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Effects of Red Blood Cell Concentration on Hemostasis and Thrombus Formation in
a Primate Model
By Yves Cadroy and Stephen R. Hanson
Because the effects of red blood cell (RBC) concentration
on hemostasis and thrombus formation have not been
studied experimentally under conditions of whole blood
flow without anti-coagulation, normal baboons were bled
or transfused t o obtain three different groups: a low
hematocrit (Ht) group (20% < Ht < 25%). a normal Ht
group ( 3 5 % < H t < 40%). and a high H t group
(50% < Ht < 55%). Measurements of platelet count, bleeding time, platelet aggregation, fibrinogen level, and coagulation time (APTT) were equivalent t o normal values in
each group. Thrombus formation was induced using a
device composed of collagen-coated tubing followed by
t w o sequentially placed expansion chambers designed t o
exhibit flow recirculation and stasis. The device was
exposed for up t o 40 minutes in an arterio-venous shunt
system. Wall shear rates in the tubular collagen segment
were 100 seconds-' and 500 t o 750 seconds-'. The
accumulation of "'In-platelets and 'asl-fibrinogen/fibrin
was measured radioisotopically: RBC incorporation was
determined from measurements of total thrombus hemoglo-
bin. Thrombus that formed on the collagen substrate was
rich in platelets and poor in fibrin and RBCs. Under high
flow conditions, thrombus composition showed no dependence on Ht. Surprisingly, under low flow conditions,
platelet thrombus volume was negatively correlated with
Ht ( r = -.73, P = .005), and was increased by greater
than twofold in the low Ht group as compared with the high
Ht group. Thrombus that formed in the disturbed flow
regions contained relatively few platelets but was rich in
fibrin and RBCs. The predominant finding was a positive
correlation between RBC incorporation and Ht at both high
and low shear rates ( r = .90, P = .oooO3; and r = .77. P =
.002, respectively), with thrombus volume increasing threet o sixfold between the low and high Ht groups. Thus, in
vivo variations in Ht ranging between 20% and 55% did not
affect hemostasis, but were found either t o promote or
inhibit the net accumulation of thrombus, depending on
local flow conditions.
0 1990 by The American Society of Hematology.
R
time. Thrombus formation was studied under conditions of
variable blood shear rate and hematocrit using a thrombogenic device that generates both platelet-rich and fibrin-rich
thrombus.20 Using this system, thrombi formed under the
different test conditions were characterized by measuring
their relative contents of platelets, fibrin, and RBCs.
ED BLOOD CELLS (RBCs) are an important component of the hemostatic mechanism. In vitro, platelet
adhesion on the subendothelium is virtually abolished in the
absence of RBCs and increases as hematocrit (Ht) values
in~rease.'.~
In vivo, anemic patients may exhibit a prolongation of the bleeding time, which usually normalizes when Ht
values exceed 30%.5-8This effect has been principally attributed to changes in the rheologic properties of the blood when
RBCs are present, ie, an increase in platelet diffusivity due to
RBC motions causing enhanced transport of platelets toward
the vessel waL9
The role of RBCs in thrombus formation has also been
investigated. Studies with in vitro perfusion systems and
anti-coagulated blood have shown that platelet thrombus
formation increases with increasing RBC concentration, but
only at high shear rates.'-3 This effect could not be demonstrated a t low shear rates because no thrombi formed under
these conditions, regardless of the H t value.3 In vivo, it has
been suggested that the risk of developing thrombosis may be
increased in patients with erythrocytosis, although clinical
studies have given contradictory results in patients with
coronary t h r o m b o ~ i s ' ~and
- ' ~ acute ~ t r o k e . ' ~ . ' ~
The aim of this study was to evaluate the effects of variable
circulating RBC concentration on hemostasis and thrombosis in a primate model. Because this experimental in vivo
system does not require the use of anti-coagulants, conditions
associated with increased platelet deposition should also
cause amplification of coagulation-related events at sites of
forming thrombus.20,2'Baboons were chosen for these studies
because this species is hemostatically similar to humans,22
and because the animals may be infused with homologous
RBCs without causing hemolysis or other adverse transfusion reactions. Thus, by selectively bleeding or transfusing
animals, steady-state hematocrit levels ranging from 20% to
55% could be readily attained. The effect of RBC concentration on hemostasis was assessed by measurements of bleeding
Blood, Vol 75, No 1 1 (June 1), 1990: pp 2 185-2 193
MATERIALS AND METHODS
Animal studies. Six normal healthy male baboons (Papio anubisl
cynocephalus) weighing 9 to 11 kg were used. All procedures were
approved by the Institutional Animal Care and Use Committee in
accordance with federal guidelines (Guide for Care and Use of
Laboratory Animals, 1985). As previously described;' all animals
had an exteriorized shunt surgically placed between the femoral
artery and vein. The shunts did not shorten platelet survival
detectably or produce measurable platelet a~tivation.'~
The animals were bled or transfused, respectively, to obtain three
different groups with Ht values in the range 20% < Ht -= 25% (group
From the Roon Research Center for Cardiovascular Disease and
Thombosis, Department of Basic and Clinical Research, Research
Institute of Scripps Clinic, La Jolla. CA; and the Division of
Hematology-Oncology, Department of Medicine, Emory University. Atlanta, GA.
