© 1998 American Heart Association, Inc.
Original Contributions |
Pretreatment of Rabbits With Either Hirudin, Ancrod, or Warfarin Significantly Reduces the Immediate Uptake of Fibrinogen and Platelets by the Deendothelialized Aorta Wall After Balloon-Catheter Injury In Vivo
From the Department of Pathology, McMaster University Health Sciences Centre, Hamilton, Ontario, Canada.
Correspondence to Dr Mark W.C. Hatton, Department of Pathology (HSC- 4N67), McMaster University Health Sciences Centre, 1200 Main St W, Hamilton, Ontario, Canada L8N 3Z5. E-mail hattonm{at}mcmaster.ca
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Abstract |
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Abstract—Fibrinogen and platelets rapidly saturate the exposed subendothelium of a freshly deendothelialized aorta in vivo. As thrombin generated within the site of injury is largely responsible for fibrin(ogen) deposition, we questioned whether various anticoagulant treatments would inhibit uptake of both fibrinogen and platelets in vivo. Rabbits were anticoagulated by pretreatment with either Warfarin, Ancrod, or recombinant hirudin. Each anesthetized, anticoagulated (or saline-injected control) rabbit was injected IV with rabbit 51Cr-platelets and 125I-fibrinogen before a balloon-catheter deendothelializing (or sham) injury of the thoracic aorta. At 10 minutes after injury, the rabbit was exsanguinated and the aorta excised. Platelet adsorption by the deendothelialized aorta surface was substantially reduced in anticoagulated rabbits (controls, 2.2x105/mm2; Warfarin-treated, 1.2x105/mm2; Ancrod-treated, 5.3x104/mm2; r-hirudin–treated [5 mg/kg], 5.3x104/mm2), and a significant reduction of fibrinogen associated with the platelet layer (from 5.3 to 1 to 2 pmol/cm2) and within the underlying intima-media layer (from 16.9 to 5 to 6 pmol/cm2) was observed in the r-hirudin–and Warfarin-treated rabbits. The pattern of aorta-deposited 51Cr-platelets and 125I-fibrin in the anticoagulated rabbits corresponded well with an assessment by transmission electron microscopy of aortic tissue samples. We conclude that
70% of fibrinogen uptake is thrombin
dependent and that 80% of platelet adsorption depends on
codeposited fibrin(ogen) during the 10-minute interval after balloon
injury. Pretreatment with an agent that interferes with either thrombin
or fibrin production will inhibit the immediate interaction of
fibrinogen and platelets with the freshly exposed
subendothelium.
Key Words: aorta • deendothelializing injury • platelets • fibrinogen • anticoagulants
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Introduction |
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In 1973, Baumgartner1 showed that circulating platelets rapidly carpeted the deendothelialized surface of a rabbit aorta after an inflated balloon catheter had been drawn along the vessel lumen. The surface of the ballooned aorta was essentially saturated with platelets by 10 minutes after injury, although deposition of fibrin was not obvious. Later, Groves et al2 quantified the deposition of 51Cr-labeled platelets on the deendothelialized aortas of rabbits that at various times after balloon-catheter injury had been preperfused, first with a heparin-containing physiological solution and then with 4% glutaraldehyde to fix the tissue. These authors found that the exposed subendothelium was saturated by
45 000 platelets per square millimeter, which, when viewed by
scanning electron microscopy, appeared as a monolayer, with most
platelets spread on the vessel surface. Later, these
authors3 reported that heparin, administered (500
U/kg IV) 10 minutes before inducing a balloon-catheter injury,
decreased slightly the accumulation of platelets at the exposed
subendothelium in vivo, and Dejana et
al4 showed that heparin significantly prevented
platelets from binding to the exposed
subendothelium of the everted rabbit aorta segments
rotated in vitro. In the latter study, it was concluded that thrombin
was involved in the accumulation of platelets at the surface of the
exposed subendothelium. Since then, several
reports5 6 have identified thrombin as a primary
agent responsible for platelets associating with the ballooned
rabbit aorta surface and promoting platelet thrombus formation in
vitro. These researchers have studied platelet behavior over a wide
range of wall shear rates using a perfusion chamber technique.
