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Electron
Microscope
Observations
Relationship
in
By
T
HE
STUDY
platelets
dation
OF
iii
of the
that
with
in explainimig
have
forms
‘
have
‘
.
‘
.
agglutination
of
normal
with
thrombocytopenic
These
tion
of the
coagulum.
blood
Since
electron
leaving
platelets
the
microscope,
the
blood
that
imi the
absence
changes6
dependent
in
there
of platelets
homogeneous
addition
of normal
intact
the mechanism
wheim mai,mtained
However,
inactive
the
system.
on
1878,
the
Hayem
been
coaguumm
has
platelets
l)eefl
fruitless.
act
as livimig
(.)f particumbar
the
the
effect
Frommm
est ruct
time
kmra
Submitted
Work
the
Moreover
presemice
is,
coagumbumm
the
led
in
this
respect,
becomes
to
the
the
more
de
Investigaci#{243}n
, Uruguay.
September
s1,p)orte(l
1)3’
3,
It
dcc-
but
that
there
is
amid
amid have
platelets
by which
supposed
because
be obtained
iii
retrac-
observations
mmot retract.
will
mno
it is a
only
w-ith
the
which
will make
the system
heterogemieous.
of the platelets
is unknown.
Platelets
become
time at room temperature
and in the icebox
has
Montevideo
Imistituto
come
of a sufficient
x-rays,
shortwaves,
a principle
which
assummptiomi
that
shaking
will cause
in
clot
work
and
syner-
so
retraction
of Tocant.ins,’#{176} who,
has shown
that some platelets
become
attached
remainimig
as kmnots at the points
of intersection.
Celular,
plate-
they
entities.’0
immterest
ultramicroscope,
fibrin
needles,
this
This
play
later
of the
are of importamice
miumerous
does
retraction
for more than three
days or are treated
with
forth.
Fh efforts
to extract
from the platelets
esis
they
and
demonstrated
coagulation,
have
The
platelets
of actiomi
for some
mecha-
miormal
stream,
imi hemostasis.
ebuci-
exact
experimemital
data
which
support
the role of the platelets
in syneresis
established
that
clot retractiomi
is dependent
on the concentration
of
in the
blood.”
8, 9
There
is a considerable
discussion
regarding
the actual
mechamiism
platelets
may influence
the retractiomi
of the fibrimi network.
It has beetm
structurally
the
to study
the disintegrating
effect of thromplays
a role in the mechanism
of physiois knowmi of the actual
interaction
of the
which
leads to the retract.iomi
of the coagu-
undergoes
then,
in
Although
morphologic
Already
purpura,
the
when,
of the
platelets.
of blood
importance
known,
there are evidences
that
by agglutinatimig
at the woumid
of blood
clotting
amid retraction
assume
.
ORGANIZATION
is of
in hemostasis.
tron microscope
has also been applied4
bin umpoli the platelets
which
probably
logical
1)bOOd clotting.
However,
little
platelets
with the fibrin clot, a process
bum amid expumisiomi of the serum.
rfh
process
of syneresis
is apparently
number
Clotting
conditions
studied,
they
a surface.’
the
they
Platelet-Fibrin
ROBERTIs
is miot entirely
of hemostasis
imi the process
imivestigators
differemit
DE
physiologic
of platelets
first stages
functions
Blood
SUBMICROSCOPIC
different.
function
nism of action
a role imi the
have
varioums
coagumlum.
Several
lets iii the
in contact
THE
E.
of the
grant.
1954;
accepted
of the
rigid,
de
elastic
Ciencias
for
Rockefeller
Foundation.
528
amid retractile.
Biol#{243}gicas,
l)ublication
by using
to and orient.
Because
of
October
Depart.ameimt.o
12,
1954.
de
Ultra-
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
E.
DE
ROBERTIS
529
In view
of these
findings
it was of particular
interest
to analyze
with
tron microscope
the structure
of the platelets
and their
relationship
to
clot in different
stages
of blood
coagulation.