Submitted May 31,1989; accepted February 14,1990.
Supported by research Grants HL 31 469 and HL 31 950from the
National Institutes of Health, US Public Health Service. This is
publication no. 5864-BCR from the Research Institute of Scripps
Clinic. La Jolla, CA.
Address reprint requests to Stephen R . Hanson, PhD. Division of
Hematology-Oncology. Department of Medicine, Emory University. Atlanta. GA 30322.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C.section I734 solely to
indicate this fact.
0 1990 by The American Society of Hematology.
0006-4971/90/7511-0020$3.00/0
2185
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2186
CADROYAND HANSON
I), 35% < Ht < 40% (group II), and 50% < Ht < 55% (group 111).
Blood counts and hemostatic parameters for the three study groups
are given in Table 1. All measurements were within normal limits,
except the RBC parameters that were varied systematically. In
particular, animals were selected to exhibit circulating platelet
counts within a relatively narrow range since platelet count has been
shown to be an important variable in this model." The low Ht group
(group I) was obtained by withdrawing from the animals a total of
200 to 300 mL of whole blood. The normal Ht value (group 11) was
obtained by transfusing group I animals with 200 mL of homologous
washed RBCs (see below). The high Ht group (group 111) was
obtained by transfusing animals having a normal Ht with 300 mL of
homologous washed RBCs. None of the animals had received prior
RBC transfusions, and no hemolysis or other adverse transfusion
reactions were observed.
Blood was drawn and collected aseptically into acid-citratedextrose (1 part ACD to 5 parts blood), and then centrifuged at
1,000 x g for 5 minutes. The supernatant was discarded and the
RBC fraction was washed twice in 5% dextrose. The concentrated
RBCs were subsequently infused into the study animals. Measurements of hemostasis and thrombus formation in the different groups
were assessed at least 12 hours after the last blood manipulation
(bleeding or transfusion). Hemostasis was assessed by measurements of bleeding time performed on the shaved volar surface of the
forearm using the standard template method.24
Thrombus formation was assessed by determining the relative
amounts of platelets, RBCs, and fibrin deposited within a thrombogenic device connected to the arterio-venous shunt as an extension
segment.20,2'The device was composed of a 2-cm length, 3.2-mm
inner diameter (id) tubing segment covalently coated with type I
collagen, followed by two sequential tubing segments of expanded
inner diameter."." The collagen was uniformly deposited and not
removed by extensive washing." The two chambers were constructed
using Teflon tubing, 9.3-mm id, connected by a I-cm length of
silicone rubber tubing (3.2-mm id). The devices were exposed to
native blood for up to 40 minutes. In each experiment, the extracorporeal circuit consisted of two devices placed in parallel. In one
branch, blood flow was freely established and measured using a
Doppler ultrasonic flowmeter (L and M Electronics, Daly City, CA);
initial blood flow rates ranged from 100 to 150 mL/min. In the other
branch, blood flow was maintained at 20 mL/min using a peristaltic
roller pump (Cole-Parmer, Model 7016, Chicago, IL) placed distal
to the thrombogenic device. While blood flow in the device was
laminar, the chamber regions were designed to produce a complex
flow pattern typically exhibiting annular vortex formation, reverse
flow along the wall, and a prolonged residence time of blood cells and
procoagulant material."
This system caused the formation of complex thrombus that was
both rich in platelets (collagen segment) and fibrin (expansion
regions).20 The tubing diameters of the device components were
chosen so that, over the range of available blood flow rates (20 to 150
mL/min), initial wall shear rates on the collagen segment would
reflect values found in arteries (100 to 750 seconds-'), while those in
the expansion regions would be more typical of venous flow conditions ((-10 seconds-' at 20 mL/min). At these tubing diameters,
an expansion region length of 2 cm was determined from previous
experimental observations showing that the overall ratio of deposited
fibrinogen to platelets was seven times higher in the two expansion
regions than on the collagen segment.2032'
The device was constructed
of silicone rubber and Teflon tubing since both have been shown to be
relatively nonthrombogenic in this primate shunt model.25
Blood flow in both branches of the circuit was pulsatile. However,
evaluation of the possible importance of differences in flow pulsatility
between branches, local flow alterations secondary to thrombus
formation, or of the effects of pulsatility per se ( v steady flow), were
considered beyond the scope of this study. Rather, each hematocrit
group was evaluated under defined conditions of high and low initial
mean blood flow rate as discussed above.
