1ACC Vol. 23 . No. 7
lunc 1994:1562-9
1562
Characterization of the Relative Thrombogenicity of Atherosclerotic
Plaque Components : Implications for Consequences of Plaque Rupture
ANTONIO FERNANDEZ-ORTIZ, MD, PHD, JUAN J. BADIMON, PHD,` EALING FALK, MD,
VALENTIN FUSTER, MD, PHD, FACC, BEAT MEYER, MD, ALESSANDRA MAILHAC, MD, PHD,
DAN WENG, MD, PREDIMAN K . SHAH, MD, LINA BADIMON, PHD
Boston, Massachusetts
Objectives . The purpeoeof tide study was t determine whether
ddereat compoaeats of huma atheraederotic plaques exposed to
0vo+ag blood resulted k dlfereutdeQemof theombeu formation .
Backgrooad. It is flkdy tint lbemtum otihesaMbrsteexpmed
after spontaneous or uugiopkRy-Iddacod plaque rupture is one
factor determining whether a valuable plaque proceeds rapidly
to an occlusive thrombus or persists m a sioaoeimlve moral
thrombus . Although observational dam show that plaque rupture
is a potent sinks for thrombosls, ad expand collagen Is
suggested to have ∎ predominant role in thrombmk, the relative
throalbogenhityofdiffereul compooedsef huma uUerooderolle
plaques Is not vied estabiblicil.
Methods. We hwealipted thrombus formation en room ce0rkb motels (obtained hem ratty dresia), eo0agea-rkb manta
(from selerote plaques), colegeaWee matrix wilioal chele tetd
crystals (from fibrdipld plaques), alto oniatass owe wish shun .
dam cholesterol crystals (from alherommom piques) ad mgments of normal lutma derived from human aorta at Recrepey.
Specimens were mounted In a tabular clamber placed within n
ex vivo extraconpooal perfusion system and expomd to hepsrtef ed porcine blood (men [*SEMI act vated partial tbrombo.
plmdo time ratio 1 .5 * 0.04) for S site under NO shear rate
conditions (1,00 s7') . Thrombus was qualeated by measurement of tedium-labeled platelets and morphometric analysis .
Under ehalar conditions, substrates were perfused with bepam Ined human blood (2 IUJd) In a in vitro system, and
thrombus formation was simgurly evaluated .
Radts. Thrombus; formation on atherismatous am was up to
unfold gsoatesr tam that on other substrates, Including coBgeurkh eWrix (P = OAMI) la both heksologom ad hamdogom
system. Although the dierormtaws core had a mare Irregular
exposed surface and thrombus formation tended to lacrese with
increasing raaghnea, the eteromatum core remained the moat
Wamhogmie mbdaee who she mbelrmm were uomdad by
the degree of lrregslsrly m defused by she rouglusiss Miss (p
0.002).
CosetraonJ. The mherommoue on Is the moot thrombogdc
compouml d Yanonu Nberosdowde p aqua . Therefore, phalluses
wish a large MYaummem care content me at bier risk of /ad rag
to never corauvy syudromee after spentrmm or mee6adc*
induced rupture becauae oftbe teamed tkwtaogddsy eftbeir
.