Recently, however, Gast et al7 have concluded,
from perfusion chamber experiments, that thrombin is not involved in
platelet thrombus formation immediately after injury but is
involved later in thrombogenesis on the exposed aortic
subendothelium. The behavior of plasma 125I-fibrinogen has also been studied in rabbits after a balloon-catheter injury to the aorta.8 9 Fibrinogen saturated the deendothelialized aorta surface in vivo too rapidly (<5 minutes after injury) for a precise time curve to be measured. Subsequently, we demonstrated from studies in vitro that the quantity of fibrinogen bound by the exposed subendothelium was dependent directly on the amount of active thrombin associated with that surface.10 Indeed, an enhanced uptake of fibrinogen was maintained throughout the reendothelialization process, an event that endured for at least 20 months in the rabbit aorta.11
As fibrinogen supports the aggregation of thrombin-activated platelets, we questioned to what extent the deposition of plasma fibrinogen and the deposition of platelets at the ballooned aorta surface in vivo were interdependent. Is a single factor, namely thrombin, responsible for the deposition of both fibrinogen and platelets? In this study, we compare the uptake of fibrinogen with that of platelets over the first 10 minutes after balloon-catheter injury to the rabbit aorta in vivo using a quantitative and a morphological approach. An interval of 10 minutes between balloon injury and exsanguination was chosen because it allows sufficient time for saturation of the ballooned aorta by both fibrin(ogen) and platelets. As we are uncertain of the ratio of adsorbed fibrinogen and deposited fibrin by the aorta wall, we have referred to this material as fibrin(ogen). In addition, we have compared the adsorption of fibrin(ogen) and platelets to the deendothelialized aorta by using rabbits that had either not received an anticoagulant treatment or had been anticoagulated using one of four different procedures to discover any relationship that directly links these responses to injury. The chosen anticoagulants were recombinant hirudin, a highly specific thrombin inhibitor12 ; Ancrod, which acts by effectively removing fibrinogen from the circulatory system; and Warfarin, which selectively inhibits production of active vitamin K–dependent proteins by the liver.
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Methods |
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Preparation and Labeling of Rabbit Fibrinogen
Fibrinogen was isolated from ACD-anticoagulated13 plasma from NZW rabbits using first a ß-alanine precipitation method14 and purified further by DEAE cellulose chromatography with a Ca2+-containing buffer system.15 By these means, rabbit fibrinogen with >97% thrombin clottability was obtained.8
Fibrinogen was radiolabeled using an Iodo-gen-coated glass
vial16 as follows: 100 µg of protein (in 300
µL 0.1 mol/L sodium phosphate, pH 7.4) was placed in a flat-bottomed
glass vial previously coated with 5 µg Iodo-Gen (Pierce Chemical Co)
and containing a small (5 mm) stir bar. A 10-µL vol (1 mCi) of
[125I]NaI (ICN Pharmaceuticals) was added and
the reaction was allowed to progress for 2 minutes at room temperature.
125I-fibrinogen was dialyzed for 18 hours against
4x250 mL 0.01 mol/L sodium phosphate–buffered 0.15 mol/L NaCl, pH
7.4, at 4°C, stored at 4°C, and used for experiment within 4 days
of labeling.
Preparations of rabbit fibrinogen and 125I-fibrinogen were tested for purity before and after reduction by ß-mercaptoethanol using PAGE in the presence of 0.1% SDS.17 Plasma samples from some Ancrod-treated rabbits were analyzed for fibrinogen as follows. Unreduced diluted plasmas were electrophoresed using SDS-PAGE and the gel content was electroblotted to Immobilon (Millipore Corp). The blot was developed by using first a polyspecific anti-rabbit fibrinogen antibody (raised in a laying hen and isolated from egg yolks18 ), followed by an alkaline phosphatase–linked anti-chicken IgG (Zymed Inc), and then an appropriate substrate, BCIP, and complexing agent, NBT (both obtained from BioRad). Reactive bands were scanned by using a Howteck Scanmaster "3+" (Howteck Inc) and analyzed using BioImage Whole Band Analysis software (Millipore Corp). The fibrinogen content of rabbit plasma samples was determined quantitatively using an ELISA described previously.19
Anticoagulant Drugs
Warfarin (3-[
-acetonylbenzyl]-4-hydroxycoumarin, Na salt)
was purchased from Sigma Chemical Company. Ancrod (also known as
"Arvin") was obtained from Knoll Pharma Inc as a sterile solution
(70 IU/mL). Recombinant desulfato-hirudin (r-hirudin; CGP39393) was a
gift from Ciba Pharmaceuticals (Horsham, Sussex, UK).
Preparation and 51Cr Labeling of Rabbit
Platelets
For each experiment that required
51Cr-platelets, the platelet fraction,
obtained from exsanguinated blood of a donor NZW rabbit (weight range
2.3 to 3.0 kg), was radiolabeled with 51Cr before
injecting into a second NZW rabbit (designated the experimental
rabbit). Procedures used for isolating, washing, and radiolabeling
platelets from freshly drawn, ACD-anticoagulated rabbit blood were
described previously.20 21 After a fourth wash,
the radiolabeled platelets were suspended in 10 mL of
platelet-poor rabbit plasma. For Ancrod-treated rabbits,
51Cr-labeled platelets were suspended in 10
mL of freshly prepared, Ancrod-treated rabbit plasma (after removal of
the fibrin clot). Measurement of the
51Cr-platelet count (before injection) was
determined using a Coulter counter, and radioactivity content using a
Minaxi-Autogamma 5000 counter (Canberra-Packard Ltd). The specific
radioactivity (ie, number of platelets per
51Cr count per minute) ranged between 2000 and
8000 platelets per cpm (mean: 4900±1385 platelets per cpm;
n=40).