Platelet deposition was determined using autologous platelets
labeled with "'In-oxine as previously described." The average
labeling efficiency was greater than 85%. Platelet accumulation was
measured using a Picker DC 4/ 11 Dyna scintillation camera (Picker
Corp, Northford, CT) interfaced with a Medical Data Systems A'
image processing system (Medtronic, Ann Arbor, MD). Good
spatial resolution was achieved by acquiring only the low (172 keV)
energy peak of "'In with a 10%energy window and high sensitivity
collimator. Dynamic images were acquired at 5-minute intervals
over the study period. Immediately after each experiment, standards
consisting of 3 mL of whole blood (blood standard) as well as an
identical thrombogenic device filled with autologous blood (device
standard) were imaged for 5 minutes. Deposited "'In-platelet
activity was determined by counting the radioactivity in the regions
corresponding to the collagen-coated tubing and to the chamber
portions of the device, and subtracting the corresponding device
standard activity for each region. The total number of deposited
platelets (labeled plus unlabeled) was calculated by dividing the
deposited platelet activity (cpm) by the blood standard platelet
activity (cpm/mL) and multiplying by the circulating platelet count
Table 1. Blood and Hemostatic Parameters in the Animal Study Groups
Group I
22.5
3.0
7.9
74.9
26.3
10.2
301.0
70.0
4.0
2.4
32.0
n
i 0.3
* 0.1
i 0.1
f 0.9
i 0.5
f
i
k
f
i
i
5
1.6
30.0
4.0
0.2
0.2
1.0
Group II
38.7
5.2
13.0
74.5
24.9
9.1
290.0
69.0
3.2
2.5
35.0
f
0.8
i 0.1
f 0.4
i: 0.4
i 0.2
f
i
k
i
f
i
3
2.6
27.0
4.0
0.2
0.1
1.0
Group Ill
51.7
6.9
17.3
75.1
25.3
11.8
268.0
69.0
3.3
2.9
40.0
f 0.6
f 0.1
f
0.2
f 0.4
i 0.2
i
2.4
i 28.0
4.0
0.1
f 0.1
i 1.0
i
i
5
*Whole blood for A P T determinations was anti-coagulated with a fixed volume of 3.8% citrate (9:1 vol/vol). Studies in three additional control
animals with citrate/plasma ratios (0.143. 0.181. 0.230) that were the same as those in the low, intermediate, and high Ht groups, respectively, gave
A P T values (33 f 1 s, 36 i 1 s, and 41 i 2 s) which were equivalent to those obtained in groups I, (I,and Ill, respectively, indicatingthat APlT values
differed as a consequence of variable citrate dilution only. All values are mean f 1 SE of n observations.
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2187
ROLE OF RBCs IN EXPERIMENTAL THROMBOSIS
(platelets/mL) as measured in each experiment. Radioactivity
values referred to platelet activity only; the plasma (nonplatelet)
activity per milliliter of whole blood was calculated for each
experiment by multiplying the activity per milliliter of plasma by the
factor: (I-Ht). Plasma activities averaged 13.8% k 1.6% of whole
blood activities in these studies and were the same in the three study
groups (13.9% + 3.2%,15.2% f 0.7%. and 12.8% * 3.0%in groups
I, 11, and 111, respectively).
Fibrin deposition was determined using '"I-fibrinogen. Baboon
fibrinogen was purified using a @-alanine precipitation procedure,
and homologous fibrinogen preparations were labeled with '"I using
the IC1 method as described." Labeling efficiencies averaged 70%.
The thrombin clottability of the labeled fibrinogen was greater than
90%. Five microcuries of Iz5I-fibrinogenwas injected intravenously
10 minutes before device blood exposure. At the end of each study,
the thrombogenic device was thoroughly washed with isotonic saline
and then cut into segments containing the collagen-coated surface
and chamber regions." Total fibrin deposition was calculated by
dividing the deposited fibrin activity (cpm) by the clottable plasma
fibrinogen activity (cpm/mg) in samples taken immediately before
each experiment as described.20,2'The '251-emissionsassociated with
the fibrinogen samples and graft components were determined after
allowing 30 days for the "'In-radioactivity to decay (half-life: 2.8
days).
The number of RBCs deposited within each component of the
thrombogenic device was also determined. The separate segments
comprising each device were kept in distilled water at 4OC for 30
days. After determining the content of fibrin, thrombi were thoroughly mixed with the distilled water vehicle (2 to 4 mL) to ensure
complete lysis of all RBCs, with the residual material consisting only
of white fibrin strands. The hemoglobin concentration of the homogeneous mixture was then determined using an automated cell counter
(System 9000, Baker Instrument Corp, Allentown, PA). The number of RBCs was calculated by multiplying the hemoglobin concentration (g/mL) by the volume of distilled water in which the
thrombus was dissolved (mL) and dividing by the mean corpuscular
hemoglobin (g/RBC). The lower sensitivity limit for this detection
system was 0.050 x IO9 RBCs.
