(Circulation. 1996;93:327-332.)
© 1996 American Heart Association, Inc.
Articles |
Enhancement of Platelet Deposition by Cross-Linked Hemoglobin in a Rat Carotid Endarterectomy Model
From the Department of Hematology and Vascular Biology, the Department of Biometrics and Statistics, Walter Reed Army Institute of Research, and the Peripheral Vascular Surgery Service, Walter Reed Army Medical Center, Washington, DC.
Correspondence to Barbara Alving, MD, Chief, Department of Hematology and Vascular Biology, Walter Reed Army Institute of Research, Washington, DC 20307-5100.
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Abstract |
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 Top Abstract IntroductionMethods Results Discussion References |
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Background Purified human cross-linked hemoglobin, which is now being used in clinical trials, increases mean arterial pressure through binding of nitric oxide (NO). We postulated that binding of NO by cross-linked hemoglobin (
Hb)
could also increase platelet deposition at sites of subintimal
injury.
Methods and Results Male Sprague-Dawley rats were
infused with Hb (0.88 g/kg, n=8) or with the NO synthase
inhibitor
NG-monomethyl-L-arginine
(L-NMMA, 30 mg/kg, n=7) before undergoing microsurgical carotid
endarterectomy. 111In-labeled
platelets were infused after endarterectomy,
and platelet deposition was measured 20 minutes later. In control
endarterectomized rats (n=8), mean platelet deposition was
7.7±0.7x106/mm2. Platelet
deposition was significantly increased above controls in rats that
received Hb
(13.2±0.9x106/mm2,
P=.0004) and in rats infused with L-NMMA
(13.9±1.0x106/mm2,
P=.0002). The increase was prevented by infusion of
L-arginine (150 mg/kg) immediately after Hb or
L-NMMA. To determine whether aspirin (ASA) blocked the increased
deposition induced by Hb, rats received oral ASA (10 mg/kg) 18
hours before endarterectomy. Platelet
deposition in animals receiving ASA alone was
6.4±0.9x106/mm2 (n=8). This was
significantly increased to
10.8±0.8x106/mm2
(P=.002) for the ASA-treated group that received Hb
at
the time of endarterectomy (n=8). The prolonged
bleeding times induced by ASA were unaffected by the infusion of
Hb.
Conclusions These data suggest that in a rat
endarterectomy model, Hb increases
platelet deposition at sites of subintimal injury by binding NO.
Increased deposition induced by Hb can be prevented by
administration of L-arginine but not by pretreatment with
aspirin.
Key Words: hemoglobin • nitric oxide • platelets • surgery
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Introduction |
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TopAbstractIntroductionMethods Results Discussion References |
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Several different preparations of purified human hemoglobin, cross-linked to prevent renal damage, are currently undergoing clinical trials as an oxygen-carrying agent.1 In addition to binding oxygen, hemoglobin is known to have a very high affinity for NO.2 3 Infusion of hemoglobin into animals increases blood pressure,4 5 6 7 8 at least in part because of binding of NO to hemoglobin, which also results in inhibition of NO-mediated vasodilation.
In addition to moderating blood pressure, NO inhibits platelet function through increasing levels of platelet cGMP.9 10 11 12 13 Several studies suggest that this interaction can be modulated by hemoglobin. Incubation of hemoglobin with monolayers of bovine endothelial cells promotes platelet adhesion,9 and platelets obtained from rabbits that have been infused with hemoglobin demonstrate an increased response to agonists ex vivo, as measured by aggregometry.13 In vivo studies have not
been done to determine whether hemoglobin, by binding to NO, can
promote platelet deposition at the site of
endothelial injury. We therefore investigated the
effects of Hb on platelet deposition in rats undergoing
microsurgical carotid endarterectomy.