Anticoagulation of Rabbit Circulation
All proposals for experiments using NZW rabbits were first
approved by the Animal Research Ethics Board (McMaster University) and
were conducted within the guidelines recommended for recovery surgery
by the Canadian Council on Animal Care.22 Rabbits
were injected (ear vein) with one of five anticoagulant treatments, ie,
Warfarin, Ancrod, r-hirudin (low or high dose), or Ancrod followed by
r-hirudin (low dose); control rabbits were injected with sterile
saline. No serious bleeding complications were seen during or after any
of the above treatments or during or after sample injections or
balloon-catheter treatment.
Warfarin
Each rabbit was injected with Warfarin at 40 mg/kg, dissolved in
sterile saline immediately before injection. The same dose was repeated
2 days later. Warfarin-treated rabbits were taken for balloon or sham
injury on either day 4 or 5 after the start of Warfarin treatment.
Blood samples (each 1 mL) were taken from an ear artery into 0.25 mL
ACD before Warfarin was injected and again on day 4 (or 5) after the
start of Warfarin treatment for measurement of the prothrombin time.
The ratio of prothrombin clotting times, post-Warfarin plasma (mean,
24.6 seconds)/pre-Warfarin plasma (10.7 seconds), amounted to 2.3
(±0.3).
Ancrod
Rabbits were injected with Ancrod at 1 IU/kg. One hour after the
first dose, each rabbit was given a second dose (1 IU/kg), and 2 hours
after the first dose, a third dose (2 IU/kg) was given. The rabbit was
then housed for 4 hours. At 6 hours after the start of Ancrod
treatment, a blood sample was taken into a known volume of ACD and the
rabbit was anesthetized in preparation for balloon or sham
injury (see below). Platelet levels, taken before and after 6 hours
of Ancrod treatment, were measured by CBC analysis of weighed
blood samples (to determine the dilution by ACD) by the Section of
Laboratory Medicine (Chedoke-McMaster Hospitals). Also, plasmas from
the same blood samples were analyzed for fibrinogen content as
described above.
r-Hirudin
r-Hirudin (used either as a low dose of 1.2±0.2 mg/kg, or a
high dose of 5.0±0.2 mg/kg) was injected 3 minutes before the
anesthetized rabbit was subjected to either a balloon or sham
injury. Activated partial thromboplastin times were measured in
a parallel series of experiments23 on plasma
samples taken from r-hirudin–treated NZW rabbits (1 mg/kg) at 5 to 20
minutes after injection; activated partial thromboplastin times
for control (ie, prehirudin) plasmas and posthirudin plasmas were
41.8±2.9 and 84.0±19.5 seconds, respectively.
Ancrod/r-Hirudin
Rabbits were treated with Ancrod as described above. Each rabbit
was anesthetized at 6 hours after the start of Ancrod
treatment. r-Hirudin (1 mg/kg) was injected 3 minutes before the
rabbit was subjected to a balloon-catheter injury.
Saline
Rabbits, after receiving a dose of anesthetic, were injected
with 1 mL saline before balloon-catheter or sham injury.
Uptake of Platelets and Fibrinogen by the Aorta Wall
Immediately After a Balloon-Deendothelializing Injury
In Vivo
A blood sample was taken from the ear artery (1 mL) of each
NZW rabbit (range: 2.1 to 3.3 kg; mean: 2.6±0.3 kg) into 0.25 mL ACD,
weighed, and measured for platelet content.
Rabbits were anesthetized by intravenous injection
of sodium pentobarbital (up to 35 mg/kg). After shaving the front of
the neck and inner thigh areas, a carotid artery was cannulated (PE 200
tubing) and a femoral artery isolated. At 15 minutes after inducing
anesthesia, each r-hirudin–or Warfarin-treated rabbit was
injected (through the carotid cannula) with 10 mL of
51Cr-labeled platelets (containing 4 to
10x106 cpm; mean:
6.0±1.5x106 cpm). The line was then immediately
flushed with 1 mL of sterile saline before injecting 1 mL of
125I-fibrinogen (10 to
20x106 cpm; 14.9±3.3x106
cpm; 18.7±9.4 µg) in sterile saline. Again, the line was flushed
with 1 mL of sterile saline. All Ancrod-treated rabbits were injected
only with 51Cr-platelets followed by 1 mL of
sterile saline.
In all recipient rabbits, the proportion of 51Cr-platelets added to the circulation was calculated from the specific radioactivity of the preparation of 51Cr-platelets, using the known quantity of 51Cr-platelets injected and the blood platelet count and weight (and hence the blood volume) of the recipient rabbit; the proportion of donor 51Cr-platelets after injection amounted to 33.5±1.4% (n=40) of the total platelet population.
At 5 minutes after injection, a blood sample (carotid artery; 1 mL) was taken into 0.25 mL ACD and the rabbit was given either a deendothelializing injury or a sham injury to the thoracic aorta. For the balloon treatment, a Fogarty balloon catheter (Baxter Healthcare Corp; type 12-040-4F) was inserted into an exposed femoral artery and maneuvered into the aorta. The thoracic aorta was subjected to two passes with an expanded balloon volume (containing 1 mL of sterile saline) and the artery ligated as described previously.9 For the sham-injury treatment, a femoral artery was ligated but not penetrated. A timer was started as the femoral artery was ligated. At 10 minutes after femoral ligation, a second blood sample was taken before the rabbit was rapidly exsanguinated through the carotid cannula. The chest was opened and the thoracic aorta isolated, flushed with 10 mL of BSA-minimum essential medium (with Earle's salts and L-glutamine), and then excised.