The observations
which
ported
here were made
with a wide variety
of materials
prepared
for
poses,
such as controls
for the effect
of thrombin
or platelets4
and for
of the action
experiments).
of different
types
of blood
plasma
on
the
platelets
the dccthe fibrimi
will be reother
purthe study
(unpublished
EXPERIMENTAL
The
technic
electron
for
obtaimiing
stretched
has
microscope
already
out
been
platelets
described.4
and
of their
The
slides
stumdy
umider
the
with
the
covered
parlodion
film were either
incubated
in heparinized
or citrated
blood
or iii the
plasma
containing
platelets.
In this case the blood was centrifuged
for 10 minutes
at 200 r.p.m.
and the incubation
time at 37 C. was 30 minutes.
Some
of the ohservations
reported
here were from experiments
in which
the blood
clottimig
had
started
spontaneously
in the
preparation
while
the
platelets
were
incubating
in
their
owmi plasma.
In other
cases the clotting
occurred
after
the platelets
were
washed
in Tyrode
fluid and treated
with normal
plasma
from amiother
person
or
withplasmafrom
thesplenic
veinof
a caseof
idiopathic
thrombocytopemiic
purpura
(ITP).
In these
cases,
to separate
the platelets
the plasma
had previoumsly
been
cemitrifuged
at 4000 r.p.m.
for 30 to 40 minutes.
Other
details
of the preparative
technic
amid electron
microscopy
have
already
been
described.4
RESULTS
The
viously
structure
described
neighborhood
tion
of
of the
fibrin
imi a system
2, 3, 4)
chaimis
thicker
of miormal
(fig. l).
human
clot
platelets
may
may
is essemitially
of fibrinogen
one
human
platelets
The early
stages
plus
the
see
be
similar
thrombimi.
progressive
prepared
of blood
followed
morphologically
of a case
behave
intact
On
supporting
the
orientation
and
apparently
act
by
these
sectors
other
normal
platelets
radiating
disposition.
Sometimes
radial
fibrimi
fibrils,
of the platelet
elect.ronmicrographs,
but
(figs.
from the center
the emnt.ire periphery
more
frequently
1 , 2 and 3). In
one may observe
Figure
5, in which
there
is a more
advanced
1 to
parlodio,n
treated
as
centers
do
are
only
do
platelets
froni
of blood
and to
plasma
early
clotting
platelets.
The
and
seem
attached
to have
to the
surface
edges in a
is surrounded
fan-
in certain
1) a,id imi many
w-hich have pre-
present.
field (fig.
platelets,
degree,
(figs.
of short
t.actoids
with
riot.
amid beyond
the
of the platelet
out
plasma
film
of orientation
ones
are
forma-
amid Hawmm”
see that.,
in the
from
damaged
they
stage
5. The
conglomeratiomi
the same
that
the
viously
undergone
disiitegration
to art advanced
ence of fibrin
fibrils
in their
immediate
contact.
Figimres
2, 3 and 4 correspond
to stretched
5lml)Iilit.ted
to
the action
of a normal
human
technic
w-as preoccumrring
in
the
by Porter
of needle-shaped
to
platelets
condensation
of fibrin
fibrils
while
the disintegrated
such
an effect.
In figures
1 to 4 it may be observed
that fine fibrils
of the
shaped
figures
described
of ITP,
one may
quite
differently
platelets
in
to that
of particles
leading
to the formation
fibrin
fibrils.
Imi figure
1 , corresponding
from
the splenic
vein
process,
intact
platelets
with this
clotting
not
show
which
another
coagulation,
the
pres-
have
been
individumal.
belongs
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530
PLATELET-FIBRIN
1 -Elect
FIG.
l)lasna
after
with
l)lasma
fibrils
in
ronInicrogra)h
fibrin
from
fibrils;
osnmiun
of
itict,bat.ion
for
from
time splenic
radiatimmg
with
RELATIONSHIP
tet
its
DP2,
roxide
nornal
litmmnami
31) ninutes
at
veimm of a case
st,rface
va)ors
for
platelets
CLOTTING
(\IR)
obtained
figures.
l)latelets
vith
showing
no
begililmilig
action
fibri,m
:
2 and
1
platelets
tache(l
time
fibrin
l)tlt
to
treated
also
.