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Methods |
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TopAbstractIntroductionMethods Results Discussion References |
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Reagents
The NO synthase inhibitor L-NMMA was obtained from Calbiochem-Novabiochem Corp and was administered intravenously in 0.5 mL normal saline. L-Arg (Sigma Chemical Co) was administered intravenously in 1.5 mL normal saline. Generic ASA powder (Mallinckrodt Chemical Works) was dissolved at a concentration of 10 mg/mL in polyethylene glycol (average molecular mass, 4 kD; Sigma Chemical Co).
Cell-free human
hemoglobin, lot 91196, was produced at Letterman
Army Institute of Research from outdated red blood cells by methods
previously described.14 Final concentration of the
hemoglobin solution was 7.0 g/dL. The hemoglobin was cross-linked
between -chains with bis(3,5-dibromosalicyl)fumarate,
preserved in Ringer's acetate, divided into aliquots, and frozen at
-80°C until the day of use.
The endotoxin concentration, as
determined by a kinetic turbidometric
assay using limulus amebocyte lysate,14 was 0.25 endotoxin
units/mL. Two samples of this preparation of hemoglobin (Hb;
molecular mass, 64 kD) passed the rabbit pyrogenicity test as
administered at the Center for Biologics Evaluation and Research (Food
and Drug Administration). The solution had no bacterial contamination,
as shown by a sterility test conducted over a period of 1 week. The
concentration of methemoglobin was 2.6%, and the pH was 7.35. The
P50 was 25 mm Hg.
Assay Procedures
Plasma hemoglobin was measured with the
Radiometer OSM 3
Hemoximeter. Platelets were enumerated in the Baker Hematology
Series Cell Counter System 9000.
Animals
Male Sprague-Dawley rats (mean weight, 434 g) were
allowed ad libitum access to water and standard rat chow until the time
of anesthesia. Animal procedures were approved by the
Walter Reed Army Institute of Research Laboratory Animal Care and Use
Committee and were conducted in accordance with the Guide for the
Care and Use of Laboratory Animals, NIH publication 86-23, revised
1985.
Preparation of Donor Platelets
Homologous platelets for each
experiment were obtained from
a separate donor rat, which was first anesthetized with
pentobarbital (50 mg/kg IP). A femoral artery catheter was then
inserted, and the donor rat was exsanguinated. Platelets from 12 mL
whole blood (anticoagulated with 2 mL acid citrate dextrose) were
labeled with 100 µCi 111In oxine (111In, MPI
Pharmacy Inc) by a modification of the labeling technique described by
Bauman and Landry.15 Briefly, platelet-rich plasma
was centrifuged at 300g for 10 minutes, the
supernatant was decanted, and the platelets were resuspended in
acid citrate dextrose anticoagulant. 111In was then
incubated with the platelet suspension for 30 minutes at 22°C.
The platelets were then washed and resuspended in 2 mL native
platelet-poor plasma. Mean labeling efficiency was 84±7%
(mean±SD).
Experimental Protocol
The animal model, which has been
described
previously,16 is a modification of the microsurgical
carotid endarterectomy model developed by Sasaki et
al.17 The experimental rats were initially
anesthetized with pentobarbital at a dose of 50 mg/kg IP, with
subsequent doses (20 mg/kg IP) used as necessary to maintain adequate
anesthesia. Surgical procedures were performed under x5 to
x20 magnification with a Zeiss OPMI 6 series operating microscope
(Carl Zeiss, Inc, Medical Products Division).
Fig 1
outlines the experimental protocol. The
experimental rat was secured, and a catheter was placed in the femoral
vein. Normal saline was infused through this line at a
maintenance rate of 2.75
mL · kg-1 · h-1 with a model
355
Orion syringe pump (Orion Research Inc). A femoral artery catheter was
then placed, and arterial pressure was monitored through a
transducer with a Datascope 2002A hemodynamic monitor.