C=at
There is increasing evidence that acute clinical manifestations of coronary rfherosclerosis are caused by plaque
rupture and subssquent thrombus formation resulting from
exposure of plaque components to flowing blood (I). Pathologic studies of patients who died suddenly or shortly after
an episode of unstable angina or myocardial infarction have
shown thrombus anchored on ruptured atherosclerotic
plaques (2-4). Data derived from angioscopic observations
as well as postmortem studies have reported that plaque
rupture, inlimal dissection, mural thrombosis and deep
longitudinal clefts along the atheroma invariably follow
coronary angioplasty (5,6). Although there are abundant
observational, but not quantitative, data showing that spontaneous or agioplasty-induced atherosclerotic plaque rupture is a potent stimulus for thrombosis, no specific studies
under flow dynamic conditions have been performed to
investigate the thrombogenicity of different components of
the atherosclerotic vessel wall,
On the basis of in vitro and static studies, the procoagulat properties of atherosclerotic plaques have been attributed variously to their increased content of collagen, tissue
factor, fatty acids or phosphotipids (7) . However, in vivo, all
blood surface interactions are dynamic, being influenced by
fluid transport rates and local shear forces, which differ
substantially from those occurring in static systems . Our
group has previously studied thrombus formation on subendothelium, tonica media and isolated collagen type I under
than Cardiovascular Biotugy lahamsory, Cardiac Unit, Mussachoselts Gemmt Hospital, Boston, Massacaasetts . This work was supported in
part by Grant HL-38933 from the National Hun, Lung, and Blood Institute,
National Institutes at Heattb, B.11-do, MaryWtd, and CICYT Grant
91/07334. Dr. Ferndndez-Gdiz has a k11-ship form the Fondue de lnvestigaSanitaria (F.t .S.), Spain . Dr . Badinton is Professor of the High Council
of Scientific Research of Spain .
Manuscript received October 18, 1993; revised mmuncript received
January 3, 1994, accepted January 5, 5994"Present address and o idreyaforcane-&-c Dr . June Jose
.&B
mot, Cardlovascuter Biology Research Laboratories, Cardiovascular Institute, The Mount Sinai Medical Center, Ncw York,
York 140296574.
thorn
SAL-
ci6n
Now
91994 by rite American College of Cardiology
cm
(j An Call Car" 19114a3 ISQ-9)
0735-tuvytrt57-W
JACC Val. 23, No. 7
June 1994 :1562-9
THROMBUGENICITV
well characterized flow conditions using a tubular perfusion
chamber placed within an extracorpore& circulation system
(8,9). Because spontaneous or angioplasty-induced plaque
rupture can result in exposure of components different from
those of subendothelium or tunica media, the purpose of this
study was to determine whether different components of
human atherosclerotic plaques exposed to flowing blood
result in different degrees of platelet deposition and thrombus formation .
Methods
Substrate preparation and classification . Atherosclerotic
plaques were obtained from human aortas at oecropsy within
24 h after death. Lesions were preliminarily classified according to their macroscopic characteristics into four
groups: 1) fatty streaks, characterized as yellow dots or
streaks, barely raised above the intimal surface ; 2) sclerotic
plaques, characterized as raised pearly white plaques without a soft core ; 3) fibrolipid plaques, characterized as yellow
plaques larger in size than fatty streaks, raised above the
surface and without a soft core ; and 4) atherontatous
plaques, characterized as white or yellow-white plaques
raised above the surface, with a confluent soft core of
toothpaste-like consistency, separated from the lumen by a
tissue cap. We also analyzed segments of aortic vessel wall
where the intima appeared macroscopically normal .
For each type of lesion, 3- x 0 .8-cm segments were
taken . To directly expose the internal matrix of the plaque,
the superficial layers of the plaque were removed with a
scalpel ; when a tissue cap was present covering an atheromalous core, it was also removed- Superficial layers of the
intimal segments were also removed with a scalpel to expose
the subendothelial layer above the internal elastic lamina . In
all instances, the exposed substrates were part of the intimal
layer of the diseased vessel (above the internal elastic
lamina). Special care was taken to avoid irregularities on the
surface. Substrates were stored frozen at -70°C until the
day of the experiment.
Perfusion ckamber. The pley)glass perfusion chamber
used in this study has been extensively described elsewhere
(8,9). In this chamber, a portion of the circumference of the
tubular flow channel (I mm in diameter) is replaced by the
test substrate material (25 mm in length), which becomes
directly exposed to the flowing blood . To allow comparison
of thrombogenicity among different substrates, all perfusions
were performed at the same local flow conditions of high
shear rate (1,690 s - r, Reynolds number [Re) = 60, flow rate
10 ml iniu, average blood velocity 21 .2 coils) . We chose
these rheologic conditions to mimic flow in mild to moderate
stenotic coronary arteries . In addition, our previous experience shows that these rheotogic conditions result in more
consistent levels of platelet deposition (8) .
