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The effect of acetylsalicylic acid and indomethacin on rabbit platelet adherence to collagen and the subendothelium in the presence of a low or high hematocrit
THROMBOSIS RESEARCH 13 ; 971-981 Pergamon Press Ltd .1978 . Printed in Great Britain 0049-3848/78/1201-0971 $02 .00/0
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THE EFFECT OF ACETYLSALICYLIC ACID AND INDOMETHACIN ON RABBIT PLATELET ADHERENCE TO COLLAGEN AND THE SUBENDOTHELIUM IN THE PRESENCE OF A LOW OR HIGH HEMATOCRIT
Jean-Pierre Cazenave, Raelene L . Kinlough-Rathbone, Marian A . Packham and J . Fraser Mustard Department of Pathology, McMaster University, Hamilton and Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada (Received 13 .6 .1978 ; in revised form 6 .9 .1978 . Accepted by Editor K .M . Brinkhous)
ABSTRACT We have examined the effect of ASA and indomethacin on rabbit platelet adherence to collagen or the subendothelium over a range of hematocrits . Platelet adherence to the surfaces was measured in vitro using a rooting probe . Washed rabbit platelets were 5 prelabeled with Cr and resuspended in media containing 4% albumin, apyrase and physiological concentrations of calcium and magnesium . ASA, indomethacin or their solvents were incubated for 10 min with the platelet suspensions before washed rabbit red blood cells were added . Collagen-coated glass cylinders or everted segments of de-endothelialized rabbit thoracic aorta were mounted on probes and rotated for 10 min at 200 rpm at 37 ° C in a suspension of washed platelets containing various concentrations of red blood cells . The surfaces were rinsed in EDTA to remove platelet aggregates and the radioactivity associated with the surfaces was measured . Under the conditions of flow used in this system the hematocrit determined whether or not an inhibitory effect of ASA on ASA inhibited the adherence of rabbit platelets was demonstrable . platelet adherence to collagen and to the subendothelium at a low hematocrit, but at a 40% hematocrit it did not . In contrast, indomethacin showed an inhibitory effect on platelet adherence to the surfaces even at a 40% hematocrit . ASA and indomethacin were tested at concentrations that were higher than those required to block the arachidonate pathway, as measured by malondialdehyde formation . Under these conditions, ASA did not inhibit platelet adherence, indicating that the arachidonate pathway does not play a major role in platelet adherence at a 40% hematocrit . Indomethacin inhibited adherence by a mechanism that was probably independent of the These results show that inhibition of the platelet cyclo-oxygenase . the magnitude of the physical force imposed on the platelets by the red cells determines the extent of platelet adherence and modifies the effects of drugs on adherence to the surfaces .
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INTRODUCTION We have previously reported that when acetylsalicylic acid (ASA) is tested in a system without red cells it causes some inhibition of rabbit platelet adherence to collagen-containing surfaces (1,2) .
Tschopp (3) has shown that
ASA does not inhibit the adhesion of rabbit platelets to the subendothelium in the presence of red blood cells, although it inhibits the formation of plateler thrombi . The object of the experiments reported in this paper was to examine the effect of ASA and indomethacin on rabbit platelet adherence to collagen and the subendothelium in the presence of a 40% hematocrit and 4Z albumin . We ha ve • shown elsewhere that with this test system the adherence of individual platelets can be determined and platelet aggregates are not present on the surfaces
(4,5) . MATERIALS AND METHODS
Preparation of washed
rabbit platelets labeled with
51
Cr . 51 Suspension of washed rabbit platelets labeled with Cr were prepared as
described previously (5) .
The final resuspending medium was Tyrode solution or
Eagle's medium (GIBCO, Grand Island,
N.Y .,
to which was added 2 .2 g sodium
bicarbonate per liter) containing 4% bovine albumin (Pentax, Fraction V, Miles Laboratories, Kankakee, I11 .), apyrase and 5 mM Hepes buffer (N-2-hydroxethylpiperazine-N'2-ethane sulfonic acid, Sigma Chemical Co ., St . Louis, Mo .) to maintain the pH of the medium at 7 .35 . Platelet counts were adjusted to 300,000 to 700,000/mm3 in the total volume of the suspension, including the volume occupied by the washed red blood cells (see below) . The platelet suspensions were stored at 37 ° C . Preparation of washed rabbit red blood cells . Rabbit red blood cells were washed twice as described previously (5) and finally resuspended in the same medium as the platelets, except that the pH was adjusted to 9 before the addition of the red blood cells . Within 5 min of the addition of the red blood cells, the pH fell to 7 .3 and remained at this value . Just before the surfaces on the probes were to be rotated in the platelet suspension, the red blood cells were packed by centrifugation and the packed cells were added to the platelet Suspension to obtain the required hematocrit . The pH was 7 .35 and remained at this value during the rotation of the probes in the suspension . Preparation of collagen-coated segment& of glass rods . The preparation of acid-soluble collagen from bovine tendon collagen (type I collagen, reference C9879, Sigma) and the coating of 1 cm glass cylinders have been described previously (5,6) .