The initial MAP was recorded, and then blood (2 mL) was withdrawn
from the arterial line for measurement of the platelet
count and plasma hemoglobin level. The blood was replaced by an equal
volume of normal saline. An initial bleeding time was then determined
by methods described below. A preinfusion MAP was recorded, and
group-specific reagents (L-NMMA or Hb) were then administered
intravenously. The dose of Hb administered (1.25
mL/100 g body wt, or 0.88 g Hb/kg) was equivalent to 10% of the
total hemoglobin in the rat. The Hb was infused through the
venous catheter during 5 minutes, with simultaneous
withdrawal of an equal amount of blood from the arterial
catheter to avoid volume changes. Postinfusion MAPs were recorded 2
minutes after the completion of each infusion.
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The trachea and both common carotid arteries were exposed through a midline cervical incision, and a tracheostomy tube was placed. The right (experimental side) and left (sham-operated side) common carotid arteries were isolated and clamped with low-pressure Kleinert microvascular clamps. Two-millimeter longitudinal arteriotomies were made in both the experimental and sham-operated arteries. Stay sutures of 10-0 nylon were used to expose the lumen of the experimental carotid artery, and an endarterectomy of the back wall was performed by placement of two transverse and parallel score marks in the intima 0.5 mm apart with the point of a 27-gauge needle and removal of the intervening intima with microforceps, exposing the vessel media. Both arteriotomies were then closed with running 10-0 nylon sutures.
111In-labeled platelets prepared from the donor rat were given intravenously (1.5 mL volume) after arteriotomy closures. MAP was recorded 2 minutes after the platelet infusion. Platelets were allowed to circulate for 10 minutes before removal of the arterial clamps and exposure to the carotid endarterectomy site for 20 minutes until termination of the experiment. A bleeding time was obtained at the beginning of platelet circulation time, and 2 mL of blood was then withdrawn from the arterial catheter for repeat determination of the platelet count and plasma hemoglobin level.
After the 20-minute circulation time, a final MAP was recorded. The animal was hemodiluted by withdrawal of blood from the arterial catheter and injection of saline into the venous catheter and was perfusion-fixed with the injection into the arterial line of 20 mL 4% formaldehyde/1% glutaraldehyde. Three-millimeter sections of both carotid arteries that contained the suture lines were excised and placed in fixative. Occlusive thrombi were not present in the carotid arteries of any of the experimental animals.
Bleeding Time
After excision of the distal 1 mm of the tail,
a
cotton-tipped applicator was gently applied to the wound every 30
seconds for the first 5 minutes and every 10 seconds thereafter until
cessation of oozing. The time at which blood was no longer evident on
the swab was taken as the bleeding time.
111In-Labeled Platelet Counting
The radioactivity
of the experimental and sham carotid specimens
and of blood samples was determined in a TM Analytic Gamma Trac 1193
gamma well-counter programmed for 111In photon
peak.
Endarterectomy Area Measurement
After measurement of
radioactivity, the experimental carotid
artery was opened longitudinally through the arteriotomy suture line
and mounted on a silicon rubber block with minutien pins. The specimen
was postfixed with osmium tetroxide, critical point–dried with
carbon dioxide, and coated with gold palladium. The artery was then
examined with scanning electron microscopy at x25 to x50
magnification, and an electron photomicrograph that contained the
entire endarterectomized segment was generated. This photomicrograph
was analyzed with computer image analysis software
(Optimus Bioscan Inc), and the surface area of the
endarterectomy was determined.
Primary Outcome Measure
The effect of the experimental
treatments on platelet
deposition was measured as the number of adherent
111In-labeled platelets per square millimeter of
the carotid endarterectomy. The radioactivity of
whole blood (cpm/µL) and platelet count were measured just before
euthanasia, and the specific activity of circulating platelets
(cpm/platelet) was calculated. The 111In-labeled
platelet activity per square millimeter of the
endarterectomy site was calculated by subtracting
the activity of the sham specimen from the activity of the experimental
specimen and dividing the net radioactivity by the area (in square
millimeters) of the endarterectomy sites.