Experimental procedure. The hypothesis was tested in
two different experimental approaches : 1) ex vivo, exposing
human arterial wall to porcine blood, which allowed us to
FERNANDEZ-ORTtZ ET AL.
OF ATHEROSCLEROTIC PLAQUES
1563
use low levels of in vivo blood anticoagulation (activated
oa-rial thromboplastin time ratio 1
.5), and 2) in vitro, exposing human arterial wall to human blood, which required
higher levels of anticoagulation but allowed us to test comparisons in a homologous human system.
Ex viva perfusion model (human arterial wall and pig
blood). Yorkshire albino pigs were used as blood donors
(body weight 32 -- 2 kg). All procedures performed in this
study were in accordance with the appropriate institutional
guidelines and followed the "Position of the American Heart
Association on Research Animal Use" adopted by the
Association in November 1984. The pigs were sedated with
intramuscular ketamine (20 mg/kg body weight, Ketalar,
Parke-Davis) followed by intravenous injection of sodium
pentobarbital (t0 mglkg, sodium pentobarbital injection C) .
Adequate anesthesia was maintained with pentobarbital infusion as needed and confirmed by the absence of a limb
withdrawal reflex . An exttacorporeal circuit (carotid arteryperfusion chambe •jugular vein) was established as previously described (8). Briefly, the artery was connected to the
input of the chamber; the output was connected to a peristaltic pump (Master-flex, model 7013) ; and blood that
passed through the chamber was recirculated back into the
pig by the contralateral jugular vein . After canoulation and
baseline blood sample collection, pigs were anticoagulated
with iotravenous unfractionated heparin (50-tUikg bolus plus
50-]U/kg per h continuous infusion (mean 1±SEM] activated
partial thromboplastin time L5 ± 0 .04) . The specimens were
placed in the chamber and perfused initially with phosphatebuffered saline solution (0 .01 moUliter, pH 7 .4) at 37°C for
60 s. Thereafter, blood at 10 mllmin was perfused through
the chamber for 5 min . At the end of the Llood perfusion
period, buffer was again passed for 30 s and discarded . In
total, 29 substrates were perfused ; mean hematocrits (30.5'
0 .0%), mean platelet counts (465 . 15 x 10'!µq and mean
heparin plasma levels (0.10'- 0 .02 lUlmq were shoilar in all
perfusions .
In vitro perfusion model (human arterial wall and human
blood) . Perfusion conditions were as described earlier .
Mounted specimens were perfused with phosphate-buffered
saline solution at 37°C for 60 s . Thereafter, 25 ml of heparinized blood (2 IU/mi) obtained from healthy volunteers
was perfused in a closed circuit over the surface for 5 min . In
total, 15 substrates were perfused with blood from two
donors. Donors had given informed consent, and the study
protocol was approved by the institutional committee on
human research. Mean hematocrits (40 .6 ± 0 .4), mean
platelet counts (187 ± 2 x 10 3/pl) and activated partial
thromboplastin time ratio (>5) were similar in all perfusions .
To analyze segments with a homogeneous composition,
after perfusion, each 25-mm substrate was divided in five 3to 6-mm segments according to the macroscopic characteristics of the surface . Every segment was analyzed for
deposition of radiotabeled platelets and processed for microscopic examination .
1564
Table 1.
FERNANIEZORTIZ ET AL .
THROMBOGENICITY OF ATHEROSCLEROTIC PLAQUES
Classification of Substrates
Type
Normal intma
Foam cell-slob
mama
Collagen-rich matrix
Collagen-poor matrix
Atheromatoos core
Mkroscopl Iksmiptios of Exposed Components
Loper composed mostly of eofagre . proteoglycaas,
elastic fibers and smooth muscle cells
One or more layers offoam ceus
Mace matrix rich in collagen with few or no cells
Collagen-poor motels with variable cefularity and
without cholesterol crystals or core cavity
Soft core with abaodan cholesterol crystals
Radioactive labeling of platelets. For the ex vivo perfusion model, 24 h before the experiment, autologous platelets
were labeled with indium-III (tropolone) in plasma, as
previously described (8,10) . The labeling procedure had an
average labeling efficiency of 70 ± 3% with a mean indium
plasma activity of 4.9 ± .