Laboratory procedures. Blood parameters were determined on
whole blood collected in vacutainer tubes (Becton Dickinson, Rutherford, NJ) containing disodium EDTA. The white blood cell count
(WBC), RBC count, hemoglobin level (HGB), Ht value, mean
corpuscular volume (MCV), mean corpuscular hemoglobin (MCH),
and platelet count were determined using an automated cell counter
(Baker System 9000). Fibrinogen levels were measured as total
thrombin-clottable protein using the method of Jacobsson.z0,26
Activated partial thromboplastin times (APTT) were determined
on citrated plasmas (9 vol blood into 1 vol 3.8% sodium citrate).
APTT (activated PTT reagent, Ortho Diagnostic Systems, Raritan,
NJ) measurements were performed using a fibrometer (Fibrosystem, Becton Dickinson, Cockeysville, MA).
Platelet aggregation was measured in citrated plasma (9 vol blood
into 1 ~013.2%sodium citrate). Platelets in platelet-rich plasma were
adjusted to a final count of 250,000 platelets/pL. Aggregation was
induced by the addition of 19.2 pg/mL collagen (Hormon, Munich,
FRG). Aggregometer tracings were quantitatively analyzed to
determine the maximum increase in light tran~mittance.".~~
Statistical evaluations. Statistical analyses were performed using the CLINFO programs provided by the US Department of
Health and Human Services. All data are given as the mean k 1 SE.
The student's t-test (two-tailed) for unpaired sample groups was
used when the data were normally distributed (Wilk-Shapiro test).
Least-squares linear regression analysis was used to determine the
correlation coefficient ( r ) and the significance level (P)for relationships between variables.
RESULTS
Blood counts and hemostatic parameters for the three
study groups are given in Table 1. H t values averaged
22.5% 0.3%, 38.7% k 0.8%, and 51.7% k 0.6% in groups I,
11, and 111, respectively. While RBC counts and total HGB
varied in proportion to Ht, measurements of MCV and MCH
were not different between the groups (P> S ) . Other
measured parameters were also equivalent between the three
groups, including the platelet count, extent of platelet aggregation (Agg) in response to collagen, APTT, and fibrinogen
level (Fg).
Hemostasis. Hemostasis was assessed by measurements
of bleeding time (BT), with results given in Table 1. The
average bleeding time value in the animals with normal Ht
0.2 minutes. Bleeding times were
(group 11) was 3.2
minimally prolonged (4.0 f 0.2 minutes) in animals with low
Ht as compared with the high (3.3 + 0.1 minutes) and the
normal Ht groups (P< .05 in both cases), although bleeding
times in all groups were within normal limits (range: 3.0 to
4.5 minutes)
Effect of variablePow conditions on thrombus formation.
Thrombus formation was assessed by determining the relative amounts of platelets, fibrin, and RBCs deposited within
the thrombogenic device. The device was exposed to blood for
a maximum of 40 minutes. Under high flow conditions,
occlusive thrombus formation with flow stoppage predictably
occurred within 30 to 40 minutes. Under low flow conditions,
the device was exposed for 40 minutes; no occlusions were
observed over this time period.
Platelet deposition onto the collagen segment increased
linearly with time under both high and low flow conditions
(Fig 1, A and B). However, the rate of deposition was
markedly higher at high flow. For example, in the normal Ht
group (group 11) after 10 minutes of blood exposure, five
times more platelets were deposited on the collagen at high
flow (5.93 f 1.17 x 10' platelets) than at low flow (1.06
0.18 x 10' platelets). However, levels of fibrin deposition
onto collagen measured after blood exposure were similar at
both low flow (0.46 0.03 mg at 40 minutes) and high flow
(0.39 f 0.08 mg at 30 minutes) (Fig 2, A and B). Thus, in
the animals with normal Hts, the overall ratio of deposited
fibrin to deposited platelets was two times higher at low flow
than at high flow. Few RBCs were deposited onto collagen
regardless of the blood flow rate (Fig 2).
As compared with the collagen segment, the chamber
regions accumulated fewer platelets but more fibrin and
RBCs (Figs 3 and 4). The total number of platelets deposited
in the chambers was reduced modestly under low flow versus
high flow conditions (Fig 3, A and B). Thus, in the studies
with animals having normal Hts, total platelet deposition
after 30 minutes averaged 4.92 0.35 x lo8 platelets at low
flow, a n d 7.69 * 0.95 x 10' platelets at high flow (P> .lo).
Conversely, the accumulation of fibrin and RBCs in the
chambers was 3 to 4 times higher at low flow than at high
flow (Fig 4).