Platelet deposition, defined as total platelets per square
millimeter on the endarterectomy site was computed
as platelet deposition
(platelets/mm2)=cpm/mm2xplatelets/cpm.
Experimental Groups
The experimental protocol was performed
in two series. Series 1
(n=40) consisted of six experimental groups: (1) control (n=8);
(2)
infusion of L-NMMA alone (30 mg/kg, n=7); (3) infusion of L-NMMA
followed by L-Arg (150 mg/kg, n=7); (4) infusion of Hb alone
(0.88 g/kg, n=7); (5) infusion of Hb followed by L-Arg
(n=7); and
(6) infusion of L-Arg alone (n=4).
Series 2 (n=16)
included two additional experimental groups: (1) oral
administration of ASA alone (10 mg/kg, n=8) and (2) oral administration
of ASA followed by intravenous Hb (0.88 g/kg). ASA
was administered orally through a gastric tube the evening before the
experiment to both donor and experimental animals, and Hb was
administered before the endarterectomy as in series
1. In each series, to control for possible time effects, block
randomization was used to assign animals to experimental groups. For
series 1, each block contained 6 rats (1 from each experimental group),
and for series 2, each block contained 2 rats (1 from each of the two
experimental groups).
Exclusions
A total of 59 rats underwent carotid
endarterectomy. Three rats died during the surgical
procedure and were excluded from the analysis. One rat
developed copious pulmonary secretions and respiratory distress
after platelet administration, and 1 rat became hypotensive after
Hb and L-Arg infusion. The third rat, a recipient of L-NMMA and
L-Arg, developed hypotension and died after the carotid arteries were
unclamped.
Statistical Analysis
Summary results for continuous outcomes
are reported as
mean±SEM. ANOVA for independent groups was used to determine the
overall statistical significance of differences in mean platelet
deposition.18 19 Analysis was done on transformed
(square root and rank) and untransformed data with similar results. The
unpaired t test was used for individual comparisons in mean
platelet deposition between two groups by use of the ANOVA mean
square error for untransformed data. Overall differences between the
initial and final bleeding times (logarithmically transformed) among
groups were assessed by repeated measures ANOVA. Paired t
tests were used for mean changes in bleeding time for a single
experimental group (by repeated measures mean square error). All
reported probability values are two-sided; no adjustment was made
for multiple testing. A value of P<.05 was considered to be
significant. Statistical calculations were carried out with Minitab
statistical software.20
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Results |
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TopAbstractIntroductionMethods Results Discussion References |
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Effect of L-NMMA and
Hb on Subintimal Platelet
Deposition We hypothesized that in animals infused with
Hb,
platelet deposition could be increased because of binding of NO to
circulating free hemoglobin. To determine whether increased
platelet deposition could be detected in this animal model if NO
were inhibited, one group was infused with L-NMMA, a known
inhibitor of NO synthase. In control rats, mean
platelet deposition at the endarterectomy site
was 7.7±0.7x106/mm2 (Fig 2),
and in the group receiving L-NMMA, platelet
deposition was significantly increased by 81% to
13.9±1.0x106/mm2
(P=.0002). In the group infused with Hb, platelet
deposition was
13.2±0.9x106/mm2, an
increase of 71% above controls (P=.0004).
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L-Arg, a
substrate for NO synthase, was infused immediately after
L-NMMA or Hb to determine whether sufficient NO could be
generated to prevent increased platelet deposition. In one group
that received L-Arg alone, platelet deposition was less than that
of the control group, although the reduction was not significant
(6.1±1.1x106/mm2 versus
7.7±0.7x106/mm2,
P=.42). In the group receiving Hb followed by L-Arg,
platelet deposition was
4.8±0.9x106/mm2, a value
less than that of the control group (P=.028). Platelet
deposition was 7.5±0.6x106/mm2
in the group infused with L-NMMA and L-Arg and was not different from
the control group (P=.88). Thus, infusion of L-Arg into
groups that had received Hb or L-NMMA successfully blocked the
increase in deposition, resulting in platelet deposition that was
either the same as or less than that of the control group.