1 1%. The mean injected activity
was 256 t 20 pCi, and 2.6 x 106 ± 0.2 x 10s/pl of
indium-labeled platelets were reinjected .
For the in vitro perfusion model, 2 h before the experiment, 43 ml of blood from a healthy volunteer donor was
collected into 7 ml of modified acid citric dextrose solution
(pH 5) . Platelets were labeled with indium-I I (tropolone) in
plasma (11). The final pellet of labeled platelets was resuspended in 200 ml of fresh blood collected from the same
donor. The average efficiency of the labeling procedure and
the indium plasma activity were 58 ± 3% and 1 .5 ± 0%,
respectively. The mean final activity was 54 t 7 pCi, and
0 .8 x 106 ± 0.1 x 106/µ1 of indium-labeled platelets were
resuspended in fresh blood .
The number of platelets deposited on each specimen was
calculated from the platelet count and the indium activity on
the perfusion area and in whole blood with a method
previously described (8,10). Results were normalized by
area of exposed surface .
Microscopic and morphoaaetrlc exaalnadon . All sub
strates were processed for microscopic examination, From
each specimen, 10 step sections were taken at 100-µm
intervals parallel to the detection of the flow and stained with
hematoxylin-eosin and trichrome stain . The single section
with the greatest amount of thrombus, coincident with the
longitudinal central line of the surface exposed to blood, was
systematically chosen from each segment to evaluate, by
light microscopy, characteristics of the substrate-thrombus
interaction. Microscopic examination confirmed the tentative naked-eye classification of the exposed substrates (Table I) . The "normal intima" segments exposed the subendothelial layer above the internal elastic lamina, composed
mostly of collagen, proteoglycans, elastic fibers and smooth
muscle cells (12) (Fig . LA) . Foam cells were the predominant
plaque component exposed in "fatty streak" segments (Fig.
1B). Dense collagen-rich matrix with few or no sells was
exposed in "scierotic plaque" segments (Fig. IC) . Collagenpoor matrix with variable cellularity and without cholesterol
crystals or atheromatous core cavity was exposed in "fibro-
JACC Vat . D, No. 7
June 1994 :1567-9
lipid plaque" segments (Fig. ID). A nearly acellular collagenpoor soft core consisting of pultaceous debris with abundant
cholesterol crystals was exposed in "atheromatous plaque"
segments (Fig . lE and F) .
On each section, the area of thrombi >5 pm was measured, as was the substrate surface profile (13) . Calculations
were done using a computerized planimetry system and
normalized by the length of the segment exposed . The total
substrate surface covered with thrombus was also calculated
(percent of the surface covered by thrombus) . Because the
exposed surface of different substrates could vary in the
degree of irregularity (toughness), with a consequent effect
on local flow disturbances and platelet deposition, a "roughness index" (RI) of the exposed surface was calculated as
follows :
[(SP-L)IL] x 106=RI (%),
where SP = length of the surface profile ; and L = length of
the straight line drawn between the proximal and distal edges
of the surface (Fig. 2) . This index is directly proportional to
the degree of roughness of the surface . For further analysis,
surfaces were classified into three categories of irregularity :
surfaces with roughness index <5%, roughness index between 5% and 10% and roughness index >10%, respectively .
A total of 45 intimal segments, 6 foam cell rich, 24
collagen-rich matrix, 35 collagen-poor matrix and 31 atheromatous core segments, were analyzed in the ex vivo perfusion experiments; 21 intimal segments, 19 foam cell rich, 15
collagen •rich matrix, 7 collagen-poor matrix and 11 atheromatous core segments were analyzed in the in vitro perfusion experiments .