Fixation
5,30()X
3.-Electronmicrograpim
‘ith
imornal
. .,
.;r,
of
Imunman
normal
plasmima
imi the
orientation
of the
clotting
is
time simrface
amid emmi in the iieighborimood
fibrils.
37 C.
fibrin
I
2
FIGS.
at
fine
clot
-u.
figure
citrated
mninimtes
simowimig
disintegration
the
011
from
60
C.
1)P1,
platelets
all
BLOOD
amm(l then
t reated
for
of ITP.
NP,
normal
platelets
37
(arrows).
(lisimitegrated
IN
.
human
(1.I).I1
visible.
of
I
.
platelets
.)
for
I
.--
(\LR)
60 miimutes.
The
radiating
the chrononere
7,300X
.01
..‘,
obtained
The
effect.
fibrimi
fibrils
(chr)
; with
as
of
in
the
are
at
arrows,
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
E.
FIG.
4.-l’lectronmicrograph
lrocess
su))ortimig
of
arrows;
(iir,
FIG.
clotting
film.
of
in
of a disintegrated
to a preparation
platelet
stretching.
chromomere
is
of
a l)rel)aratioml
platelet;
are
a
platelets
mammy :tcict,lar
omi the
more
of normal
Chr,
2 amid
as
tactoids
are I)eimig
fornmed
the fibrimi is nmarked
of
in
figures
treated
orientation
advanced
platelets.
chromomere.
3. The
on
the
vit.ii
stage
See
of
1)100(1
(lescril)tiomi
ill
clottilig
t
he
text..
occt,rring
H,
residue
6,500X.
in which
clotting
occumrred
spontaneously
(luring
the l)rOcess
of
A large
platelet.
showing
the
numerous
gramnmles
of the
crossed
in
all
direct.iomms
Most
of these
fibnils
reach
the
imimier part of its surface.
In these
platelets
imilact
and
l)latelet
531
IIOBERTIS
7,300X.
5.-Electronmicrograph
sl)ontalmeol,slv
three
is nore
advanced
The act.iolm of the
chromomere.
DE
morphobogically
platelet
advanced
intact,
hut
by long
submicroscopic
fibrin
fibrils.
with
a radial
orientation
and cross
the
stages
of normal
1)100(1 clotting,
many
a fev
of them
have
disintegrated
and
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532
PLATELET-FIBRIN
some
of the
fibni,i
clot
residual
(fig.
RELATIONSHIP
material
can
IN
be observed
BLOOD
CLOTTING
as demise
bodies
attached
to the
5).
DISCUSSION
The
complex
chemical
transformatiomis
ting are fimmalby expressed
studied
under
the electron
with
some
of the
the
close
was
described,
and
stages
of the
relatiomiship
but
conversion
existing
there
imivolved
imnt.o macromolecular
microscope.
The
between
were
on the nature
of this relatiomiship.
The results
reported
here suggest
that
as cemmt.ers
which,
taking
brumogen
occurs
at the
going
l)y
place
sumrface
of
while
imi the
With
some
other
unknown
platelets
platelets
may
the
undergo
early
orient
does
crossed
disint.egratiomi,
place
iii
‘
process
platelets
act
apparently
by
probably
of fi-
platelets
process,
directions
thrombin,4
and their
fragmemits
remain
attached
to the
The action
of the platelets
itt the retract.iomm
of the clot
“ . 13 So far
network7
polymerization
cbot.tiiig
all
clotcan be
mainly
of this
mechamnism
take
in
‘
fibrimi
imitact
This
not.
that
deals
stages
the
of a normal
become
of blood
fibrimi.”
morphologically
mediumm.
and
progress
may
amid the
mechaimism,
platelet.
the
imito
platelets
omi the
surrounding
of the
disintegrat.io,i.
surface
an
process
of fibrinogemi
the
observations
no
in the
and cellular
changes
information
available
by
under-
the
entire
fibrin
the
fibrils,
action
of
fibnimi reticulum.
amid syneresis
may
prob-
ably l)e explained
on the basis of these
observations
amid of the existing
kmiowledge
about
the mechanical
properties
of gels. It is well kmiowmi that ebomigated
mobeculan chains,
even
at low concentration,
have a marked
imiteraction.
