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Effect of heparin and thrombin on platelet adherence to the surface of rabbit aorta
ccl49_3848:i9i0701a69
THRO~CG~~SRESEARCH Voi. 13, PP. 69-78. 0 Papmaa Raa Ltd. 1978. FTintcdin Grtot Britain.
ScmxYo
EFFECT OF HEPARIN AND THROMBIN ON PLATELET ADHERENCE TO THE SURFACE OF RABBIT AORTA E.M. Essien, J.-P. Cazenave, S. Moore and J.F. Mustard Deparment of Haematology, University of Ibadan, Ibadan, Nigeria and Department of Pathology, McMaster University, Hamilton, Ontario, Canada
(Received 2.2.1976; in revised foral 12.5.1978. Accepted by Editor S. Nieuiarouski)
ABSTRACT Platelet adherence to both the damaged and undamaged surfaces of rabbi! aorta in perfusion experiments in vitro was inhibited by heparin. The preparation of the vessel segments for these perfusion studies require< 30 to 60 minutes because of the need to tie off all of the small vessels. In other experiments in which damaged or undamaged vessel segments were everted on a probe, the preparation of the vessels could be done more quickly and heparin did not inhibit platelet adherence. However, exposure of either the damaged or undamaged surfaces used in the latter type of experiment to thrombin markedly promoted platelet adherence to the surfaces. Heparin inhibited this effect. It seems likely that during the preparation of the aortas for the perfusion studies, thrombin generation occurred in the vessels. Thus, in preparing aortic segments for studies of platelet adherence, thrombin generation must be avoided. Since thrombin enhanced platelet adherencr to undamaged endothelial surfaces, it may be that thrombin formation in vivo could cause platelet adherence to vessel walls from which endothelial cells have not been lost.
INTRODUCTION Platelet adherence to the surface of damaged arteries is one of the first events in the initiation of arterial thrombi.
A variety of factors influence
the formation of thrombi, and thus it is difficult to establish quantitative methods to study the effects of agents and conditions that modify platelet adherence to the surface of damaged vessels.
Several techniques for studying
platelet adherence to the constituents of the subendothelial surface of the vessel wall have been described.
Segments of aorta have been exposed to flow-
ing anticoagulated blood or platelet-rich plasma in a perfusion system and 69
70
PLATELET
ADHERENCE:
RABBIT
AORTA
Vol.13,No.l
platelet deposition on the surface estimated by morphometric analysis (1). 51 Other methods have been used to quantitate the adherence of Cr-labeled platelets to damaged aorta (2,3) or to the constituents of the vessel wall such as collagen (3.4).
The adherence of platelets to collagen has been also esti-
mated using sedimentation techniques (5), radiolabeled collagen (6), and affinity chromatography on sepharose-collagen (7). In a preliminary communication (8), we reported that in the perfusion system described here, pretreatment of aorta surfaces with heparin significantly reduced the number of platelets that adhered to them.
The effect of heparin
could be due to inhibition of platelet,adherence to the subendothelium or to inhibition of thrombin which might have formed on the surface of the vessels during preparation of the segments for the perfusion experiments.
We have,
therefore, examined the effect on platelet adherence of exposure of the surface of the vessel to thrombin.
MATERIALS AND METHODS Preparation of suspensions of washed rabbit platelets. Suspensions of washed rabbit platelets were prepared by a modification of the method of Ardlie et al. and labeled with 51Cr, as described previously (9). The final suspending medium was Eagle's medium (Grand Island Biological Company, Grand Island, N.Y.) at pH 7.35, containing apyrase and 0.35% bovine albumin (Pentex, Fraction V, Miles Laboratories, Kankakee, Ill.).
The platelet
count was adjusted to 400,000 or 700,000 per mm3. Preparation of washed rabbit red blood cells. Rabbit red blood'cells were washed as described previously (3) and resuspended in the same medium as the platelets. Experimental animals and isolation of aorta for perfusion experiments. New Zealand White rabbits weighing 2-3 kg. were anaesthetized, exsanguinated and then flushed with 50 ml of Eagle's medium through a cannula in the carotid artery as described previously (9).
The intercostal and other branches
of the thoracic aorta were securely tied with 000 surgical silk. be done in situ and required 30 to 60 minutes.
During this time the vessels
were not kept filled with Eagle's medium under pressure. to be used was demarcated externally.
This had to
The length of aorta
A slit was made in the proximal portion
of the wall of the thoracic aorta and a 5F balloon catheter (Edwards Laboratories, Santa Anna, California) was introduced into the lumen. was inflated
to
a pressure of 450 mm Hg and withdrawn.
This procedure was
repeated twice to ensure that endothelial cells were removed. elastic lamina remained intact.
The catheter
The internal
A segment of aorta distal tc the damaged
PLATELET
v01.13,w0.1
ADHERENCE:
RkBBIT
AORTA
segment and the same length as the damaged segment, was considered to be the "undamaged" segment.
In alternate experiments, the distal segment was injured
by introducing the balloon catheter through an incision in the femoral artery. The aorta, consisting of a damaged and an "undamaged" segment, was flushed with 10 ml of Eagle's medium to remove loose endothelial cells:
the "undamaged"
segment was exposed to the washing fluid before the damaged segment in each aorta.