ShMMied a,alysia, Results were analyzed statistically for
the best bivariate model in distribution-free data. Group
differences among indium platelet deposition, thrombus
area, roughness index and percent of thrombus covering
were tested by the Kruskal-Wallis H test. The MannWhitney U test and linear regression analysis were used
when applicable . Results are mean values t SEM unless
otherwise stated. A p value < 0.05 was considered significant for hypothesis testing . Statview 11 (Abacus Concepts,
Inc .) running on a Macintosh Ilsi computer system was used
for all statistical analyses.
Results
En vivo perfusion model (human arterial wag and pig
blood) . Indium platelet deposition. When porcine heparinized blood (activated partial thromboplastin time 1 .5) was
drawn over human atherosclerotic plaques, platelet deposition was significantly higher on segments with exposed
atheromatous core (658 t 95 platelets x 106/cm) than on all
other substrates (normal intima 66 t 9, foam cell rich 128 ±
35, collagen-rich matrix 108 ± 12 and collagen-poor matrix
segments 134 ± 21 platelets x l06/cat, p = 0.0001) (Fig . 3,
left). In addition, platelet deposition on normal intima was
I
C VA. 23, No_ 7
1991:1567^9
FERNANOEZ VRTI2 ET AL.
THROMHOOENICI Y OF ATHEROSCLEROTIC PLAQUES
I ~f S
looms 1. Representative photomicrographs from the different types of substrates exposed to flowing blood in the ex vivo experiments . A.
Lo9giludiaat section of an intimal segment showing a rich cellular layer without lipid infiltration . , Foam cell-rich matrix. C, Collagea •r ich
Matrix. D, Cvllagen •puor matrix without cholesterol crystals, E and F, Acritular collagen•p oor soft core with abundant cholesterol oryslair .
Thrombus formation on the surfaces is stained in red . Note larger thrombi deposited on athcromatoas cum (E and F) . Trichrorne x IO[F.
1566
FERNANDEZ-ORTIZ ET AL~
THROMBOGENICITY OF ATHEROSCLEROTIC PLAQUES
SP
L
SP
L
L
Figure 2. Schematic representation of the three categories of surface irregularity defined in the study : RI = (SP - L)/L x 100, where
Rl = roughness index ; SP = surface profile ; L = length .
significantly lower than on foam cells, collagen-rich and
collagen-poor matrix segments (p = 0 .007) .
Morplrometric analysis. Thrombus area measured on
segments with exposure of atherematous core (Table 2) was
larger than on the other substrates (10 .0 ± 0.8 µm 2/mm on
atherematous core versus 0 .8 ± 0.1 µm2lmm on normal
intima, 2.0 ± 0.5 µm2/mm on foam cell-rich matrix, 1 .1
0.2 µam/mm on collagen-rich matrix and 1 .7 ± 0.2 punt/mm
on collagen-poor matrix, p = 0 .0001). Thrombus area measured by computerized planimetry and normalized by surface length correlated well with the amount of platelet
deposition calculated by indium platelet labeling (r = 0.80,
slope = 0 .97, SEM = 4 .2%n, n = 139, p = 0.8001).
The thrombus-covered surface was larger in foam cellrich matrix, atheromatous core and collagen-poor matrix
segments (80 ± 0.7%, 63 ± 0.69o and 60 t 0.4%, respectively), than in collagen-rich matrix and normal intima segments (47 ± 0.5% and 33 ± 0.5%, respectivell) (p = 0 .0001)
(Table 2).
Surface exposed in atheromatous core segments was
more irregular than surface exposed in all other segments
(mean roughness index in atheromatous core was 19 .0
0.2% vs. 5.6 ± 0.1% in collagen-poor matrix, 4 .8 ± 0.1% in
collagen-rich matrix, 4.1 s 0.1% in normal intima and 3.9 ±
0.1% in foam cell-rich segments, p = 0.0001) (Table 2). The
average roughness index of the different plaque components
preclassified into surfaces with roughnc ., index <5%,
Figure 3. Mean platelet deposi .
don calculated by indium labeling
on normal intima, foam cell-rich
matrix, collagen-rich matrix, collagen-poor matrix and atheromataus core segments after ex vivo
perfusion experiments (kill and
after in vitro perfusion experiments (right). Error bars indicate
SEM . Platelet deposition was significantly higher on atheromatous
core segments than all other segments (Kruskal-Wallis, p =
0.0001) .