Additional
studies were performed to determine whether ASA blocked the
increased deposition noted with Hb. Two groups received ASA 18
hours before carotid endarterectomy; one group also
received Hb just before the surgery. In the ASA-treated group
that also received Hb, platelet deposition was
10.8±0.8x106/mm2. This was
significantly higher than the value of
6.4±0.9x106/mm2 for the group
receiving ASA alone (P=.002) and for the control group
(7.7±0.7x106/mm2,
P=.016). There was no difference in platelet deposition
between the control group and the rats receiving ASA alone
(7.7±0.7x106/mm2 versus
6.4±0.9x106/mm2,
P=.28).
Bleeding Times and Platelet Counts in Experimental
Groups
Comparison of initial and final bleeding times within groups
showed that for the group receiving L-NMMA, the final bleeding time was
decreased by 10% from that of the initial bleeding time (673±19
versus 604±26 seconds, P=.012). In all other groups,
including those receiving ASA alone and ASA and Hb, the final
bleeding time did not differ significantly from the initial bleeding
time (Table 1).
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A comparison of bleeding times among the
groups showed that the initial
bleeding time in the two groups receiving ASA was significantly higher
than the initial bleeding time for the control group
(P<.01). For the group receiving ASA followed by Hb
at the time of endarterectomy, the initial and
final bleeding times were not different from the group receiving ASA
alone (P>.5).
Although no significant differences in
platelet counts were
obtained between the beginning and end of the experimental protocol in
any of the groups, platelet counts tended to be lower at the end of
the experiment in those groups that received Hb (Table
1
).
Hemodynamic Effects of L-NMMA and of
Hb
In control rats, the MAP ranged from 88 to 102 mm Hg
throughout
the experiment (Table 2). In the group receiving
Hb and L-Arg, the MAP increased from 94±5 to 118±6
mm Hg after
infusion of Hb (P=.0042); after the infusion of
L-Arg,
the MAP was not significantly different from the baseline value
(P=.079). Similarly, for rats that received both L-NMMA and
L-Arg, MAP was significantly increased after infusion of L-NMMA
(P=.0012), but shortly after the infusion of L-Arg, it was
reduced to values that were not different from baseline
(P=.206). Pretreatment of rats with ASA before the infusion
of Hb did not affect the hemodynamic
response.
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In the Hb-treated group, the MAP measured after the
infusion
of 111In-labeled platelets was not different from
that measured at the termination of the experiment. In all other
groups, the MAP decreased during this time. However, the MAP did not
appear to influence platelet deposition. There was no correlation
between platelet deposition and the MAP measured immediately after
the infusion of 111In-labeled platelets
(r=-.172, P=.22) or between platelet
deposition and the absolute decrease in MAP that occurred during the
time between the infusion of the 111In platelets and
the termination of the experiment (r=.096,
P=.48).
Plasma Hemoglobin Levels
In the three groups of rats
receiving Hb (n=22), the
mean preinfusion plasma level of hemoglobin was 86±18 mg/dL. When
measured at the end of the experiment (ie, 100 minutes after infusion
of Hb), the mean plasma hemoglobin level was 618±49 mg/dL
(P<.0001). The mean plasma hemoglobin level for rats (n=14)
not receiving Hb was 71±16 mg/dL at the beginning of the
experiment and 157±89 mg/dL at the end; the increase was not
statistically significant (P=.143).
