From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
Human
Thrombocytopenia
Endothelium
Is Associated
That
Are
With
Ameliorated
By Craig
breaks.
may
Platelets
have
a role
S. Kitchens
A
DEQUATE
NUMBERS
necessary
thelial
for
breaks
normal
in the event
of
functional
hemostasis.
of rupture.
in
supplying
a mean
Claiming
seal
that
are
endolack
of
this sealing
function
alone
does not account
for capillary
fragility
seen
in severe
thrombocytopenia,
it has
been
hypothesized
that
platelets
additionally,
in some
manner,
may play a role in maintaining
endothelial
integrity.”2
This
laboratory
has published
morphological
thrombocytopenia
in rabbits
supporting
have additionally
demonstrated
that
data in experimental
this hypothesis.3
We
endothelial
structural
alterations
in this experimental
model
were
the
administration
of glucocorticosterioids.4
reported
observations
made on tissue obtained
with
severe
ameliorated
by
Herein
are
from humans
thrombocytopenia.
MATERIALS
AND
METHODS
Strict criteria
for patient
accession
accounts
for the
few patients
accrued
over a five-year
period. Patients
had to have
severe
thrombocytopenia
(15,000/L)
not due to a process
Patients.
(chemotherapy
or radiation
therapy)
that
conceivably
could
directly
damage
a variety of tissues,
including
endothelium.
The platelet
count had to remain
15,000/ML
after four days of daily administration of prednisone,
I mg/kg,
in order to minimize
endothelial
structural
changes
related
primarily
to resolution
of thrombocytope-
nia. Before their entry into the study, they could not have received
glucocorticosteroids.
Because of our strong feelings that glucocorticosteroids
are effective
for hemorrhage
associated
with severe
thrombocytopenia,
we could not ethically
accept transfer
of thrombocytopenic
patients
from other cities without
first recommending
the administration
of such agents. This greatly restricted
the number
of patients
who were available
to serve in this study comparing
determinations
made before and during prednisone
administration.
Finally, patients had to agree to serve in the experiment,
all parts of
which were approved by the Internal Review Board of the University
of Florida.
Tissue.
As rapidly as possible after initial evaluation
and understanding
of
the
protocol,
biopsies
were
performed
after
which
prednisone,
1 mg/kg/d,
was begun.
Biopsy material
was also
obtained on the fourth day ofsuch treatment
ifthe platelet count was
still
15,000/ML.
If the platelet
count was
15,000/L,
the original
biopsy material
was not further
processed.
Biopsy material
was
obtained
from skin and muscle. Skin biopsies were taken from the leg
in areas near fresh petechiae
but not of petechiae
themselves.
This
area was chosen to ensure that capillary
fragility
was present.
Our
previous
animal studies demonstrated
that capillary
thinning
was
not confined
to actively
hemorrhaging
areas, being just as pronounced
in normal-appearing
tissue.34 An elliptical
incision 6 x 2
Blood,
Vol 67, No
1 (January),
1986:
pp 203-206
Administration
areas.
including
toward
some
normal
at a time
equally
severe.
fenestrations.
after
the
These
findings
days
degree
findings
All
of prednisone
of thrombocytopenia
four
are
hypothesis
that platelets
are necessary
ture and function
of endothelial
cells
costeroid
administration
may ameliorate
ogy of thrombocytopenia.
© 1986
by Grune
& Stratton.
Inc.
consistent
for
and
with
normal
the
struc-
that glucocortithe pathophysiol-
very
platelets
Platelets
was
of the
Pendergast
administration
findings
frequent
J.F.
reverted
has
demonexperimental
have
and
thinned
in maintaining
endothelial
hemeostasis.
This
laboratory
strated
thinnning
of capillary
endothelium
in
thrombocytopenia.
We
now
report
similar
human
thrombocytopenia.
Capillary
endothelium
either
skin or skeletal
muscle
was
found
to
thickness
only half that of normal
as well as
Abnormalities
by Glucocorticosteriod
Capillary
fragility
is characteristic
of severe
thrombocytopenia.