1ACC Vol . 2:, No .7
lace 19941562-9
5-10% or >10% was not different (fable 3), and further
analysis was done to elucidate the cooperative effects of
surface roughness and composition its thrombus formation .
Results are presented in Figure 4 . Although platelet deposition tended to be lower on smooth than on irregular atheromatous core surfaces (317 ± 45 vs. 809 ± 123 platelets x
10°lcm2 , p = 0.09), platelet deposition on smooth atherematons core segments was still significantly higher than that
observed on smooth foam cell-rich (76 ± 42 platelets x
10°/em2), smooth collagen-rich matrix (92 t 12 platelets x
t0°/cm2) and smooth collagen-poor matrix segments (123 1
23 platelets x l0°Icm2 , p = 0.002) (Fig. 4).
In vitro perfuslau model (human arterial wan and human
blood). Indium platelet deposition . The in vitro perfusion
experiments gave comparative results similar to those obtained in the ex vivo perfusion experiments (Fig . 3, right).
When human heparinized blood was perfused over human
atherosclerotic plaques, platelet deposition on segments
with exposure of the atheromatous core was fourfold to
sixfold greater than on other components (53 ± 12 platelets x
10%m2 on atheromatous core segments vs . 9 ± 2 platelets x
105/cm' on normal intima, I I ± I platelets x 10°/cm 2 on foam
cell-rich matrix, IO ± I platelets x 10/cm' on collagen-rich
matrix and 14 ± I platelets x 10°/cat on collagen-poor
matrix segments, p = 0.0001) .
Specimens were similarly processed for light microscopic
examination . Morphomefic analysis was not performed in
specimens from the in vitro experiments because most of the
thrombi were <5 pan.
Discussion
The major finding of this study was that the atheromatous
core of human atherosclerotic plaques is associated with the
greatest platelet deposition and largest thrombus formation
compared with other components of human atherosclerotic
lesions. Similar relative differences were observed in heterologolrs (ex vivo) and homologous (in vitro) perfusion systems. The study was performed under well defined experimental conditions, modeling mild to moderate stenotic
IJACC Val . 23, No.7
... 1934:1562-9
FERNANDEZ45RTIZ ET AL .
THROMROGENICIrY OF ATHEROSCLEROTIC PLAQUES
1567
Table 2. Morphometric Substrate Chamcterisics
Normal Inlet Foam Cell-Rich Colleges-Rich Collagen-l'aor Afxromatouo Corn
=431
In=6)
(r.=23)
(n=35)
(n=30)
Thrombus
Area %malmm)
0.8 t G.1
2.0 x 0.5
1 .1 ` 0 .2
17 t 0 .2
19.0 x 0 .5'
Covering (%)
33 x 0.5'
80 9 0.7
47 7 0 .5'
60 m 0 .4
63 t 0 .6
RI I%)
4.1x0.1
3.9_0.1
4890.)
5 .610.1
19.090.2'
'p = 0.0001 (Kmskel-Wallis). Data presented as mean values _ SEM. RI = rougbaess index.
coronary vessels (high shear lap- 1,690 s') and perfusing
the substrates for a constant time (5 min) with heparinized
blood (ex vivo, activated partial thromboplastin time ratio
1 .5 ; in vitro 2 (U/rap . The amount of thrombus formed on
atheromatous core surfaces (collagen-poor matrix with
abundant cholesterol crystals) was up to sixfold greater than
on foam cell-rich matrix, collagen-rich matrix or Collagenpoor matrix without cholesterol crystals, as measured by
indium platelet deposition and thrombus morphometry .
Effect
o substrate composition .
Although abundant ob-
servational data suggest that internal atherosclerotic plaque
matrices are potent stimuli for thrombosis (2-6), no studies
have been performed to compare thrombogenicity, among
the different matrices that may be exposed to flowing blood
after spontaneous plaque rupture or after angioplasty performed on different types of atherosclerotic lesions . It is well
known that lipid incorporation into the arterial wall is the
key event in initiation atherosclerosis . and formation of a
soft lipid core is a determinant in the process of spontaneous
plaque rupture (!4,15), whereas the hard fibrous (collagen)
component is a determinant in the growth of the plaque (16) .