The diameter of each segment was measured in two planes at right angles
to each other. Heparin treatment of aorta for perfusion studies. Heparin obtained from hog mucosa (Connaught Laboratories, Toronto, Canada) was added to Eagle's medium at a concentration of 50 units per ml.
In some
experiments, the aorta was flushed with this solution (50 ml) and then with 30 ml of Eagle's medium, before the vessels were tied off and the aorta injured by the balloon catheter. The posterior branches of the aorta were then tied off and the aorta segments were removed from the animal. Platelet interaction with aorta surfaces in perfusion experiments. In these studies, the undamaged segment of the aorta was placed first in the circuit chamber containing Eagle's medium pH 7.35 at 37°C.
The circuit of
plastic tubing was completed and the system filled, without air bubbles, with the 51Cr-labeled platelet suspension that had been prewarmed to 37°C contained 10% washed red blood cells.
and
A diagram and a description of the
perfusion system have been published elsewhere (9).
The pulsatile perfusion
pump was preset at 50 pulses per minute to deliver 150 ml of fluid per minute at a pressure of 90 mm Hg.
The pH of the bathing medium was maintained at 7.35
by adding carbon dioxide (Titrator'll, Radiometer, Copenhagen).
After the
platelet suspension has been pumped through the apparatus and recirculated for 10 minutes, it was replaced in the system by an equal volume of Eagle's medium at 37"C, pH 7.35.
The Eagle's medium was pumped through the system for one
minute to dislodge any loose platelet aggregates that might have formed on the wall of the aorta.
The aorta was then fixed in situ with 2.5% glutaraldehyde
in cacodylate buffer, removed from the bath and cut into previously demarcated damaged and "undamaged" segments.
Approximately 2 mm was removed from each end
of the aorta corresponding to the portion that was used to fasten the aorta in the circuit.
These pieces were discarded to exclude the possibility of using
an area damaged by manipulation in the computation of results for an "undamaged" segment, and to minimize variation in the damaged area.
Each remaining segment
of aorta was placed in 2 ml of 2.5% glutaraldehyde and the radioactivity determined in a well type gamma counter (Packard Instruments).
72
PLATELET
ADBEREKCE:
RIBBIT
AORTA
v01.13,s0.1
Experiments with the rotating probe system. Further experiments were carried out using a system which has been described in detail elsewhere (2,3).
Immediately after exsanguination of the
rabbits through a cannula in the carotid artery, the abdomen was opened to expose the aorta from the arch to below the diaphragm.
The abdominal aorta was
transected, the aortic arch was incised and the aorta rinsed with 30 to 50 ml of modified Tyrode solution (no calcium, magnesium or glucose).
A balloon
catheter was introduced through the incision in the aorta and pushed down below the diaphragm. withdrawn.
The balloon was inflated to 450 mm of Hg and the catheter was
This procedure with the catheter was done five times to ensure
complete removal of the endothelium.
Samples of undamaged rabbit thoracic
aorta, or samples damaged in situ with a balloon catheter were everted on a metal probe and rotated for 1 min in a Tyrode-albumin solution containing 20 units/ml of bovine thrombin (Parke Davis and Co., Detroit, Mi.) and then rinsed twice for 15 set each time in 10 ml portions of Tyrode solution.
The
probe was then rotated mechanically at 200 rpm for 10 min in a suspension of 51 Cr-labeled platelets containing 10% washed red blood cells. Samples of undamaged rabbit aorta which had not been deliberately exposed to thrombin were used in control experiments.
In this study the platelets were resuspended at
37°C in Tyrode solution at pH 7.35 containing 0.35% albumin and apyrase.
In
experiments in which samples were to be taken for scanning electron microscopy, the platelets were suspended in a medium containing 4% albumin, apyrase and 40% washed red blood cells. Calculation of platelet adherence to aortic segments in both studies. The number of platelets that adhered per square mm of exposed aorta surface was calculated by the method of Cazenave (3).
Preliminary experiments estab-
lished that the radioactivity that became associated with the aorta represented adherent platelets. Electron microscopy. Specimens of aorta were prepared for scanning electron microscopy (SEM) and transmission electron microscopy (TEM) essentially as described by Gerrity et al. (10).
Specimens were fixed in Karnovsky's fixative containing 2% para-
formaldehyde and 2% glutaraldehyde in 0.l.M sodium cacodylate buffer and 0.25 mg/ml CaC12, pH 7.35, 900 mOsm for 6 hours at 4°C.
The tissue blocks were
washed for 1 hour in two changes of O.lM sodium cacodylate buffer, pH 7.35, containing 7.5% sucrose and 0.5 mg/ml CaC12, then postfixed in 1% osmium tetroxide at 4°C for 1.5 hours.
For TEM the blocks were then dehydrated in
ethanol, embedded in Spurr's resin, and sectioned on a Reichert ultramicrotome.
v01.13,x0.1
PIA-I'ELET ADHERENCE:
RABBIT
AORTA
73
Cltrathin sections were stained with 5% methanolic uranyl acetate followed by lead citrate, and examined In a Philips 301 electron microscope. For SEM, samples were fixed and dehydrated
as
above, then critical point
dried from CO2 in a Bomar SPC-SOOEX critical point dryer.
They were then
mounted on specimen pegs, coated with gold/palladium (60:40) and examined in an A%-1000
scanning electron microscope.