This
mechanical
weakness
may
not
be solely
accounted
for by decreased
ability
of platelets
to repair
endothelial
Structural
mm was made with a surgical scalpel deep enough to reach subcutaneous tissue. The wound was closed with three sutures, and pressure
dressings
were applied. Tissue from the gluteal muscle was obtained
using a Lee soft-tissue
biopsy needle (Becton
Dickinson,
Atlanta),
which was placed roughly
along the axis of the indicated
bone
marrow
aspirate
and biopsy tract. Biopsy yielded
muscle in both
pretreatment
and posttreatment
attempts
in only three patients;
therefore,
muscle data are complete
for only three patients.
Data
regarding
skin biopsy is complete
for all five patients.
Tissues were
then immediately
diced in cold 3% glutaraldehyde
in phosphate
buffer and postfixed
in 0504.
It was then embedded,
sectioned,
and stained
with lead citrate
and uranyl
acetate
as previously
described.3
Electron
microscopy.
All material
was examined
using a Philips
300 electron
microscope
(Eindhoven,
The Netherlands).
All photomicrographs
were made at a fixed magnification
(50,000)
without
knowledge
of patient or status of prednisone
administration.
It was
impossible
to be unaware
of whether
the biopsy source was skin or
muscle. Several
blocks from each patient
sample
were examined
until approximately
100 capillaries
from each biopsy sample
had
been
photographed.
All
vessels
regardless
of photogenicity,
which vessels to photograph.
Collection
ofdata.
encountered
in order
Before
were
to minimize
breaking
the
photographed,
bias in choosing
patient
)
and
preparation
code, each photomicrograph
was examined
for ( 1 “thin spots,” (2)
fenestrations,
and (3) mean thickness
of the capillary
endothelium.
Figure 1 demonstrates
these terms. A thin spot is defined as an area
in which the endothelium
attenuates
to a thickness
ofonly 700 to 800
A, the thickness
of an endothelial
vesicle. This degree of thickness
is
admittedly
arbitrarily
selected
but being markedly
thinner
than
normal endothelium
(4,000 to 6,000 A), such areas were readily
identifiable.
Fenestrations
are not found in normal endothelium
of
capillaries
supplying
muscle or skin (except capillaries
supplying
sweat glands).3
Mean capillary
thickness
was determined
using
planometric
methods
has
patients
before
they
the
Veterans
From
University
cine,
a
previously
method
Pathology.
Veterans
Submitted
Address
Administration
described.4
of
±2.7%.
developed
In our
Because
College
and
Statistics.
was
Medical
of Medicine,
experience,
tissue
thrombocytopenia
Administration
of Florida
The work
ofthe
variability
not
Center
Departments
this
from
these
available,
and
of
the
Medi-
Gainesville.
was done when Dr Kitchens
was a Clinical
Investigator
Administration.
Feb
reprint
4, 1985;
accepted
requests
Medical
to
Center,
July
Dr
Craig
Medical
24,
1985.
S.
Service
Kitchens,
Veterans
(1 1 1). Gainesville,
FL 32602.
©
I 986
by Grune
& Stratton,
Inc.
0006-4971/86/6701.-0032$03.00/0
203
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
KITCHENS
204
AND
PENDERGAST
Fig 1 .
Definition
of morphological
terms.
(A) demonstrates
normal
endothelium
with a thickness
of 3.000
to 5.000
A.
(B)
shows
attenuation
to a point of 700 A and is referred
to as a
“thin spot” (arrow).
A fenestration
is shown
in (C). All figures
oriented
with capillary
lumen to the right and all x 33.000.
determinations
using
from
human
normal.6’7
The
ticosteroid
lary
other
skin
and
methods
studies
were
administration
endothelial
this
muscle
laboratory
capillaries
identical.
To our
normal
The
were
data
performed
used
thrombocytopenia
the initial
biopsy
to define
knowledge,
does not affect
structure.4’8
has
were
glucocor-
mammalian
capil-
analyzed
using
the
Statistical
an IBM
University
Analysis
System (SAS)9 computer
software
package
on
4341 machine
in the Faculty
Support
Center
at the
of Florida. Significance
of changes in capillary
thickness
measures
was
determined
weighted
to adjust
between
the
two-sample
for the
study
test,
t
within-subject
using
variability
group
again
a one-sided
and
after
paired
within
normal
Comparisons
were
the
made
measures
using
to adjust
a
for
variability.’0
and
all
fulfilled
all
tration.