Adventitial fat was removed and the thoracic aorta divided into eight 1-cm-long segments (segment 1, arch end; segment 8, diaphragm end). Segment 4 from each aorta was placed into 4% (wt/vol) paraformaldehyde for TEM studies. The remaining segments were processed as follows: After measuring the surface area of each segment using a digital planimeter (Lasico), the luminal surface, which for sham-injured aortas included the endothelium and associated basement membrane24 and for deendothelialized aortas included the platelet layer,8 was stripped cleanly from the aorta using cellulose acetate paper as a Häutchen preparation.25 The underlying subendothelium, referred to as the intima-media layer, was then stripped from the vessel wall using Bergh forceps.26
The vessel layers of seven individual segments from each aorta were each placed in a plastic-capped vial (Biovial, Beckman) and measured for radioactivity content. The 125I counts were corrected for crossover of 51Cr into the 125I channel. The corrected 125I radioactivity per unit area, ie, cpm/cm2 of aorta layer, was compared with the respective radioactivity content of 1 mL of blood at exsanguination. The quantity of bound fibrinogen per square centimeter was calculated assuming a hematocrit of 42% and a blood concentration of 3.58 µmol/L fibrinogen19 for a healthy NZW rabbit. By using the known proportion of 51Cr-platelets in the circulation for each 51Cr-platelet preparation and their specific radioactivity (ie, number of platelets per cpm), the mean number of adsorbed platelets per square millimeter could be calculated from the 51Cr content of the cellulose acetate layer obtained from each of the seven 1-cm-length segments of each thoracic aorta.
Uptake of 125I-Fibrinogen by the Aorta Wall During
Exsanguination
Rabbits that had not received an anticoagulant treatment were
anesthetized and a carotid cannula was inserted as described
above. A balloon or sham injury was performed, and the clock was
started at the moment of femoral ligation as the balloon (or sham)
injury was completed. An intravenous dose of
125I-fibrinogen (19 µg in 1 mL of sterile
saline) was injected at either 7 minutes, 8 minutes, or 9 minutes after
the clock was started. An intra-arterial blood sample (2
mL into 0.5 mL ACD) was taken at 10 minutes, and the rabbit was
exsanguinated immediately through the carotid cannula. The aorta was
isolated, flushed with BSA-minimum essential medium Eagle, and excised
as described above. The surface area and radioactivity content of the
Häutchen preparation of the platelet layer (of
balloon-injured aortas) or endothelium (of the
sham-injured aortas) and of the underlying intima-media were measured,
and the fibrinogen content per square centimeter of aorta wall was
calculated relative to the radioactivity content of blood at
exsanguination as described above.
Measurement of Prothrombin Time
After collection, blood samples were weighed (to determine the
dilution due to ACD) and centrifuged (10 000 rpm, 2 minutes)
to separate the plasma fraction. An automated plasma coagulation
procedure described by Organon Technica, the supplier of Simplastin
Excel (a form of thromboplastin), was used with a coagulation
analyzer (Behring) to determine the prothrombin time for the
ACD-plasma samples.
Morphology of the Platelet Layer
One-millimeter-thick, full-circumference rings of
paraformaldehyde-fixed aorta wall were prepared for TEM
as described previously.2 3 The samples were
postfixed in buffered 1% osmium tetroxide for 1 hour, stained in 2%
aqueous uranyl acetate for 1 hour, dehydrated through graded ethanol,
and embedded in Spurr's resin. One-micrometer-thick
sections, stained with toluidine blue, were examined by light
microscopy, and areas showing maximal platelet accumulation with
the luminal surface were selected for examination by TEM. Thin sections
(60 to 90 nm), stained with lead citrate, were viewed on a Philips TEM
301 transmission electron microscope (Eindhoven, the Netherlands).
Statistical Calculations
When appropriate, data are given as the mean (±SEM). Data in
Tables 1
and 2 were compared by ANOVA
(QuattroPro version 7.0; one-way).
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Results |
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Properties of 125I-Fibrinogen
Rabbit 125I-fibrinogen, as shown by SDS-PAGE (Fig 1
), consisted essentially of one
single band, which barely entered the 7.5% polyacrylamide gel.
After reduction by ß-mercaptoethanol, the three component chains of
fibrinogen, A, Bß, and 
, appeared wholly separated as bands of
Mr estimated at 65 kD, 58 kD, and 56 kD,
respectively. The Coomassie-stained bands matched well with those of
the autoradiograph (compare lane 1 with 2 and lane 3 with 4 in Fig 1).