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Discussion |
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TopAbstractIntroductionMethods Results Discussion References |
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The present study demonstrates that in the rat microsurgical model, the infusion of the NO synthase inhibitor L-NMMA or
Hb before endarterectomy significantly
increases platelet deposition. Administration of L-Arg after L-NMMA
or Hb abrogated this response, strengthening the association of
platelet deposition with NO availability and indicating a possible
role for L-Arg in modifying the Hb-associated increase in
platelet deposition.
Platelet deposition in ASA-treated rats infused with Hb was
increased above that of control rats or rats receiving ASA alone,
indicating that ASA does not block the proaggregatory effects of
Hb. These findings are consistent with recent studies by
Broekman et al,21 who demonstrated that NO-mediated
inhibition of platelet reactivity was ASA independent.
ASA caused a significant increase in bleeding time, which was sustained
even with the infusion of Hb. Thus, the bleeding time did not
accurately reflect the increased platelet deposition at the site of
the endarterectomy. These data are
consistent with other reports demonstrating that the bleeding
time does not predict the hemostatic function of platelets in
animal models22 or in patients subjected to
surgery.23 24 25
MAPs paralleled those expected with changes in NO availability,
with significant increases occurring shortly after infusion of L-NMMA
or Hb, followed by restoration to preinfusion values after the
administration of L-Arg. Infusion of L-Arg alone caused no significant
change in MAP. The effect of Hb on MAP in this model was similar
to that found in conscious rats.4
Rats received Hb at a dose that was 
10% of the whole blood
hemoglobin; by the end of the experiment, the plasma values were
equivalent to 5% of the total hemoglobin concentration. The relatively
short half-life is consistent with that found in other
studies.5 In control rats, plasma hemoglobin levels
remained at 
1% of the total hemoglobin throughout the experiment.
Similar plasma hemoglobin values have been reported in control rats in
other studies; this may be due to an increased fragility of rat
erythrocytes, which results in lysis either in vivo or ex
vivo.26
The role of NO in regulating platelet function has been suggested in several in vivo and in vitro studies. In vitro studies have confirmed that NO can partially inhibit platelet aggregation27 or platelet adhesion to endothelial cells.9 These effects can be reversed by the addition of hemoglobin.2 9 Hemoglobin does not appear to have a direct effect on platelet function in vitro.27 28
In one study, platelet deposition, as assessed by electron microscopy, was increased on injured endothelium of rabbits infused with L-NMMA.29 Yao et al30 found that infusion of L-NMMA into dogs with a mechanical injury and stenosis of either a coronary artery or femoral artery resulted in recurrent platelet aggregation as assessed by cyclic flow variation; the aggregation was prevented by infusion of L-Arg. Groves et al31 performed bilateral carotid angioplasty on anesthetized pigs and compared aggregation of 111In-labeled platelets between controls and animals receiving the NO donor SIN-1. Compared with controls, SIN-1 significantly reduced platelet aggregation at sites of both superficial and deep arterial injury. The use of SIN-1 was further accompanied by a significant increase in platelet cGMP.
It is unknown whether the increased platelet deposition associated
with the infusion of Hb is a dose-dependent phenomenon, since
only one concentration of Hb, which was calculated to be
equivalent to a one-unit transfusion in a human recipient, was used
in this study. The lowest doses at which NO will be completely
inhibited have not been established. It is also unknown whether the
effect of hemoglobin on platelet deposition is a property of the
hemoglobin preparations that are now in clinical trials. The
significance of these findings therefore remains to be investigated in
human recipients of commercial products.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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The authors gratefully acknowledge the assistance and support provided to this project by the following individuals at the Walter Reed Army Institute of Research: Edward Asafoadjei, William Mondy, and Marcos Gomez, Department of Ultrastructural Pathology; Stephen Van Albert, Division of Biometrics; and William H. Baker, LTC, DVM, Division of Pathology.
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Footnotes |
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The opinions expressed herein are solely those of the authors and do not reflect official views or policies of the Department of the Army or the Department of Defense.
Received July 26, 1995; revision received November 10, 1995; accepted November 15, 1995.
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