RESULTS Scanning electronxaicrographsof the "undamaged" surface of the aorta before perfusion showed the endothelial surface to be intact.
The damaged
surface after perfusion showed a layer of platelets adinerent to the denuded subendothelial surface (9).
Strands of what appeared to be fibrin were
observed from time to time on segments of aorta from the perfusion experiments. In the perfusion system, fewer platelets adhered to the undamaged aorta than to the surface of the aorta that had been damaged vith the balloon catheter (Table I).
This difference was similar to that found when platelet
adherence to the surface of damaged and "undamaged" aortas was studied using the rotating probe method (2).
Similar differences were reported by
Baumgartner (11) between damaged and undamaged surfaces.
Fewer platelets
adhered to the "undamaged" or to the damaged aortas that had been exposed to heparin than to vessels that had not been treated with heparin (Table I).
TABLE
I
Platelet adherence to surface of perfused rabbit aorta Surface of aorta
Damaged "Undamaged"
2P*
Number of platelets per mm* (geometric mean) No heparin
Heparin
199,000
14,200
(6)
4,000
(4)
co.1
16,200
(7)** (7)
*
Significance of difference between the means using an unpaired two-tailed t-test.
**
Numbers in brackets indicate the number of aortas examined. Damaged and "undamaged" segments of the aorta were obtained from the same animal. In tuo experiments with heparin, no undamaged section was studied.
72
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PLATELET
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AORTA
v01.13,x0.1
Figure 1.
Scanning electron micrograph of the endothelial surface of a segment of aorta that had not been damaged by a balloon-catheter before it was everted on a probe and rotated at 200 rpm for 10 min in a platelet suspension containing 4% albumin in the presence of a 40% hematocrit. 9 focal small area of endothelial loss is indicated by the arrow. Small craters on the endothelial cell surface are indicated by the arrow heads. The absence of adherent platelets should be noted (x5000).
Figure 2.
Transmission electron micrograph of a segment of aorta that had not been damaged by a balloon-catheter before it was everted on a probe and exposed to the same conditions as described in Figure 1. The endothelium is intact but shows subendothelial vesicles. NO platelets are adherent to the surface (X9800).
PLATELET
Vol.l~,So.f
hDHERE?iCE: RABBIT
AORTA
73
SEM examination of the rabbit aorta which had not been deliberately damaged and was everted on the probe showed some damage characterized by focal small areas of endothelial loss estimated as being 5 to 10% of the surface area.
In
addition, small craters were observed on the luminal surface of the endothelial cells (Figure 1).
On TEM, the endothelial covering was largely intact but
spaces were frequently observed between the endothelial cells and the subendothelium (Figure 2). We next examined the effect of heparin on the adherence of platelets to the surface of damaged and undamaged vessels everted on a rotating probe. Table II shows that heparin had very little effect on platelet adherence to the damaged vessel surface in this system.
When the damaged aorta placed on
the probe was exposed to thrombin for 1 minute and washed, platelet adherence to the surface was enhanced and aggregates formed (Table II). platelet deposition to this thrombin-treated surface.
Heparin inhibited
When "undamaged" aortas
were similarly exposed to thrombin, platelet adherence to the endothelial surface was also enhanced (Table III).
Treatment of the aortic segments with
heparin in the suspending fluid inhibited platelet adherence to the "undamaged" endothelium (Table III).
TABLE II Platelet adherence to surface of damaged rabbit aorta Treatment of aorta surface*
Addition to platelet suspension*
(n)**
Number of platelets adherent per mm2 (mean f S.E.M.)
1
Tyrode
Tyrode
(10)
59.500 + 7,200
2
Tyrode
Heparin (50 u/ml)
(10)
54,000 2 9,800
3
Thrombin (20 u/ml)
Tyrode
(10)
4
Thrombin (20 u/ml)
Heparin (50 u/ml)
(10)
* ** *** ****
2p***
co.7
471,000 t 37,500****
44,200 + 3,100
Final concentrations of thrombln and heparin used are given in brackets. The aorta was rotated at 200 rpm for 10 min in a 5lCr-labeled platelet suspension (7OO,OOO/mm3) containing red blood cells (hematocrit 10%). Number of segments of aorta Significance of difference between means using an unpaired two-tailed t-test: 1 versus 2 and 3 versus 4. Presence of platelet aggregates on the surface.
76
PLATELET
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Vol.13,No.l
TABLE III Platelet adherence to surface of "undamaged" rabbit aorta
Treatment of aorta surface*
Addition to platelet suspension*
W**
Number of platelets adherent per mm2 (mean + S.E.X.)
1
Tyrode
Tyrode
(10)
4,200 + 700
2
Tyrode
Heparin (50 u/ml)
(10)
2,900 + 400
3
Thrombin (20 u/ml)
Tyrode
(10)
4
Thrombin (20 u/ml)
Heparin (50 u/ml)
(10)
2p***
co.1
53,100 + 4,600****
5,200 + 1,200
For legend see TABLE II
DISCUSSION The effect of heparin in diminishing platelet adherence to the segments of damaged or undamaged rabbit aorta in the perfusion experiments appears most likely to be due to inhibition of thrombin that formed while the segments were being prepared.