Effectof variable RBCconcentration. Thrombus formed
on collagen under high flow conditions was not influenced by
variable RBC concentration: the rate of platelet accumula.21323.24
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CADROYAND HANSON
A: High Flow
B: low Flow
T
20
20
15
15
10
10
5
51
0
0
I
I
Time lmin)
3
T
I 4- I
T
30
Time (min)
tion and the total amount of platelets, fibrin, and RBCs
deposited onto the collagen at the end of the blood exposure
period were equivalent in the three study groups (Figs 1A
and 2A). In contrast, under low flow conditions, platelet
deposition onto the collagen-coated segment decreased as the
H t value increased (Figs 1B and 2B). Thus, after 40 minutes
of blood exposure, the number of platelets accumulated on
the collagen substrate averaged 15.57 3.45 x lo8 platelets
and 7.03 * 1.77 x 10' platelets in the low Ht and high Ht
A: High Flow
20
10
40
Fig 1. Total platelet deposition onto collagen at high (A)
and low (B) blood flow rates.
The time course of platelet deposition is shown for animals
having low Ht (0).normal Ht
( 0 ) .and high Ht (0)values as
given in Table 1. Data are mean
f 1 SE of observations in three
( 0 ) or five IO,0 )animals.
groups, respectively (P = .06). Figure 5 shows the inverse
relationship observed ( r = -.58, P = .04)between Ht and
the number of platelets deposited on collagen at the low blood
flow rate.
A previously reported linear relationship between blood
platelet count and platelet deposition after 40 minutes of
blood exposure at low flowz0was also seen in the present
study with animals from each H t group (Fig 6 ) . Platelet
accumulation onto collagen at high flow was also positively
6: tow Flow
I
I
I
- 80
I
I
I
I
I
I
I
I
I
T
X
50
I
I
I
W
0
40
lu
c
I-0
I
I
TI
W
30
0
a
W
I
I
20
I
I
I
p
a
I
I
-
-
I-0
10
I
0
Fig 2. Effect of Ht on platelet, fibrin. and RBC deposition onto collagen at high (A) and low (B) blood flow rates. Endpoint measurements
were taken after blood exposure for 30 minutes (A) or 40 minutes (e),in animals having Ht values that were low (I),normal (11). or increased
(111). Time course measurementsof platelet deposition are given in Fig 1.
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2189
ROLE OF RBCs IN EXPERIMENTAL THROMBOSIS
B: Low Flow
A: High Flow
10
v)
c
W
Fig 3. Total platelet deposition in the chamber regions of
flow expansion at high (A) and
low (6) mean blood flow rates.
The data illustrate the time
course of platelet deposition in
animal groups having low Ht
(0).normal Ht (0).
and high Ht
(0)values as given in Table 1.
Data are mean i 1 SE of obseror five (0.
vations in three (0)
0 )animals.
.zX
5
0"
E
"0
10
20
25
-
c
10
Time (mid
correlated with platelet count ( r = .69, P = .03). Therefore,
it was important that all study groups exhibited comparable
circulating platelet counts (Table 1). Under the assumption
that platelet deposition onto collagen at low flow was linearly
related to platelet count, the data comprising Fig 5 were
normalized as previously described" to correct for modest
platelet count differences and to estimate values of platelet
deposition that would have resulted had all animals exhibited
a fixed platelet count equalling the overall group mean
(286,000 plateletslpl). The empirical inverse relationship
between platelet deposition and H t was substantially improved after correcting for variable platelet count ( r = - .73,
P = .005).
The formation of fibrin on collagen was also dependent on
H t under low flow conditions. Thus, the total amount of fibrin
deposited on collagen a t low flow was modestly higher a t low
A: High Flow
30
20
40
30
Time (min)
H t than at high Ht (0.66 0.08 mg v 0.46 * 0.06 mg,
P = .07; Fig 2). While it is expected that plasma fibrinogen
levels should directly influence fibrin formation, and perhaps
blood cell accumulation, the fibrinogen level per se was not
evaluated as an independent variable in these studies since
plasma fibrinogen concentrations varied little between individual animals or animal groups (Table 1).
Platelet deposition in the chamber regions of flow expansion was not affected significantly by RBC concentration at
either low flow ( P > .10 a t 40 minutes) or high flow (P> .SO
a t 30 minutes) (Fig 3), but was found to depend on platelet
count. Platelet accumulation after 30 to 40 minutes was
positively correlated with platelet count at both low flow
( r = .64, P = .02) and high flow ( r = .68, P = .03). Similarly, levels of fibrin deposition were comparable regardless
of the H t value (Fig 4). However, the RBC concentration
B: Low Flow
r
T
I
- 80
O'-
0
-
I
-60
X
-50
-M
W
u
-40
p
LT
U
.z
W
-30
0
W
U
-20
-
-
0
t-0
10
A n
Fig 4. Effect of Ht on platelet, fibrin, and RBC deposition in the chamber regions of flow expansion at high (AI and low ( 6 )blood flow
rates. Endpoint measurementswere taken after blood exposure for 30 minutes (A) or 40 minutes (6) in animals having Ht values that were
low (I), normal (11). or increased (111). Time course measurementsof platelet deposition are given in Fig 3.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
2190
CADROYAND HANSON
20
-
15
-
10
-
1
9D
0
c
X
v)
al
al
c
c
m
h
-0
al
.-c
v)
0
n
0)
0
m
4-
r-”
5 -
u
+
~~
“
20
30
50
40
Hematocrit
60
(%I
Fig 5. Effect of Ht on platelet deposition onto collagen under
low flow conditions. After 40 minutes of blood exposure, platelet
deposition was related to H t in an inverse linear fashion
(y = - 0 . 2 9 ~ 21.9, r = -.58, P = .W).