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Effect of Ancrod on the Clearance of 125I-Fibrinogen
From the Rabbit Circulation
A three-dose regimen was used in the study of the effect of Ancrod
pretreatment of rabbits used for studying the behavior of
51Cr-platelets after balloon-catheter injury
(see below). Blood samples were taken before and at 6 hours after the
start of Ancrod treatment. ELISAs of the fibrinogen content of the
plasmas were performed in the presence of appropriate quantities of
purified rabbit fibrinogen on the same 96-well plates, and corrections
were made for ACD content (Fig 2a). From
the ELISAs, plasma fibrinogen levels in the Ancrod-treated rabbits was
37% of that in normal rabbits. However, as shown by the
immunoblot in Fig 2b, the polyspecific anti-fibrinogen
antibody could not distinguish between intact fibrinogen and
fibrinogen-related degradation products. Densitometric measurements
of similar immunoblots of plasmas taken from five rabbits
before and at 6 hours after Ancrod treatment showed that the content of
intact fibrinogen in plasma before Ancrod treatment ranged from 91.0%
to 99.8% (mean: 95.0±1.7%) of all stained bands, whereas
"intact" fibrinogen in plasma at 6 hours after Ancrod treatment
amounted to 38.0±0.6%. Thus, the plasma level of intact fibrinogen in
plasma at 6 hours after starting the three-dose regimen of Ancrod
treatment is calculated to be 14% of normal plasma levels (ie, 38%
of 37% of normal plasma level), equal to 1 µmol/L.
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Measurements of platelet levels indicated that at 6 hours after the start of Ancrod treatment, the platelet level (3.3±0.2x1011/L) was insignificantly different from that before treatment (3.7±0.3x1011/L).
Uptake of 51Cr-Platelets and
125I-Fibrinogen by the Surface of the Aorta Wall After a
Deendothelializing Injury to Saline-Treated
Rabbits
At 10 minutes after a balloon-catheter injury to the aorta of a
saline-treated rabbit, the aortic surface contained an abundance of
adsorbed platelets and fibrinogen, as determined by the
51Cr- and 125I
radioactivity contents, relative to the aortic surface of the
sham-operated rabbit. The platelets adsorbed by the
deendothelialized aortas of eight rabbits
amounted to a mean of 222 800/mm2 (Table 1
). By
contrast, platelets adsorbed by the aorta surface in the
sham-injured rabbit (ie, the endothelium) amounted to
768/mm2. The fibrin(ogen) content of the
platelet layer was calculated to be 5.3
pmol/cm2 in aortas from balloon-injured rabbits,
and 0.7 pmol/cm2 with the aortic
endothelium of sham-injured rabbits (Table 2).
After the platelet layer was removed, the underlying
subendothelium (isolated as the intima-media layer) was
found to contain significant quantities of 51Cr
and 125I radioactivities. As the platelet
layer had been cleanly removed by cellulose acetate
paper8 and the specific radioactivity of the
51Cr-labeled platelet preparation was known,
the 51Cr data were calculated as "platelet
equivalents" per square millimeter, ie, the number of platelets
equivalent to the measured 51Cr radioactivity per
square millimeter. The intima-media layer of the eight balloon-injured
aortas contained 237 000 platelet equivalents per square
millimeter in contrast to 616/mm2 determined for
that of the sham-injured aortas (Table 1). The mean fibrin(ogen)
content of the balloon-injured aortas amounted to 16.9
pmol/cm2, in contrast to 0.4
pmol/cm2 of sham-injured aorta (Table 2).
Effect of Pretreatment with r-Hirudin on Uptake of
51Cr-Platelets and 125I-Fibrinogen by the
Aorta Wall After a Deendothelializing Injury
Deendothelialized aortas from
r-hirudin–treated rabbits contained significantly fewer adsorbed
platelets compared to those from saline-treated rabbits, decreasing
to 121 000/mm2 (ie, 55% of the value
measured for saline-injected, ballooned aortas) in rabbits pretreated
with low dose, and to 53 000/mm2 (24%) in
rabbits pretreated with high dose of r-hirudin (Table 1). Fibrin(ogen)
deposition in the platelet layer was similarly decreased to 2.0
pmol/cm2 (38% of the saline-injected ballooned
aorta) and 0.9 pmol/cm2 (17%) in the low- and
high-dose r-hirudin–treated rabbits, respectively (Table 2).
Compared with aortas from saline-treated rabbits, the platelet
equivalents per square millimeter of intima-media from ballooned aortas
of low- and high-dose rabbits was decreased significantly to 41 000
(17% of the saline-injected, ballooned aorta) and 16 000 (7%),
respectively (Table 1). Fibrin(ogen) associated with the intima-media
layer (6.7 and 6.4 pmol/cm2) of low- and
high-dose rabbits was decreased significantly to 40% and 38%,
respectively, of the values measured for the intima-media from
saline-treated rabbits (Table 2). Compared with the low dose, the use
of a high dose of r-hirudin did not significantly decrease further the
uptake of fibrinogen by the deendothelialized
aorta.