The bomids involved
in those
to hydrogeim
properties
bonds.
interactions
bonds
of gels
If there
may
and van der
are dependent
is a continuous
of interaction,
a contraction
vary
Waal
on
in strength
forces.’4
The
the number
increase
of the
suit.
The fumnctiomi of the platelets
number
of int.eractimig
junctions.
.
in the
gel and
from covalemit
or salt. linkages
stability
and other
mechamiical
and varying
stremigth
of these
number
amid
expulsion
in clot
It seems
retraction
possible
strength
of the
of the
liqumid phase
is probably
that
at the
poimits
may
re-
to increase
the
sumrface of the in-
tact. platelets,
forces
of adhesiomi
develop
which
hold amid orient
the molecular
chains
of fibrimi amid that
the total
miumher
of bonds
increases
with the progress
imi the polymerization
of fibrin.
On the contrary,
platelets
which
are undergoing
disintegration
or have
suffered
an injury
which
alters
these
suirface
adhesive
properties,
tractiomm
is
do miot interact
impaired.
with
the
fibrimi
fibrils
and
the
mechanism
of clot.
re-
SUMMARY
The
l)ehavior
clotting
has
coagulation
of stretched
been
the
observed
morphobogically
out
with
humami
the
platelets
electromi
intact
in
different
microscope.
In
platelets
apparently
stages
the
early
act
as
of
blood
stages
of
centers
of
oriemmtat.iomm amid comidensatiomi
of the fibrin fibrils.
This effect is not found
in platelets which
have
previously
undergomie
extensive
damage
or disimitegrat.ion
In
more
advanced
stages
of clottimig,
many
platelets
remaimi
normal,
showing
their
attachment
to the fibrin,
while
others
have disintegrated
and appear
a residual
demise bodies.
The probable
action
of platelets
in the retraction
amid syneresis
of the blood
.
clot
is discumssed.
It
is suggested
that
at
the
sumrface
of the
morphologically
intact
I
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E.
platelets,
adhesiomi
forces
fibrin
increasing
On the contrary,
the
the
surface
adhesive
the mechanism
DE
develop
number
platelets
BOBERTIS
which
Esseva
observate
per
medio
de fibrimia.
suffrite
mente
Ist.e
extense
coagulation,
mult.e
IN
del
in
amid orient
with
the
niicroscopio
fibrin
chains
fibrils
of
syneresis.
alters
these
and,
as a result,
electronic
le activitate
de exten-
del coagulation
del sanguine.
que es morphologicaniemite
de orientation
e condensation
effecto
mion se manifesta
quando
dammios
o disintegratiomi.
In
remniane
molecular
and producimig
imijury which
INTERLINGUA
vane
stadios
be plachettas,
como
centros
plachett.as
the
junctions
suffered
an
properties
do riot interact
of clot retraction
is impaired.
sionate
plachet.t.as
humami
prime
stadios
del coagulation
imitacte,
age apparentement.e
fibnilbas
hold
of interacting
which
have
SUMMAIu0
533
normal
le plachettas
plus
ha previe-
avantiate
e demonst.ra
br
In le
ancora
del
stadios
del
attachaniento
al
fibrina.
Alteres,
,ionobstamite,
es disintegrate
e appare
como
dense
corpores
residimab.
Es discutite
le probabibe
action
del plachettas
in
be retract.iomn
e synerese
del
coagubo
sanguimiee.
Xos postula
que al superficie
del plachettas
niorphobogicamente
imitacte,
umi fort.ia
de adhesiomi
es disveboppate
que retemie
e orienta
le
eatenas
molecular
del fibrimna e qUe augnnent.a
le numero
del junctiomies
interactive
assi promove
le processo
syneretic.
Del abt.ere batere,
Ic plachettas
que ha suifrite
besiomies
capace
a impedir
ible fortia
de adhesiomi
superficial,
non establi
ulle
interaction
(‘011
be fibriblas
de fibnina,
e comisequemitememite
be mnechanismo
del
C
retract
ion
del
coagumbo
es distu,
rhate.
REFERENCES
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C.
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and
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1955 10: 528-533
Electron Microscope Observations of the Platelet-Fibrin Relationship in
Blood Clotting
E. DE ROBERTIS
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