Previous studies have ascribed a predominant role to exposed collagen in activation of platelets and the coagulation
cascade after plaque rupture (17,13) . However, our results
suggest that the lipid core component of the plaque is a more
powerful stimulus for thrombus formation than the fibrous
collagen component. The collagen-riot: matrix exposed to
flowing blood in our experiments was up to sixfold less
thrombogenic than the atheromatous core and only twice as
thrombogenic as normal intimal matrix .
We have found that although thrombus area and platelet
deposition were low in segments with exposed foam cell-rich
matrix, the thrombus-covered surface was the largest . This
finding suggest. that different substrate-related stimuli may
be responsible for thrombus spreading (platelet adhesion)
and thrombus growth (platelet aggregation) when atherosclerotic components are exposed to flowing blood .
Effeet of substrate roughnon. Besides the nature of the
exposed substrate, the interaction of platelets with reactive
surfaces in the vasculature strongly depends on in vivo local
rheologic conditions (9,19-21) . After spontaneous or mechanically induced plaque rupture, when the vascular lumen
is reduced by stenosis or by intimal disruptions, wall shear
rate is increased over values typical of unobstructed vessels .
Our experiments were canted out at high shear rate conditions, but the roughness of the surface may additionally
increase platelet deposition. In fact, as surface irregularity
(roughness index) increased, the thrombogenicity increased
in all four types of atherosclerotic surfaces studied . However, although the atheromatous core segments had greater
surface irregularities, thrombus formation on this substrate
was still significantly higher than that on other substrates
with similar degrees of surface irregularity, suggesting that
composition of the substrate together with surface roughness
were responsible for the high thrombogenicity of the atheromatous core .
'rhe thrombogenic properties of the atheromatous core
found in our study could be attributed to one or more of its
constituents, such as the crystaline lipids, soft lipids, phospholipids or cellular degradation products within the core
(22). Of the latter, tissue factor could be a major candidate
(23). Differences in the thrombogenicity of different cultured
cell types and their matrices have been correlated with the
presence of tissue factor activity, and incubation with antibodies against tissue factor has inhibited 70% to 90% of the
prothrombotic activity of these cells (24,25) . Furthermore,
pathologic studies have identified tissue factor protein in tha
atheromatous core surrounding cholesterol crystals (26).
This may well contribute to the high thrombogenicity of the
atheromatous core found in our experiments . Other potential
Table 3 . Roughness Index Versus Plaque Components
RI
<5%
5-10%
>0%
Nernul notion
Foam Cell-aich
Collagen-Rich
1 .8x0.2
n=33
7,5 ` 0.7
=7
22x6.9
n-3
1.8x0.3
n=4
1 .7x0.3
8.1 ` I.8
0.5
0 =5
17.4*_4.7
n=3
n=2
n=0
0
6.5
=15
9
Collagen-Poor
Atheromatous Core
x 0.3
n=21
6.9 x 0.6
2.9 t 1 .0
n=4
8.4 t 0 .6
n=5
24.7 x 2 .2
n=21
1 .8
n=s
17.1 x 4.1
n=6
Data presented are mean values x SEM or number of substrates. RI = roughness index.
FERNANDEZORTr2 ET AL.
THROMEOOENICITY OF ATHEROSCLEROTIC PLAQUES
1569
IM
Atherematoua
core
JACC VoL 25, No. 7
June 1994 :156719
exposed atherosclerotic plaque components obtained at autopsy, and, therefore, there may be a difference with respect
to in vivo plaque rupture : and 2) the use of porcine blood
with human atherosclerotic plaques (ex vivo perfusion experiments) may be questioned because of the species difference. However, this study was designed to test and to
compare thrombogenic capabilities of different substrates on
a relative basis, not an absolute basis . Additionally, our
homologous it. vitro experiments with human atherosclerotic
W.