The correlation wes
improved by correcting for differences in platelet count between
animals, as described in Results (y = - 0 . 2 5 ~ 19.8, r = -.73.
P = .005). Values are mean k1 SE of observations in three
animals (normal Ht) or five animals (low Ht and high Ht groups).
+
+
markedly influenced the extent of RBC accumulation in the
chambers as shown by positive linear relationships found
between Ht and RBC deposition at both high flow (r = .90,
P = .00003) and low flow (r = .77, P = ,002) (Fig 7). As
compared with the low H t group, RBC deposition in the high
Ht group was increased sixfold a t high flow (2.75 0.26 x
IO’ v 0.44 k 0.14 x lo9, P = .0001) and threefold at the
lower flow rate (7.66 k 1.14 x lo9 v 2.57 f 0.44 x lo9,
P = .006) (Fig 4).
blood-vessel wall interface under some circumstances, thereby
increasing their interactions with subendothelial tissue
components.’.’ The enhancement of platelet adhesion is
probably unrelated to the availability of RBC adenosine
diphosphate (ADP) since platelet adhesion is normal in the
presence of RBC ghosts in perfusion studies2’ and is unaffected by the presence of ADP-scavenging enzymes (eg,
apyrase).29
In contrast to previous clinical studies, we did not find an
increased bleeding tendency at reduced Ht. However, in the
studies with anemic patients, some individuals also exhibited
a reduced platelet count.6 In uremia, there may be additional
hemostatic defects unrelated to RBC con~entration,’~~’l
which
may be corrected in part by therapies that increase plasma
levels of factor VIII/von Willebrand factor (eg, desmoIn addition, our study baboons were only moderpre~sin).’~
ately anemic (Ht > 20%) with no other clinical or biologic
abnormalities. Under these circumstances, Ht values in the
ranges studied presumably were sufficient to effectively
augment platelet transport and prevent abnormal bleeding.”
Thrombosis. A clinical relationship between elevated H t
and thrombotic risk is generally admitted, although not
clearly demonstrated. Clinical studies have yielded contradictory results regarding a possible correlation between erythrocytosis, angina pectoris, and the occurrence of coronary
3[
21
DISCUSSION
Hemostasis. Hemostasis was assessed in animals with
variable RBC concentrations by the measurement of bleeding times performed using the standard template method.24
Bleeding times showed no dependence on Ht, averaging 3 to
4 minutes in the different study groups. These values were
within the reported normal range for baboon^.*^**'^^^*^'
It is well-established that the presence of adequate numbers of RBCs supports hemostatic plug formation in vivo.
Early studies by Duke’ and Hellem et aI6 showed that
patients with severe anemia also exhibited a prolonged
bleeding time that could be corrected by RBC transfusion.
Similar observations have been reported for anemic, uremic
patients in which prolonged bleeding times were corrected by
increasing H t values above 25% to 30%.’,’ In vitro, blood
perfusion over subendothelium has also shown that platelet
adhesion increases as H t values increase between 10% and
70%, particularly a t high shear rates.’-4 These observations
have been attributed to the physicochemical effects played by
RBCs for promoting platelet transport and platelet-surface
interactions.’.’ Thus, due to their small size relative to RBCs,
platelets may be concentrated in a peripheral layer a t the
11
200
300
400
Blood Platelet Concentration
(PIateIetslpL x 10-3)
Fig 6. Effect of circulating platelet count and Ht on platelet
deposition onto collagen under low flow conditions. After 40
minutes of blood exposure, platelet deposition and platelet count
were linearly related for each Ht group. Group I (low Ht group):
y = 0 . 0 9 7 ~- 13.5, r = .84. P = .08; group II (normal Ht group):
y = 0.099~- 18.3, r = .99, P = .OB; group 111 (high Ht group): y =
0.063~- 9.9, r = .99, P = .001.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
ROLE OF RBCs IN EXPERIMENTALTHROMBOSIS
cox
0
"
Y
801
I
6ol
C
50
40
Hematocrit
(%I
Fig 7. Effect of Ht on RBC deposition in the chamber regions.