During the 30 to 60 minute period that the vessel remained in
situ while the intercostal vessels and other branches were tied off, some blood seeped into the lumen of the aorta; the observation of strands of fibrin on the surface of the vessel indicates that thrombin must have been generated in the aorta. In the experiments in which segments of the vessel were removed immediate1 after the vessel was flushed free of blood, heparin had little influence on platelet adherence.
This observation indicates that little, if any, thrombin
formation occurred in vessels prepared in this way.
Thus, in preparing vessel
segments for perfusion studies, measures should be taken to prevent thrombin formation. The observation that thrombin treatment of damaged vessels enhances platelet accumulation on their surface may have some relevance to the process of thrombus formation since constituents of the subendothelium can activate blood coagulation (12). more thrombogenic.
Exposure of the subendothelium to thrombin makes it
In addition, when the undamaged aorta surface has been
exposed to thrombin, platelets can adhere to it.
It is recognized that the
Vol.13,No.l
PLATELET
ADHERENCE:
RABBIT
AORTA
endothelial cells on the surface of the everted aorta are altered.
77
They are,
.however, still effective in preventing platelet adhesion to the vessel wall. Awbrey and associates (13) have shown that in endothelial cell tissue cultures, exposure of the cells to thrombin alters the surface so that platelets adhere to it.
r\shfordand Freiman (14) observed the formation of thrombi at sites of
minimal injury, even though the endothelial cells were still present.
The
presence of fibrin with these platelet thrombi indicates that thrombin must have formed at the sites of injury and may have had a role in the formation of platelet aggregates.
ACKNOWLEDGEMENTS We wish to thank Dr. Mary Richardson for the electron microscopy and Mrs. D. Blondowska for technical assistance. a World Health Organization Fellowship, 1974.
E.M. Essien was the recipient of J.-P. Cazenave is a Senior
Research Fellow of the Ontario Heart Foundation.
This work was supported by
grant MT1309 from the Medical Research Council of Canada.
REFERENCES 1.
BAUMGARTNER, H.R. and MUGGLI, R. Adhesion and aggregation: morphological demonstration and quantitation in vivo and in vitro. In: Platelets in Biology and Pathology. J.L. Gordon (Ed.). ElsevierfNorth-Holland Biomedical Press, 1976, p.23-60.
2.
CAZENAVE, J.-P., PACRHAM, M.A., GUCCIONE, M.A. and MUSTARD, J.F. Inhibition of platelet adherence to damaged surface of rabbit aorta. 3. Lab. Clin. Med. 86, 551-563, 1975.
3.
CAZENAVE, J.-P., PACKHAM, M.A., DAVIES, J.A., KINLOUGH-RATHBONE, R.L. and MUSTARD, J.F. Studies on platelet adherence to collagen and subendothelium. In: PlateletFunction H.J. Day, H. Holmsen and M.B. Zucker (Eds.). U.S. Department of Health, Education and Welfare. DHEW Publication No. (NIH) 78-1087, 1978, p.181-198.
4.
CAZENAVE, J.-P., PACKHAM, M.A. and MUSTARD, J.F. Adherence of platelets to a collagen-coated surface: development of a quantitative method. J. Lab. Clin. Med. 82, 978-990, 1973.
5.
LYMAN, B., ROSENBERG, L. and RARPATRIN, S. Biochemical and biophysical aspects of human platelet adhesion to collagen fibers. J. Clin. Invest. 50, 1854-1863, 1971.
6.
GORDON, J.L. and DINGLE, D.T. Binding of radiolabelled collagen to blood platelets. J. Cell Sci. 16, 157-166, 1974.
7.
BRASS, L.F., FAILE, D. and BENSUSAN, H.B. Direct measurement of the platelet: collagen interaction by affinity chromatography on collagen sepharose. J. Lab. Clin. Med. 87, 525-534, 1976.
78
PLATELET
ADHEREXCE:
UBBIT
AORTA
VOl.13,NO.l
8.
ESSIEN, E.H., KI.tZOUGH-RATHBONE, R.L., MORE. S. and ,wSTARD, J.F. The role of heparin on the inhibition of platelet adhesion to damaged arterial endothelial surface. Thromb. Diath. Haemorrh. 34, 600, 1975.
9.
ESSIEN, E.M. and ?iUSTARD, J.F. Inhibition of platelet adhesion to rabbit aorta by sulphinpyrazone and acetylsalicylic acid. Atherosclerosis 27, 89-95, 1977.
10.
GERRITY, R.G., RICHtUIDSON,M., SOMER, J.B., BELL, F.P. and SCHWARTZ, C.J. Endothelial cell morphology in areas of in vivo Evans blue uptake in the aorta of young pigs. II. Ultra-structure of the intima in areas of differing permeability to proteins. Amer. J. Pathol. 89, 313-334, 1977.
11.
BAWGARTNER, H.R. Platelet interaction with vascular structures. Diath. Haemorrh., Suppl. 51, 161-176, 1972.
12.
NIEWIAROWSKI, S., BANKOWSKI, E. and ROGOWICICA, I. Studies on the adsorption and activation of the Hageman factor (factor XII) by collagen and elastin. Thromb. Diath. Haemorrh., 14, 387-400. 1965.
13.
AWBREY, B.J., OWEN, W.G., FRY, G.L., CHENG, F.H. and HOAK, J.C. Binding of human thrombin to human endothelial cells and platelets. Blood 46, 1046, 1975.