Effect of Pretreatment With Ancrod on the Uptake of
51Cr-Platelets by the Aorta Wall After a
Deendothelializing Injury
The plasma levels of fibrinogen were reduced by Ancrod treatment
to 14% of normal plasma fibrinogen at the time of balloon-catheter
treatment. In parallel, the adsorption of platelets by the exposed
subendothelium was decreased significantly to 24% of
the value measured for the saline-treated, ballooned rabbits, ie, from
222 800/mm2 (saline-treated) to
52 700/mm2 (Table 1). After removal of the
platelet layer, the platelet equivalents associated with the
underlying intima-media had decreased to
15 400/mm2, 7% of the value for
saline-injected, ballooned aortas.
The effect of injecting r-hirudin into Ancrod-treated rabbits decreased
the deposition of platelets on the ballooned aorta
subendothelium to 42 000/mm2,
which is 19% of the mean value determined for saline-injected
ballooned aortas (Table 1). This decrease was similar to that observed
with either Ancrod alone or high-dose r-hirudin alone. However, the
platelet equivalents associated with the intima-media layer
decreased to 8800/mm2 (3.7%) in the
Ancrod/r-hirudin–treated rabbits, a value substantially less than that
in either Ancrod or low-dose r-hirudin pretreatments.
Effect of Pretreatment With Warfarin on the Uptake of
51Cr-Platelets and 125I-Fibrinogen by the
Aorta Wall After a Deendothelializing Injury
In four Warfarin-treated balloon-injured rabbits, platelet
deposition on the surface of the
deendothelialized aorta was decreased to
121 600/mm2 (ie, 55%) and fibrin(ogen) to 1.4
pmol/cm2 (26%) compared with aortas from
saline-treated, balloon-injured rabbits (Tables 1 and 2).
From the 51Cr radioactivity, the platelet
equivalents associated with the intima-media were decreased
significantly in the Warfarin-pretreated rabbit to
30 000/mm2 (ie, 12.5% of the saline-treated;
Table 1). Fibrin(ogen) associated with the intima-media was decreased
to 5.5 pmol/cm2 (30%; Table 2).
Uptake of 125I-Fibrinogen by the
Deendothelialized Aorta Wall During
Exsanguination
An intravenous dose of
125I-fibrinogen was injected into
nonanticoagulated rabbits at either 7, 8, or 9 minutes after inflicting
a balloon (or sham) injury to quantify the fibrin(ogen) associated with
the aorta surface (cellulose acetate layer) and underlying intima-media
at 3 minutes, 2 minutes, and 1 minute before exsanguination (Fig 3). These data allowed extrapolation to
the time of the start of exsanguination so that the quantity of
fibrin(ogen) deposited on and within the aorta wall during the
exsanguination process could be assessed. Compared with the
fibrin(ogen) content at 10 minutes after balloon injury (ie, 5.3
pmol/cm2 of platelet layer; 16.9
pmol/cm2 of intima-media; Table 2), the
accumulation of fibrin(ogen) by the platelet layer (amounting to
<5% of the total fibrinogen) and by the intima-media (<10%) of the
aorta wall during exsanguination was considered to be negligible.
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; ballooned, 
)
and intima-media layer (sham, 
; ballooned, 
) was compared with
that present in 1 mL of blood at exsanguination. Fibrinogen
concentration was calculated assuming that rabbit blood contains
3.58 µmol of fibrinogen per liter.
From the slope of each curve (Fig 3) and the mean level of fibrin(ogen)
saturation from the data in Table 2, the rates of turnover of
fibrin(ogen) at the platelet layer and the intima-media were
estimated to be 6 minutes and 10 minutes, respectively, during
the 10-minute interval after injury.
Assessment of the Luminal Surface of Aortas using TEM
The quantitative measurements of fibrin(ogen) and platelet
adsorption by the sham- and balloon-injured aorta surface were
supported by TEM inspection of the fourth segment from each aorta (Fig 4a through 4d). The luminal surfaces of
aortas from sham-injured rabbits were essentially devoid of
platelets associated with the endothelium (data not
shown).
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Platelets accumulated in deposits ranging in depth from <5 to >20
platelets on the deendothelialized aortas
from saline-treated rabbits (Fig 4a). The platelets adsorbed
directly to the subendothelium were invariably
degranulated and spread. As the depth of platelets increased,
platelets were more rounded and less shape changed as their
distance from the aorta wall increased. Polymerized fibrin with a
periodicity of 25 nm (see inset, Fig 4a) was seen filling the
interplatelet spaces. By contrast, platelets adsorbed to
ballooned aortas from hirudin-treated (5 mg/kg) rabbits were
comparatively sparse, the deendothelialized
aortic surface covered generally with only a monolayer of
shape-changed, vacuolated, and spread platelets adhering closely to
the exposed subendothelium (Fig 4b). The luminal
surfaces of the ballooned aortas of Ancrod-treated rabbits (Fig 4c),
Warfarin-treated rabbits (Fig 4d), and Ancrod/r-hirudin–treated
rabbits (data not shown) after balloon-catheter injury were similar in
appearance to those from r-hirudin–treated rabbits with respect to the
platelet layer.
