similar

for the

percentage

v01.6,~0.5

an in vivo

experimentally

in human

venous

as

adhesiveness

platelets

and

OCCLUSION

occlusion

et al.

with

the

case,

underwent

of venous

FUSTER

during

samples

blood

in dogs

coagulation

AND VENOUS

plasma

stasis and

that fibrinolytic ,

(1,3,17-19)

proactivator)

in

.

assistance

of

Mr V.

CUPOLA

.

REFERENCES 1. PONARI,O.,CIVARDI,E.,MEGHA,A.,PINI,M.,POTI1,R. drugs Effect of alpha- and beta-blocking fibrinolytic 2,463,1973 26 SARAJAS,H.S.S. adhesiveness.

response

to

venous

stasis

and DETTOR1,A.G.: on the clotting and

in man.

Brit.J.Haematol.:

e Stasis-induced and MYLLYd,G.: Experientia : 2l,223,1965 .

3. NILSSON I.M. and ROBERTSON,B. : Effect coagulation and fibrinolytic components Thrombos.Diathes.Haemorrh.: 20,397,1968

changes

of venous in normal .

in platelet

occlusion subjects

on .

4. NERI SERNERI,G.G.,MASOTTI,G.,SILVBSTRINI,E. and PAOLETTI,P.: Ricerche sui meccanismi patogenetici delle emorragie da stasi venosa provocata.IX.Alterazione dell'adesivit8 piastrinica in vivo dopo stasi venosa nei soggetti con prova de1 laccio positiua. Riv.Clin.Med.: 3,226,1969 .

PLATELET

Vo1.6,No.5

AND VENOUS

5. HOLZKNECHT,F.,SPGTTL,

F., CONSTANTINI,R., and BRAUNSTEINER,H. : Platelet adhesion occlusion. Atherosclerosis : ll,105,1970

6. HLADOVEC,J.,KOLEILAT,Z. occlusion on the Diathes.Haemorrh.:

7. BRECHER,G. human

blood

449

OCCLUSION

and PREROVSKY,Io sequestration of blood

KNAPP,E., before and .

: The effect of venous platelets. Thrombos.

~8,383,1972 o

and CRONKITE,E.P. : Morphology and enumeration platelets. J.Appl.Physiol.: Q,365,1950.

: The adhesiveness of human blood Scand.J.Clin.Lab.Invest.: l2,supp1,51,1,1960.

8. HELLEM,A,J.

9. SALZMAN,E.W. Med.:

HERBST,M. after venous

: Measurement

@,724,1963

of platelet

platelets

adhesiveness.

of

in vitro.

J.Lab.Clin.

.

10. O'BRIEN,J.Ro,HEYWOOD,J~B~ platelet relation

and HEADY,J.A.: The quantitation of : a study in aggregatinn induced by four compounds to myocardial infarction. Thrombos.Diathes.Haemorrh.:

&752,1966. 11.

SPAET,T;H. and CINTRON,J.: Studies Brit.J.Haematol.: l&269,1965.

Eine l.2. DEUTSCH,E.: Thrombozytenfaktor

einfache

Methode

on platelet

zur

Bestimmung

factor-3

availability

von

4.Thrombos.Diathes.Haemorrh0:4,93,1960.

Les inhibiteurs de la VRIES,S.I. and DIK,H.J.: fibrinolyse et le dosage de l'activite antiplasminique physiologique et pathologique. Thrombos. Diathes. Haemorrh.:

13. MORIAU,M.,de

x,335,1963.

14. HOLEMANS,R. fibrinolysis.

15. EGEBERG,O. clotting

Influence of blood platelets on and GROSS,R.: Thrombos.Diathes.Haemorrh.: &196,1961 o

: The system.

effect of venous congestion on the Scand.J.Clin.Lab.Invest.: l&20,1963

16. BIZZI,B.

and LEONE,G.: Stasi coll'incremento dell'attivith

blood o

venosa e fattore VIII.Rapporti fibrinolitica . Haematologica

:

s,295,1970.

17. AMERY,A.,

VERMYLEN,J.,MAES,H. and VERSTRAETE,M.: Enhancing the fibrinolytic activity in human blood by occlusion of blood vessels. I.The appearance of the phenomenon. Thrombos. Diathes. _ _~

Haemorrh.

:2,70,1962

.

450

PLATELET

AND VENOUS

OCCLUSION

18. HOLEMANS,R. : Increase in fibrinolytic occlusion. J.Appl.Physiol. :g,ll23,1963. 19.

activity

v01.6,~0.5

by venous

IATRIDIS,S.G.,IATRIDIS,P.G. and FERGUSON,J.H.: The role of HF (Factor XII) in the pathogenesis of the thrombolytic state induced by venous occlusion. Thrombos.Diathes.Haemorrh.:l6, 207,1966 .

20. FUSTER,V.,BOWIE,E.J.W.,KAZMIER,F.J. and OWEN,C.A.,Jr.: Platelet consumption in chronically induced plasma platelet factor &like activity reflecting rate of intravascular coagulation in dogs. Thrombos.Res.:&,247,1974 . 21. FUSTER,V.,CASH,J.D.,OWEN,C.A.,Jr.,KAZMIER,F.J. and BOWIE,E.J.W.: Plasma platelet factor-4 in diagnosis of intravascular coagulation (Abstr.). 3rd Congr.Internat.Soc.Thrombosis and Haemostasis. Washington, DC, 1972,p.41.

Platelet function following venous occlusion in man.

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