14.
ASHFORD, T.P. and FREIMAN, D.G. Platelet aggregation at sites of minimal endothelial injury. An electron microscopy study. Amer. J. Pathol. 53, 599-607, 1968.
Thromb.
THRO~CG~~SRESEARCH Voi. 13, PP. 69-78. 0 Papmaa Raa Ltd. 1978. FTintcdin Grtot Britain.
ScmxYo
EFFECT OF HEPARIN AND THROMBIN ON PLATELET ADHERENCE TO THE SURFACE OF RABBIT AORTA E.M. Essien, J.-P. Cazenave, S. Moore and J.F. Mustard Deparment of Haematology, University of Ibadan, Ibadan, Nigeria and Department of Pathology, McMaster University, Hamilton, Ontario, Canada
(Received 2.2.1976; in revised foral 12.5.1978. Accepted by Editor S. Nieuiarouski)
ABSTRACT Platelet adherence to both the damaged and undamaged surfaces of rabbi! aorta in perfusion experiments in vitro was inhibited by heparin. The preparation of the vessel segments for these perfusion studies require< 30 to 60 minutes because of the need to tie off all of the small vessels. In other experiments in which damaged or undamaged vessel segments were everted on a probe, the preparation of the vessels could be done more quickly and heparin did not inhibit platelet adherence. However, exposure of either the damaged or undamaged surfaces used in the latter type of experiment to thrombin markedly promoted platelet adherence to the surfaces. Heparin inhibited this effect. It seems likely that during the preparation of the aortas for the perfusion studies, thrombin generation occurred in the vessels. Thus, in preparing aortic segments for studies of platelet adherence, thrombin generation must be avoided. Since thrombin enhanced platelet adherencr to undamaged endothelial surfaces, it may be that thrombin formation in vivo could cause platelet adherence to vessel walls from which endothelial cells have not been lost.
INTRODUCTION Platelet adherence to the surface of damaged arteries is one of the first events in the initiation of arterial thrombi.
A variety of factors influence
the formation of thrombi, and thus it is difficult to establish quantitative methods to study the effects of agents and conditions that modify platelet adherence to the surface of damaged vessels.
Several techniques for studying
platelet adherence to the constituents of the subendothelial surface of the vessel wall have been described.
Segments of aorta have been exposed to flow-
ing anticoagulated blood or platelet-rich plasma in a perfusion system and 69
70
PLATELET
ADHERENCE:
RABBIT
AORTA
Vol.13,No.l
platelet deposition on the surface estimated by morphometric analysis (1). 51 Other methods have been used to quantitate the adherence of Cr-labeled platelets to damaged aorta (2,3) or to the constituents of the vessel wall such as collagen (3.4).
The adherence of platelets to collagen has been also esti-
mated using sedimentation techniques (5), radiolabeled collagen (6), and affinity chromatography on sepharose-collagen (7). In a preliminary communication (8), we reported that in the perfusion system described here, pretreatment of aorta surfaces with heparin significantly reduced the number of platelets that adhered to them.
The effect of heparin
could be due to inhibition of platelet,adherence to the subendothelium or to inhibition of thrombin which might have formed on the surface of the vessels during preparation of the segments for the perfusion experiments.
We have,
therefore, examined the effect on platelet adherence of exposure of the surface of the vessel to thrombin.
MATERIALS AND METHODS Preparation of suspensions of washed rabbit platelets. Suspensions of washed rabbit platelets were prepared by a modification of the method of Ardlie et al. and labeled with 51Cr, as described previously (9). The final suspending medium was Eagle's medium (Grand Island Biological Company, Grand Island, N.Y.) at pH 7.35, containing apyrase and 0.35% bovine albumin (Pentex, Fraction V, Miles Laboratories, Kankakee, Ill.).
The platelet
count was adjusted to 400,000 or 700,000 per mm3. Preparation of washed rabbit red blood cells. Rabbit red blood'cells were washed as described previously (3) and resuspended in the same medium as the platelets. Experimental animals and isolation of aorta for perfusion experiments. New Zealand White rabbits weighing 2-3 kg. were anaesthetized, exsanguinated and then flushed with 50 ml of Eagle's medium through a cannula in the carotid artery as described previously (9).
The intercostal and other branches
of the thoracic aorta were securely tied with 000 surgical silk. be done in situ and required 30 to 60 minutes.
During this time the vessels
were not kept filled with Eagle's medium under pressure. to be used was demarcated externally.
This had to
The length of aorta
A slit was made in the proximal portion
of the wall of the thoracic aorta and a 5F balloon catheter (Edwards Laboratories, Santa Anna, California) was introduced into the lumen. was inflated
to
a pressure of 450 mm Hg and withdrawn.
This procedure was
repeated twice to ensure that endothelial cells were removed. elastic lamina remained intact.
The catheter
The internal
A segment of aorta distal tc the damaged
PLATELET
v01.13,w0.1
ADHERENCE:
RkBBIT
AORTA
segment and the same length as the damaged segment, was considered to be the "undamaged" segment.
In alternate experiments, the distal segment was injured
by introducing the balloon catheter through an incision in the femoral artery. The aorta, consisting of a damaged and an "undamaged" segment, was flushed with 10 ml of Eagle's medium to remove loose endothelial cells:
the "undamaged"
segment was exposed to the washing fluid before the damaged segment in each aorta.
