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.