After 30 minutes (high flow) or 40 minutes (low flow) of blood
exposure, RBC deposition was linearly related to Ht at both high
flow (A) (y = 0 . 0 8 ~- 1.4, r = .90, P = .00003). and low flow (B)
(y = 0 . 1 7 ~- 1.2, r = .77, P = .0021. Values are mean +1 SE of
observations in three animals (normal Ht) or five animals (low Ht
and high Ht groups).
t h r o m b o ~ i s , ' ~or- ' the
~ role of H t and hemodilution in acute
~ t r o k e . ' ~ .While
'~
thromboembolic complications occur in
polycythemia vera,34this myeloproliferative disorder may be
associated with other abnormalities such as thrombocytosis.
In a recent review, Ht was not found to be a risk factor for
thrombosis in these patients.35 This conclusion is also supported by the observation that patients with secondary
polycythemia are known to be relatively free of vascular
complication^.^^
In our studies, the role of RBCs in thrombus formation
was assessed by determining the relative incorporation of
platelets, fibrin, and RBCs into thrombi produced under
conditions of variable flow and Ht. A thrombogenic device
containing a segment of collagen-coated tubing followed by
two tubular expansions was exposed to native blood using an
arterio-venous shunt system as described previously.20*21In
each experiment, two devices were placed in a parallel
branching design. In one branch, blood flow was spontaneously established a t rates between 100 and 150 mL/minute
with initial wall shear rates in the collagen segment ranging
from 500 to 750 seconds-'. In the other branch, flow was
constantly maintained at 20 mL/minute using a roller pump;
the initial wall shear rate a t the collagen surface was 100
seconds-'. Flow past the collagen segment was unidirectional, while regions of flow expansion typically exhibit flow
reversal near the wall with recirculation of blood cells and
procoagulant material. As shown previously, this device
generates a complex thrombus having components that are
both platelet-rich (collagen segment) and fibrin-rich (chamber regions), and which may be selectively blocked by
anti-platelet agents or anti-coagulating amounts of heparin.20.2'
In the normal H t group, platelet deposition on the collagen-
2191
coated segment increased monotonically a t both high and
low flow rates, but the rate of platelet accumulation was
reduced under low flow conditions. Thus, after 10 minutes of
blood exposure, five times fewer platelets were deposited at
low flow than a t high flow (Fig 1). This result is in general
agreement with data obtained in perfusion test systems
where increased shear rates produced increased platelet
adhesion as well as thrombus f~rmation.'~
In contrast, fibrin
deposition was similar a t both high and low flow rates (Fig
2). The shear-rate dependence of fibrin deposition, ie, decreasing fibrin deposition with increasing shear rate, has been
previously demonstrated when subendothelium is exposed in
an annular perfusion chamber.36 However, in our study fibrin
deposition was measured as an endpoint value only, after 30
to 40 minutes of device blood exposure. I t is conceivable that
a shear-rate dependence of fibrin deposition might be demonstrable at the shorter exposure periods typically used in
earlier studies (eg, 5 to 10 minutes). Overall, the ratio of total
deposited fibrin to total deposited platelets was twice as high
at low flow than at high flow, with relatively few RBCs
deposited on collagen regardless of blood flow conditions.
The chamber regions of flow expansion produced a venoustype thrombus, ie, one that was rich in fibrin and RBCS.~'In
animals with normal Hts, the overall ratio of deposited fibrin
to deposited platelets was about eight times higher in the
chambers than on collagen, a value comparable with results
reported
Similarly, thrombus formed in the
disturbed flow regions was approximately 65 times richer in
RBCs. Under high flow conditions, the accumulation in
thrombus of fibrin and RBCs was reduced three- to fourfold
versus the studies performed a t the lower shear rate. Interestingly, the extent of platelet deposition in the chambers was
comparable a t both high and low flow rates. In an earlier
study using a similar flow geometry, Karino and Goldsmith3'
also observed that initial platelet attachment on the wall of a
tubular expansion showed little dependence on shear rate.
Thrombus formation on collagen under high flow conditions was not affected by variations in H t ranging from 20%
to 55%. The accumulation of platelets, fibrin, and RBCs was
equivalent in the three study groups (Figs 1A and 2A). In
contrast, in previous studies performed under comparable
flow conditions but with anti-coagulated blood and a short
exposure period (5 minutes), both platelet adhesion and
thrombus formation on subendothelium increased continuously as H t values were increased from 10% to 70%.' In our
study, a good correlation was found between platelet deposition onto collagen and H t after 5 minutes of blood exposure
( r = .64, P = .04),but no such relationship was found a t
later time points. This result suggests that the effects of
variable H t may be less apparent a t longer exposure times, ie,
that platelet thrombus formation a t low H t may be only
transiently reduced.
In contrast, under low flow conditions, platelet deposition
onto collagen was decreased as H t values increased. Previous
in vitro perfusion studies performed at comparable low shear
rates (50 and 200 seconds-') showed no thrombus formation
after 10 minutes for Ht values between 10% and 70%; ie,
while increased RBC numbers promoted increased platelet
adhesion, this effect was not associated with enhanced
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
2192
CADROYAND HANSON
thrombus f ~ r m a t i o nIn
. ~ the present study, platelet thrombus
formation at low flow was significantly reduced as the RBC
concentration was increased, and in a manner that also
depended strongly on the circulating platelet count (Fig 6).