The diameter of each segment was measured in two planes at right angles
to each other. Heparin treatment of aorta for perfusion studies. Heparin obtained from hog mucosa (Connaught Laboratories, Toronto, Canada) was added to Eagle's medium at a concentration of 50 units per ml.
In some
experiments, the aorta was flushed with this solution (50 ml) and then with 30 ml of Eagle's medium, before the vessels were tied off and the aorta injured by the balloon catheter. The posterior branches of the aorta were then tied off and the aorta segments were removed from the animal. Platelet interaction with aorta surfaces in perfusion experiments. In these studies, the undamaged segment of the aorta was placed first in the circuit chamber containing Eagle's medium pH 7.35 at 37°C.
The circuit of
plastic tubing was completed and the system filled, without air bubbles, with the 51Cr-labeled platelet suspension that had been prewarmed to 37°C contained 10% washed red blood cells.
and
A diagram and a description of the
perfusion system have been published elsewhere (9).
The pulsatile perfusion
pump was preset at 50 pulses per minute to deliver 150 ml of fluid per minute at a pressure of 90 mm Hg.
The pH of the bathing medium was maintained at 7.35
by adding carbon dioxide (Titrator'll, Radiometer, Copenhagen).
After the
platelet suspension has been pumped through the apparatus and recirculated for 10 minutes, it was replaced in the system by an equal volume of Eagle's medium at 37"C, pH 7.35.
The Eagle's medium was pumped through the system for one
minute to dislodge any loose platelet aggregates that might have formed on the wall of the aorta.
The aorta was then fixed in situ with 2.5% glutaraldehyde
in cacodylate buffer, removed from the bath and cut into previously demarcated damaged and "undamaged" segments.
Approximately 2 mm was removed from each end
of the aorta corresponding to the portion that was used to fasten the aorta in the circuit.
These pieces were discarded to exclude the possibility of using
an area damaged by manipulation in the computation of results for an "undamaged" segment, and to minimize variation in the damaged area.
Each remaining segment
of aorta was placed in 2 ml of 2.5% glutaraldehyde and the radioactivity determined in a well type gamma counter (Packard Instruments).
72
PLATELET
ADBEREKCE:
RIBBIT
AORTA
v01.13,s0.1
Experiments with the rotating probe system. Further experiments were carried out using a system which has been described in detail elsewhere (2,3).
Immediately after exsanguination of the
rabbits through a cannula in the carotid artery, the abdomen was opened to expose the aorta from the arch to below the diaphragm.
The abdominal aorta was
transected, the aortic arch was incised and the aorta rinsed with 30 to 50 ml of modified Tyrode solution (no calcium, magnesium or glucose).
A balloon
catheter was introduced through the incision in the aorta and pushed down below the diaphragm. withdrawn.
The balloon was inflated to 450 mm of Hg and the catheter was
This procedure with the catheter was done five times to ensure
complete removal of the endothelium.
Samples of undamaged rabbit thoracic
aorta, or samples damaged in situ with a balloon catheter were everted on a metal probe and rotated for 1 min in a Tyrode-albumin solution containing 20 units/ml of bovine thrombin (Parke Davis and Co., Detroit, Mi.) and then rinsed twice for 15 set each time in 10 ml portions of Tyrode solution.
The
probe was then rotated mechanically at 200 rpm for 10 min in a suspension of 51 Cr-labeled platelets containing 10% washed red blood cells. Samples of undamaged rabbit aorta which had not been deliberately exposed to thrombin were used in control experiments.
In this study the platelets were resuspended at
37°C in Tyrode solution at pH 7.35 containing 0.35% albumin and apyrase.
In
experiments in which samples were to be taken for scanning electron microscopy, the platelets were suspended in a medium containing 4% albumin, apyrase and 40% washed red blood cells. Calculation of platelet adherence to aortic segments in both studies. The number of platelets that adhered per square mm of exposed aorta surface was calculated by the method of Cazenave (3).
Preliminary experiments estab-
lished that the radioactivity that became associated with the aorta represented adherent platelets. Electron microscopy. Specimens of aorta were prepared for scanning electron microscopy (SEM) and transmission electron microscopy (TEM) essentially as described by Gerrity et al. (10).
Specimens were fixed in Karnovsky's fixative containing 2% para-
formaldehyde and 2% glutaraldehyde in 0.l.M sodium cacodylate buffer and 0.25 mg/ml CaC12, pH 7.35, 900 mOsm for 6 hours at 4°C.
The tissue blocks were
washed for 1 hour in two changes of O.lM sodium cacodylate buffer, pH 7.35, containing 7.5% sucrose and 0.5 mg/ml CaC12, then postfixed in 1% osmium tetroxide at 4°C for 1.5 hours.
For TEM the blocks were then dehydrated in
ethanol, embedded in Spurr's resin, and sectioned on a Reichert ultramicrotome.
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Cltrathin sections were stained with 5% methanolic uranyl acetate followed by lead citrate, and examined In a Philips 301 electron microscope. For SEM, samples were fixed and dehydrated
as
above, then critical point
dried from CO2 in a Bomar SPC-SOOEX critical point dryer.