Table
patients
of this study (Table
immune
thrombocytopenia
administration.
having
amegakaryocytic
quently
developed
their
1). Three
were
purpura
basis
prednisone
and
Two
were
results
anemia,
the
diagnosed
as having
(ITP)
resistant
to
initially
diagnosed
boc”
aplastic
form
whereas
as
one subseother’s
dis-
2;
the
ease evolved
into acute nonlymphocytic
leukemia.
The response
of each patient
to prednisone
administration
was noteworthy.
Whereas
each
patient
manifested
fresh
petechial
rhagic
formation
and
phenomena
cocorticosteroid
epistaxis
resolved
on
with
administration,
Table
1 . Patient
admission,
such
hemor-
the commencement
although
of glu-
the
patient’s
Characteristics
Platelet
Count!
1O34oL
1 mg/kg/d
Age
Sex
Diagnosis
58
F
AMT
73
M
AMT
3
3
18
F
ITP
2
10
88
M
ITP
2
6
71
AMT,
topenic
M
amegak
purpura.
Base Line
4
ITP
aryocytic
Prednisone
4
11
thrombocytopenia;
x Four d
were
<9.0
pooled
number
and
immune
during
muscle
and
human
to revert
tion
of glucocorticosteroids
for
muscle)
clinically
at
a
statistical
when
abating.
were
from
capillary
five skin
normal.
mune
the
was
biopsies
Mean
.05)
three
<
Additionally,
these
after the administrafor skin and P = .055
patients’
and
after
normal.
each
individual
specimens.
There
thrombocytopenia
karyocytic
Despite
bleeding
muscle
prednisone
were
(P
thinner
during
capillary
<
also
.05)
was
capillary
endo-
administra-
than normal
endothelium
for muscle).
All interendothelial
thickness
cantly
(P
two of the
2.2
±
adminis-
analysis.
the
Skin
in thrombocytopenia
is still thinner
and P = .008
observed
Data
16.5
data.
Data from the five
and three pairs of muscle
endothelium.
toward
normal
(P = .001
time
markedly
thelium
(mean,
prednisone
the capillary
endothelium
of both
is thinner
(P = .001
and
P = .013,
shows
more thin spots
and fenestrations
than
normal
changes
tend
and
with
minutes
for
(P
=
for
.001
junctions
analyzed.
in all
three
Mean
muscle
thrombocytopenia
compared
thickness
increased
signifi-
after glucocorticosteroid
muscle
samples
and
administration
in all five of the
in
skin
was no difference
in the data whether
the
was due to decreased
production
(amega-
thrombocytopenia)
or increased
destruction
(im-
thrombocytopenia).
15
ITP.
Likewise,
those made
of subjects,
respectively)
tion
skin
<20
minutes)
2 gives our morphological
(five pairs of skin biopsies
were
small
criteria
which
normal,
skin
patients
of
biopsies)
RESULTS
Five
improve.
whereas
on the fourth
day bled only little more than similar
biopsies
performed
on patients
having
normal
platelet
counts.’3
These
events
correlated
with their
template
bleeding
times,
all of
which
were >20 minutes
before
prednisone
administration
minutes;
on measures
subjects.
tissue
weighting
test
t
did not substantially
sites bled significantly,
DISCUSSION
thrombocy-
The
obtained
results
of this
in our
studies
study
are
remarkably
of experimental
similar
to those
thrombocytopenia
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
ENDOTHELIUM
IN THROMBOCYTOPENIA
205
Table
2.
Obs#{149}rvation of Human
Material
(n
normal
Skin:
thrombocytopenia
(n =
Skin:
thrombocytopenia
and prednisone
36)
(n -
120)
(n
Muscle:
normal
Muscle:
thrombocytopenia
(n
Muscle:
thrombocytopenia
and prednisone
#{149}Per100
thickness,
43% and
100)
109)
In each
becomes
and thin spots and
6%, respectively,
prednisone
reverts
original
(n = 147)
situation,
roughly
normal,
while
the
assuming
thin spots
endothelial
scanning
electron
of the capillary
Endothelial
alterations
were also found in dogs
are
2,832
±
933
4,040
±
1,702
4,180
±
1,321
2,074
±
737
2,552
±
1,013
A
A
A
A
A
A
Fenestrations
0.0
0.0
43.3
6.7
5.0
1.0
0.0
0.0
44.9
5.5
20.4
1.3
mechanical
at a time
when
strength
capillary
found
in
thrombo-
and
endothe-
hemorrhage
mechanism
by which either platelets
may
support
normal
endothelial
hence
strength,
possibilities
endothelial
is
exist.
cells
surface
area.
cells changes
not
known
at
Endothelium
could
were
available
to
It is unlikely
that the
as rapidly
as necessary
observed
in either
this clinical
mental
studies.