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Discussion |
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The present data support evidence that thrombin, generated spontaneously at or within the exposed aortic subendothelium after injury, is involved directly with the rapid deposition of fibrin(ogen) and platelets on the exposed surface of the subendothelium in vivo (Fig 4a
);
evidence of polymerized fibrin with a periodicity of 25
nm27 was frequently observed between aggregated
platelets (Fig 4a, inset). We also conclude that pretreatment with
various anticoagulants that either inhibited thrombin
production or activity or fibrin(ogen) deposition reduced
substantially the accumulation of aggregated platelets and
associated fibrin(ogen) at the surface of the exposed
subendothelium. Thus, pretreatment of the rabbit with
r-hirudin led to a parallel decrease in the quantities of adsorbed
fibrinogen and platelets in the platelet layer (Tables 1 and 2), the higher dose of r-hirudin (5 mg/kg) reducing the uptake of
fibrinogen by 70% and platelets by 80%. Kelly et
al28 reported a similar dose-dependent effect of
r-hirudin on platelet deposition on endarterectomized aortic tissue
and on platelet and fibrin deposition on collagen-coated tubing
when used as arteriovenous shunts in baboons. We conclude that the
deposition of fibrin(ogen) and the deposition of platelets after
endothelial injury are not independent events.
The fact that Warfarin treatment also caused a decrease in fibrinogen
and platelet uptake after injury further strengthens the concept
that thrombin plays a pivotal role in recruiting fibrinogen and
platelets to the wound site. Warfarin acts as a vitamin K
antagonist in vivo, inhibiting the synthesis of
biologically active, vitamin K–dependent coagulation factors, such as
factors VII, IX, X, and prothrombin.29 From ELISA
measurements of plasma samples taken before treatment and on day 4 or 5
since the start of treatment, Warfarin induced a sharp decline in
rabbit plasma prothrombin concentration from 1.8 µmol/L to
0.2 to 0.3 µmol/L (A. Parshad and M.W.C. Hatton, unpublished
data, 1996). From this information and our knowledge of the fractional
distribution of prothrombin in the rabbit,9 we
conclude that Warfarin causes the plasma prothrombin concentration, and
consequently the resident concentration of prothrombin within the
extracellular space of the aortic intima-media, to be decreased by up
to 10-fold. This depleted reserve of prothrombin (and, presumably, of
factors VII, IX, and X) within the intima-media may be insufficient to
provide an adequate concentration of thrombin during the crucial first
few minutes after injury, and the compromised plasma levels of these
vitamin K–dependent factors would be inadequate to provide a
sufficient flux to meet the prothrombinase requirement within the
injured intima-media.
The similar effect of hirudin and Warfarin on the behaviors of
fibrinogen and platelets toward the aorta wall after balloon injury
suggests strongly that most of the platelet and fibrin(ogen)
deposition is linked directly with thrombin production at the
wound site. Why, then, should pretreatment with Ancrod, a snake-venom
protease, which selectively depletes plasma
fibrinogen30 and which does not affect the plasma
levels of prothrombin or other vitamin K–dependent factors in vivo or
interfere with thrombin activation,31 decrease
platelet accumulation at the
deendothelialized aorta wall (Table 1, Fig 4c)
to a monolayer similar to the effect of r-hirudin? Ancrod cleaves the
A chain of mammalian fibrinogens, releasing only
fibrinopeptide A to yield des-A-fibrinogen (or
Ancrod-fibrin monomer).32 In vitro,
des-A-fibrinogen polymerizes poorly compared with thrombin-derived
fibrin monomer,33 34 and, for this reason,
Ancrod-fibrin clots are believed to be relatively easily dispersed and
cleared from the circulation. As a result, after 6 hours of Ancrod
treatment (Fig 2), the intact fibrinogen level measured 14% of the
normal plasma level, a value similar to that previously reported in
rabbits after 6 hours of Ancrod treatment.35 We
also observed that the platelet level had not changed significantly
after 6 hours of Ancrod treatment.