They were then
mounted on specimen pegs, coated with gold/palladium (60:40) and examined in an A%-1000
scanning electron microscope.
RESULTS Scanning electronxaicrographsof the "undamaged" surface of the aorta before perfusion showed the endothelial surface to be intact.
The damaged
surface after perfusion showed a layer of platelets adinerent to the denuded subendothelial surface (9).
Strands of what appeared to be fibrin were
observed from time to time on segments of aorta from the perfusion experiments. In the perfusion system, fewer platelets adhered to the undamaged aorta than to the surface of the aorta that had been damaged vith the balloon catheter (Table I).
This difference was similar to that found when platelet
adherence to the surface of damaged and "undamaged" aortas was studied using the rotating probe method (2).
Similar differences were reported by
Baumgartner (11) between damaged and undamaged surfaces.
Fewer platelets
adhered to the "undamaged" or to the damaged aortas that had been exposed to heparin than to vessels that had not been treated with heparin (Table I).
TABLE
I
Platelet adherence to surface of perfused rabbit aorta Surface of aorta
Damaged "Undamaged"
2P*
Number of platelets per mm* (geometric mean) No heparin
Heparin
199,000
14,200
(6)
4,000
(4)
co.1
16,200
(7)** (7)
*
Significance of difference between the means using an unpaired two-tailed t-test.
**
Numbers in brackets indicate the number of aortas examined. Damaged and "undamaged" segments of the aorta were obtained from the same animal. In tuo experiments with heparin, no undamaged section was studied.
72
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Figure 1.
Scanning electron micrograph of the endothelial surface of a segment of aorta that had not been damaged by a balloon-catheter before it was everted on a probe and rotated at 200 rpm for 10 min in a platelet suspension containing 4% albumin in the presence of a 40% hematocrit. 9 focal small area of endothelial loss is indicated by the arrow. Small craters on the endothelial cell surface are indicated by the arrow heads. The absence of adherent platelets should be noted (x5000).
Figure 2.
Transmission electron micrograph of a segment of aorta that had not been damaged by a balloon-catheter before it was everted on a probe and exposed to the same conditions as described in Figure 1. The endothelium is intact but shows subendothelial vesicles. NO platelets are adherent to the surface (X9800).
PLATELET
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hDHERE?iCE: RABBIT
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73
SEM examination of the rabbit aorta which had not been deliberately damaged and was everted on the probe showed some damage characterized by focal small areas of endothelial loss estimated as being 5 to 10% of the surface area.
In
addition, small craters were observed on the luminal surface of the endothelial cells (Figure 1).
On TEM, the endothelial covering was largely intact but
spaces were frequently observed between the endothelial cells and the subendothelium (Figure 2). We next examined the effect of heparin on the adherence of platelets to the surface of damaged and undamaged vessels everted on a rotating probe. Table II shows that heparin had very little effect on platelet adherence to the damaged vessel surface in this system.
When the damaged aorta placed on
the probe was exposed to thrombin for 1 minute and washed, platelet adherence to the surface was enhanced and aggregates formed (Table II). platelet deposition to this thrombin-treated surface.
Heparin inhibited
When "undamaged" aortas
were similarly exposed to thrombin, platelet adherence to the endothelial surface was also enhanced (Table III).
Treatment of the aortic segments with
heparin in the suspending fluid inhibited platelet adherence to the "undamaged" endothelium (Table III).
TABLE II Platelet adherence to surface of damaged rabbit aorta Treatment of aorta surface*
Addition to platelet suspension*
(n)**
Number of platelets adherent per mm2 (mean f S.E.M.)
1
Tyrode
Tyrode
(10)
59.500 + 7,200
2
Tyrode
Heparin (50 u/ml)
(10)
54,000 2 9,800
3
Thrombin (20 u/ml)
Tyrode
(10)
4
Thrombin (20 u/ml)
Heparin (50 u/ml)
(10)
* ** *** ****
2p***
co.7
471,000 t 37,500****
44,200 + 3,100
Final concentrations of thrombln and heparin used are given in brackets. The aorta was rotated at 200 rpm for 10 min in a 5lCr-labeled platelet suspension (7OO,OOO/mm3) containing red blood cells (hematocrit 10%). Number of segments of aorta Significance of difference between means using an unpaired two-tailed t-test: 1 versus 2 and 3 versus 4. Presence of platelet aggregates on the surface.
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TABLE III Platelet adherence to surface of "undamaged" rabbit aorta
Treatment of aorta surface*
Addition to platelet suspension*
W**
Number of platelets adherent per mm2 (mean + S.E.X.)
1
Tyrode
Tyrode
(10)
4,200 + 700
2
Tyrode
Heparin (50 u/ml)
(10)
2,900 + 400
3
Thrombin (20 u/ml)
Tyrode
(10)
4
Thrombin (20 u/ml)
Heparin (50 u/ml)
(10)
2p***
co.1
53,100 + 4,600****
5,200 + 1,200
For legend see TABLE II
DISCUSSION The effect of heparin in diminishing platelet adherence to the segments of damaged or undamaged rabbit aorta in the perfusion experiments appears most likely to be due to inhibition of thrombin that formed while the segments were being prepared.