Endothelium
could appear
thinner
ence
increased.
of the
study
time,
appear
cover
but
thinner
a fixed
several
if fewer
luminal
number
of endothelial
to explain
the changes
or the
previous
microcirculation.
just as likely
among
groups
work) with respect
if the
We
of patients
to diameter
mean
perfectly
round
as deterproximal
or fenestrated
in a small,
flat
to
endothelium
(ie, “closed”)
capillary.
As capil-
cell investiture,
blood flow. The
overinflation
due to proximal
pressure.
Most likely these alterations
are caused
effects
that platelets
and glucocorticosteroids
endoperoxides
King
their
law of
and
induced
vations
have
in
or
cells
for
demonstrated
metabolism.’5”6
a factor
in platelets
by glucocorticosteroids.’9
may in part play a role
Any or all of these obserin endothelial
cell homeosta-
sis.
Shepro
structural
et al#{176}
were unable
to find
changes
in nonquantified
in thrombocytopenic
and
appear
observe
are imperfectly
understood
and
Platelets
can provide
prostaglan-
to endothelial
Buchwald’7
by the metabolic
have on endo-
that promotes
endothelial
cell growth
and is distinct
from
platelet-derived
growth
factor.
Blajchman
et al’8 have demonstrated
that hydrocortisone
administration
decreases
yessel wall prostacylin
production
in vivo. Profound
alterations
in protein
synthesis
by endothelial
cells studies
in vitro are
mal
circumfer-
do not
or “closed”
of arterioles
smooth-muscle
by proximal
mental
thrombocytopenia.
thickness
observed
in the
experi-
(or animals
of capillaries
“open”
Thinned
to be found
as in a nearly
vessels
microcirculation
any differences
experimental
or glucocorticostructure,
and
this
were
or constriction
lanes
themselves
lack
“openness”
is determined
din
are
strengthened.
The
steroids
the
was
capillaries
thelial
cells.
These
effects
are now being discovered.
consistent
of the capillary
fragility
many
by relaxation
LaPlace
determines
that the thin-walled
capillary,
because
of its overall
small diameter
compared
with the rest of the
vascular
tree, is most
resistant
to passive
engorgement
or
manifest,
it is attractive
to hypothesize
a casual
relationship.
As both structural
alterations
and bleeding
are ameliorated
by glucocorticosteroid
administration,
this hypothesis
is further
2,399
capillary
These
adminis-
cytopenic
by infusion
of thrombin.8
As these results
during
thrombocytopenia
decreased
in
microsendothe-
in experimental
thrombocytopenia.
were partially
reversed
by prednisone
to those
severely
hum7
±
mined
75%
of
largely,
similar
rendered
with
Thin Spots
SD
5,881
to how
thickness
approximately
and fenestrations
but not totally,
disappeared.
Using
copy, we demonstrated
enfacement
hal
surface
changes
also
the endothelium
half the original
fenestrations
are encountered
of the capillaries
encountered.
administration,
toward
thickness,
tration.’4
humans
100)
-
+
Endothelium
capillaries.
produced
in the rabbit.4
during
thrombocytopenia
After
Thickness
Skin:
-
Microvascular
quantifying
will such
many
capillaries
thinner
than
many
changes
similar
capillary
ultraobservations
in experi-
The wide variation
in endothelial
present
study
indicates
that many
preparations
in normal
the
mean.
may
rabbits
Only
by
appear
nor-
or humans
may
observing
vessels
in a blinded
morphometric
be statistically
apparent.
and
analysis
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1986 67: 203-206
Human thrombocytopenia is associated with structural abnormalities of the
endothelium that are ameliorated by glucocorticosteroid administration
CS Kitchens and JF Pendergast
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