The Ancrod results suggest that as much as 80% of the
platelet deposit on the subendothelium is caused
directly by codepositing fibrin(ogen) and therefore only indirectly by
thrombin. If all platelet adsorption, including the monolayer, was
attributed to thrombin, then further pretreatment of the Ancrod rabbit
with r-hirudin would decrease platelet binding further. This result
was not observed (see Table 1). We conclude that the initial monolayer
of platelets is attracted to the exposed
subendothelium independently of thrombin
production. Subendothelium-associated collagen
and other adhesive proteins (eg, fibronectin, laminin, or von
Willebrand factor) have been implicated as binding sites for
the monolayer of adherent platelets immediately after
injury.36 Furthermore, in the saline-treated
rabbit, the mass of platelets that aggregate with platelets of
the initial monolayer are associated with obvious fibrin (Fig 4a), and
therefore their presence is clearly dependent on thrombin (and hence
fibrin) production at the site of injury. Presumably, the
production rate of thrombin after injury corresponds directly
with the extent of the injury and, in the presence of normal plasma
levels of fibrinogen, stimulates an appropriate deposition of
fibrin(ogen) and platelets. Ancrod pretreatment causes a
significant decrease in the availability of thrombin-derived fibrin for
significant platelet aggregation to take place on the monolayer. We
note that Chang and Huang35 reported that the
depletion of fibrinogen caused by Ancrod leads to a decreased
platelet aggregation activity in rabbits. Possibly,
subendothelium-adsorbed des-A-fibrinogen or other
Ancrod-derived fibrinogen degradation products may directly inhibit
platelet aggregation, or the polymerization of thrombin-derived
fibrin, which promotes platelet aggregation, with the platelet
monolayer. A similar suggestion to explain the effect of Ancrod-induced
hypofibrinogenemia on platelet plug formation has been made
previously by Daniel et al.37
Previous measurements of the deposition of platelets after a
deendothelializing injury to the rabbit thoracic aorta
have claimed that 42 000 to 45 000 platelets per square
millimeter of subendothelium are equivalent to a
saturated monolayer of platelets.2 38 In
their perfusion-fixation procedure, Groves et al2
perfused heparin-injected balloon-injured rabbits, first with
Locke-Ringer physiological fluid (100 mm Hg)
until the perfusate was clear of red cells, followed by a 4%
(wt/vol) glutaraldehyde solution for 1 to 2 minutes;
the aorta was excised later. In the present study, we have used
neither heparin nor a perfusion-fixation technique; rather, the rabbit
was rapidly exsanguinated through a carotid cannula, the aorta exposed,
briefly rinsed, and then excised and processed. We accept that our
procedure to deendothelialize an aorta may
conceivably cause greater medial damage and therefore greater thrombin
generation than the technique of Groves et al.2
This difference in technique probably accounts, in part, for the
greater deposition of platelets on the
deendothelialized aorta in this study and in
another recent study39 of platelet deposition
on the exposed subendothelium surface. Arguably, the
process of exsanguination and the accompanying decline in blood
pressure, followed by a partial collapse of the thoracic aorta may have
encouraged further platelet aggregation at the surface of the
artery wall. However, as shown by experiment (Fig 3), only a minor
proportion (<5%) of the total fibrin(ogen) in the platelet layer
was deposited during exsanguination. From this result, it follows that
only a minor fraction of the platelet layer would be deposited
during the exsanguination step.
After inducing a deendothelializing injury to the
rabbit iliac artery, Goldberg et al40 discovered
that a platelet -granule–specific protein, platelet factor
4, was located in increasing quantity within the intima-media during
the first 30 minutes, but was markedly diminished at 4 hours after
injury. The authors concluded that platelets in contact with the
denuded vessel release the contents of their -granules into the
subendothelial space. Later, in a study of human
platelet interaction with 35 S-labeled
extracellular matrix proteoglycan, Yahalom et
al41 discovered that heparitinase, a lysosomal
enzyme,42 was released from adherent
platelets and degraded heparan sulfate chains within the matrix.
From Table 1, it appears that 51Cr-labeled
platelets of the platelet layer release a large proportion of
their 51Cr contents into the underlying
intima-media layer, possibly as microparticles resulting from the
interaction between platelets, thrombin, and exposed
collagen.43 We rule out the possibility that the
51Cr in the intima-media is due to free
51Cr, ie, not contained by platelets, as the
quantity of 51Cr associated with the intima-media
from aortas of sham-injured rabbits or from anticoagulated,
balloon-injured rabbits was much less than that of the intima-media of
saline-injected, balloon-injured rabbits. Indeed, >90% of
51Cr deposited into the
subendothelial space was inhibited by pretreatment with
either r-hirudin, Ancrod, or Warfarin and >96% by the administration
of r-hirudin to the Ancrod-treated rabbit. We also reject the
possibility that a high proportion of residual platelets remain
adsorbed to the subendothelium after Häutchen
preparation, as a previous study8 using scanning
electron microscopy has indicated the platelet layer to be entirely
removed by cellulose acetate paper.
We conclude, similar to Gast et al,7 that direct adherence of a monolayer of platelets to the exposed subendothelium takes place rapidly after injury and independently of thrombin generation. However, as thrombin is generated during the first few minutes after injury, fibrinogen is drawn to the site of injury, interacting with subendothelium-bound thrombin and forming fibrin. Subendothelium-associated thrombin and fibrin then stimulate more platelets to interact and aggregate with the platelets that comprise the monolayer. The presence of anticoagulant conditions inhibits in part or completely the secondary stage of platelet deposition, which involves thrombin and fibrin.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by a grant from the Heart and Stroke Foundation of Ontario. We thank Andrew Parshad for his technical help with the Warfarin experiments and Rena Cornelius for help with the densitometry. We also thank Dr Hallie Groves (Department of Biomedical Sciences, McMaster University) for providing helpful comments and criticisms of the manuscript and Dr Graham Pay (Ciba Pharmaceuticals, Horsham, UK) for generously supplying recombinant desulfato-hirudin.
Received September 23, 1997; accepted December 19, 1997.
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References |
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