FoameM1kh
Collanan-poor
.e
7o-
Imam
<sx
1.55%
>In
Rougimeaa Index
Figure 4. Mean platelet devesilion, calculated by indium platelet
labeling, on the different substrate types grouped by degree of
surface irregularity . Although platelet deposition on smooth athere .
matous core surfaces (roughness index <5%) tended to be lower
compared with irregular atheromatous core surfaces (roughness
index >10%: Mann-Whitney, p - 0.09), platelet deposition on
smooth atherornatous core surfaces was still riptifcarWy higher than
that on any other smooth submits (Keuslal-Wallis, p - 0,002),
thrombogenic components of the plaque include matrixbound thrombin (27) or lipids themselves by inducing platelet aggregation as demonstrated in vitro using saturated fatty
acids and phospholipids (7,28). Finally, in vitro studies also
found that procoagulant activity exists in the nonlipid component of the atheromatous core (29). Collagen degradation
products within the core (30) could also initiate coagulation
or induce platelet aggregation on atherosclerotic plaques, or
both. Future studies should be carried out to elucidate which
component within the atheromatous core is most important
to thrombosis initiation.
As expected, normal intima was the least thrombogenic
substrate when assayed at very low levels of anticoagulation
(ex vivo experiments, activated partial thromboplastin time
ratio 1 .5); however, at high anticoagulation levels (in vitro
experiments, activated partial thromboplastin time ratio
>5), when absolute values of platelet deposition were lower,
intima) thrombogenicity was similar to that of other substrates except atheromatous core . Other investigators (24)
have fund that blood anticoagulation (heparin vs . citrate)
influences thrombogenicity of cultured cell matrices or animal vessel wall substrates (31,32) . Therefore, differences in
methodology (ex vivo vs- in vitro) and in blood anticoagulation together with substrate characteristics are important
determinants for platelet response . Systems mimicking as
closely as possible the in vivo situations, where platelet
function is minimally affected, are desirable for the study of
comparative thrombogenicity of different surfaces .
Experimental design and potential limitations . Two major
potential limitations may be addressed in our study : i) we
substrates exposed to human blood showed the same relative differences in thrombogenicity among the different atherosclerotic components, and other investigators have often
used heterologous systems to lest platelet-vessel wall interactions (19,31,33). The lower absolute magnitude of platelet
deposition observed in our in vitro experiments may be
related to the high levels of anticoagulation utilized to allow
handling and recirculation of the blood without clot formation (2 lUlml of unfractionated heparin, which results in an
activated partial thromboplactn time ratio >5), as is customary for in vitro perfusion systems (13,19 .24,31,34). Finally, it
can be questioned whether thrombosis on aortic plaques is
comparable to that on coronaries, and if the atheromatous
core from coronary arteries would have the same behavior
as the core derived from the aorta used in our experiments .
However, abundant observational, but not quantitative, data
have shown a similar relation between coronary lipid-rich
plaques and thrombosis (2,4) . Furthermore, the rheologic
conditions selected in our experiments (high shear rate)
correspond to stenosed coronary arteries, not to the aorta,
where much lower local shear rates develop .
Clbkal lmplkatiees. The increased throm)ogenicity
shown by the atheromatous core further supports the idea
that otherosclerotic plaques with an atheromatous core are
most prone to cause acute coronary events, not only because
of their vulnerability to rupture but also because of their
greater ahrombogenicity, after rupture . In addition, coronary
interventions performed in plaques with an atheromatous
core might be at higher risk to have acute thromboticmediated complications than those performed in other
plaques. A better understanding of the mechanisms underlying thrombus formation attar plaque rupture and the identification of the components responsible for the high thrombogenicity found in the core of atheromatous plaques might
,rovide useful strategies for improving the outcome of
patients with atherosclerotic vascular disease .
We gratefully acknowledge Star Sevilluw for technical enpenice in tissue
prepaation and segment staining . We also thank Dr. J . Fatae for expert
.
advice in the preparation of the manuscript
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