These findings were striking and probably not related to
possible alterations in platelets or coagulation produced by
the transfusion of washed RBCs in the normal H t and high
Ht groups. Indeed, platelet function, as assessed by measurements of bleeding time and platelet aggregation, was unchanged. Similarly, the measurements of APTT and fibrinogen level were normal in all study groups. Moreover, any
changes in platelet reactivity caused by these procedures
should have been more apparent in the studies at higher
shear rates since, under these conditions, platelet adhesion is
less diffusion-limited and more strongly governed by the
intrinsic reactivity of platelets toward the substrate surface.3938
While our findings are consistent with the possibility that
increasing Ht may simply impede platelet transport under
certain circumstances, other explanations are possible. For
example, increasing H t may have promoted the growth of
individual thrombi, which were less stable with time, such
that the overall (surface-averaged) accumulation of platelets
was reduced at higher Ht. At comparable flow rates, the wall
shear stress undoubtedly varied between study groups due to
the strong dependence of blood viscosity on Ht.38 Shear
forces will also vary with time, increasing locally as the
lumen narrows. Because the extent, rather than rate, of blood
element accumulation was measured in these studies, differences in microembolization rates due to shear stress differences could have influenced our observations, at least in part,
and may account for the observed reduction in net platelet
accumulation on collagen a t increased Ht (Fig 1B).
Alternatively, in our system the near-wall concentration of
platelets may have been reduced for Ht values exceeding
about 20%. For example, it has been shown that under some
conditions the enhanced transport of small species may at
first increase with Ht, then decrease as H t values exceed 50%
due to particle crowding.33In addition, baboons have slightly
smaller RBCs than humans (mean volume: 72 to 78 wm3).
While RBC size is thought to be important in this c ~ n t e x t , ~
the effects of such differences (v studies with human blood)
remain largely undefined. Finally, additional studies suggesting that the role of RBCs in platelet deposition is not an
entirely direct and positive one (ie, higher platelet deposition
at higher Ht), and that platelet adhesion does not necessarily
parallel thrombus formtion, have also been reported. Thus,
Baumgartner and Muggli’ showed that perfusion of rabbit
blood at 800 seconds-’ caused platelet adhesion and thrombus formation to increase with increasing H t up to a value of
approximately 30% to 40%, and that at higher Ht, thrombus
formation was abruptly reduced while adhesion continued to
increase. Local effects, including inhibition of platelet deposition on areas adjacent to growing thrombi, have also been
described by Baumgartner and Muggli2 and Sakariassen et
aI.”
In the expansion chambers, platelet deposition was largely
independent of both flow and H t (Fig 3). Previously, Karino
and Goldsmith3’ showed that the number of platelets adhering in a tubular expansion after 3 minutes may increase with
increasing Ht. Similarly, after a short blood exposure period
(5 minutes) at high flow, we observed a good correlation
between Ht and platelet deposition in the chamber regions of
flow recirculation ( r = .69, P = .03). At low flow, platelet
deposition in the tubular expansions was modestly reduced a t
high H t versus the results a t low or normal H t (Fig 3B),
perhaps as a consequence of the reduced platelet deposition
seen on the upstream collagen-coated segment (Fig 1B).
Indeed, previous experiments have shown that platelet deposition in the regions of flow expansion depends in part on the
activation of platelets produced by the proximal collagen
segment.”
Lastly, under high and low blood flow conditions, a positive
correlation was found between Ht and RBC accumulation in
the annular regions of flow recirculation. As compared with
the low Ht group, RBC deposition at high H t was increased
sixfold at high flow ( P = .0001), and threefold a t low flow
( P = .006). Thus, in low shear regions of flow recirculation,
rouleaux formation, and stasis, increased numbers of RBCs
may effectively augment the total thrombus volume.
In summary, this study in normal primates demonstrates
that hemostasis is unimpaired for Ht values ranging between
20% and 55%. In contrast, the extent of thrombus formation
was shown to depend strongly on Ht. However, variable RBC
concentration was found to either promote or inhibit the
incorporation of different blood elements into forming thrombus, depending on local flow conditions. These results may
explain in part the discordant conclusions reached in some
clinical studies regarding the role of RBCs in thrombus
formation.
ACKNOWLEDGMENT
The authors thank Andrew B. Kelly, DVM, John Dietrich,
Deborah L. White, Paul McFadden, Jill Janik, and Julius Kucsma
for their expert technical assistance.
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From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1990 75: 2185-2193
Effects of red blood cell concentration on hemostasis and thrombus
formation in a primate model
Y Cadroy and SR Hanson
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