During the 30 to 60 minute period that the vessel remained in
situ while the intercostal vessels and other branches were tied off, some blood seeped into the lumen of the aorta; the observation of strands of fibrin on the surface of the vessel indicates that thrombin must have been generated in the aorta. In the experiments in which segments of the vessel were removed immediate1 after the vessel was flushed free of blood, heparin had little influence on platelet adherence.
This observation indicates that little, if any, thrombin
formation occurred in vessels prepared in this way.
Thus, in preparing vessel
segments for perfusion studies, measures should be taken to prevent thrombin formation. The observation that thrombin treatment of damaged vessels enhances platelet accumulation on their surface may have some relevance to the process of thrombus formation since constituents of the subendothelium can activate blood coagulation (12). more thrombogenic.
Exposure of the subendothelium to thrombin makes it
In addition, when the undamaged aorta surface has been
exposed to thrombin, platelets can adhere to it.
It is recognized that the
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endothelial cells on the surface of the everted aorta are altered.
77
They are,
.however, still effective in preventing platelet adhesion to the vessel wall. Awbrey and associates (13) have shown that in endothelial cell tissue cultures, exposure of the cells to thrombin alters the surface so that platelets adhere to it.
r\shfordand Freiman (14) observed the formation of thrombi at sites of
minimal injury, even though the endothelial cells were still present.
The
presence of fibrin with these platelet thrombi indicates that thrombin must have formed at the sites of injury and may have had a role in the formation of platelet aggregates.
ACKNOWLEDGEMENTS We wish to thank Dr. Mary Richardson for the electron microscopy and Mrs. D. Blondowska for technical assistance. a World Health Organization Fellowship, 1974.
E.M. Essien was the recipient of J.-P. Cazenave is a Senior
Research Fellow of the Ontario Heart Foundation.
This work was supported by
grant MT1309 from the Medical Research Council of Canada.
REFERENCES 1.
BAUMGARTNER, H.R. and MUGGLI, R. Adhesion and aggregation: morphological demonstration and quantitation in vivo and in vitro. In: Platelets in Biology and Pathology. J.L. Gordon (Ed.). ElsevierfNorth-Holland Biomedical Press, 1976, p.23-60.
2.
CAZENAVE, J.-P., PACRHAM, M.A., GUCCIONE, M.A. and MUSTARD, J.F. Inhibition of platelet adherence to damaged surface of rabbit aorta. 3. Lab. Clin. Med. 86, 551-563, 1975.
3.
CAZENAVE, J.-P., PACKHAM, M.A., DAVIES, J.A., KINLOUGH-RATHBONE, R.L. and MUSTARD, J.F. Studies on platelet adherence to collagen and subendothelium. In: PlateletFunction H.J. Day, H. Holmsen and M.B. Zucker (Eds.). U.S. Department of Health, Education and Welfare. DHEW Publication No. (NIH) 78-1087, 1978, p.181-198.
4.
CAZENAVE, J.-P., PACKHAM, M.A. and MUSTARD, J.F. Adherence of platelets to a collagen-coated surface: development of a quantitative method. J. Lab. Clin. Med. 82, 978-990, 1973.
5.
LYMAN, B., ROSENBERG, L. and RARPATRIN, S. Biochemical and biophysical aspects of human platelet adhesion to collagen fibers. J. Clin. Invest. 50, 1854-1863, 1971.
6.
GORDON, J.L. and DINGLE, D.T. Binding of radiolabelled collagen to blood platelets. J. Cell Sci. 16, 157-166, 1974.
7.
BRASS, L.F., FAILE, D. and BENSUSAN, H.B. Direct measurement of the platelet: collagen interaction by affinity chromatography on collagen sepharose. J. Lab. Clin. Med. 87, 525-534, 1976.
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8.
ESSIEN, E.H., KI.tZOUGH-RATHBONE, R.L., MORE. S. and ,wSTARD, J.F. The role of heparin on the inhibition of platelet adhesion to damaged arterial endothelial surface. Thromb. Diath. Haemorrh. 34, 600, 1975.
9.
ESSIEN, E.M. and ?iUSTARD, J.F. Inhibition of platelet adhesion to rabbit aorta by sulphinpyrazone and acetylsalicylic acid. Atherosclerosis 27, 89-95, 1977.
10.
GERRITY, R.G., RICHtUIDSON,M., SOMER, J.B., BELL, F.P. and SCHWARTZ, C.J. Endothelial cell morphology in areas of in vivo Evans blue uptake in the aorta of young pigs. II. Ultra-structure of the intima in areas of differing permeability to proteins. Amer. J. Pathol. 89, 313-334, 1977.
11.
BAWGARTNER, H.R. Platelet interaction with vascular structures. Diath. Haemorrh., Suppl. 51, 161-176, 1972.
12.
NIEWIAROWSKI, S., BANKOWSKI, E. and ROGOWICICA, I. Studies on the adsorption and activation of the Hageman factor (factor XII) by collagen and elastin. Thromb. Diath. Haemorrh., 14, 387-400. 1965.
13.
AWBREY, B.J., OWEN, W.G., FRY, G.L., CHENG, F.H. and HOAK, J.C. Binding of human thrombin to human endothelial cells and platelets. Blood 46, 1046, 1975.
14.
ASHFORD, T.P. and FREIMAN, D.G. Platelet aggregation at sites of minimal endothelial injury. An electron microscopy study. Amer. J. Pathol. 53, 599-607, 1968.
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