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Preparation of segments of aorta damaged by a balloon catheter . Immediately after exsanguination of the rabbits, the thoracic aorta was zxposed, rinsed with modified Tyrode solution (no calcium, magnesium or ;lucose), and the endothelium was removed by 5 passages through the aorta of an inflated balloon catheter (5F, Edwards Laboratories, Santa Anna,
Ca .) .
The
,~e-endothellalized aortas were everted, mounted on metal rods and cut into 1 cm segments as described previously
(5) .
Adherence of platelets to collagen-coated glass
or to damaged aorta
segments (5) . Two metal probes were fitted with collagen-coated glass cylinders or with everted segments of rabbit aorta and rotated simultaneously at 200 rpm for 10 min in two siliconized glass test tubes, each containing 10 ml of platelet and red blood cell suspension at 37 ° C . In some experiments, washed red cells were omitted . After rotation in the platelet suspension, the segments of aorta or of collagen-coated glass were rotated in fresh modified Tyrode solution containing 10 mM EDTA, pH 7 .35 at 200 rpm for 5 min at 37 ° C . This was done to ensure that only single platelets and not platelet aggregates remained adherent to the surface . The radioactivity associated with segments of aorta or collagen-coated glass was measured in a well-type gamma-scintillation counter . The number the platelets adherent to the surfaces was calculated as described previously (2) . Treatment of platelets with drugs . Acetylsalicylic acid (ASA, British Drug Houses, Toronto) was prepared in modified Tyrode solution immediately before use . Indomethacin (Merck, Sharp and Dohme, West Point, Pa .) was prepared as a stock solution (10 2M) in 951 ethanol and further diluted with modified Tyrode solution . The drugs, or their appropriate solvents, were incubated for 10 min at 37°C with the platelet suspensions before the addition of red blood cells . In some experiments the platelets were incubated with the drugs, washed and resuspended in fresh medium before the addition of red blood cells . For each experiment appropriate controls were run so that day-to-day variations would not obscure the effects of the drugs on platelet adherence . Malondialdehyde formation . Malondialdehyde (MDA) formed by platelets exposed to 1 U/ml of thrombin (topical bovine thrombin, Parke, Davis and Co ., Detroit, Mich .) was assayed by the thiobarbituric acid method of Smith et al (7) in which the optical density at 532 nm is measured . A standard curve was constructed using malondialdehyde bis-(dimethyl acetal)(Aldrich Chemical Company Inc ., Milwaukee, Wis .) dissolved in a mixture of equal parts absolute alcohol and 0 .2 N HC1 .
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Scanningelectron microscopy . Segments of aorta were examined by scanning electron microscopy by the method of Gerrity et al (8) . RESULTS Table I shows the effect of the hematocrit on platelet adherence and on As the hematocrit was increased up to 20% the
the inhibitory effect of ASA .
number of platelets adhering to the collagen-coated surface also increased .
At
hematocrits of 10% or 20%, the presence of ASA at a concentration of 0 .25 mM significantly reduced the number of platelets adherent to the surface . However, at a 40% hematocrit, ASA (even at a concentration of 10 mM) did not have a significant effect on adherence .
In a separate experiment, it was shown
that concentrations of ASA of 0 .25 mM and greater blocked MDA formation in response to 1 U/ml of thrombin . TABLE I Effect of ASA on the adherence of rabbit platelets to a collagen-coated glass surface when tested in the presence of various red blood cell concentrations .
Exp . 1.
2.
3. 4.
ASA (MM)
Uct (%)
(n)*
Number of platelets per mm 2 (mean + S .E .)
2P**
0 0 .25 0 0 .25 0 0 .25 0 0 .25
0 0 10 10 20 20 40 40
(16) (16) (16) (16) (16) (16) (16) (16)
5,400 6,400 25,600 14,800 63,700 48,200 65,300 60,400
+ + + + + + + +
600 500 1,300 1,200 2,500 1,800 1,900 1,700
0 0 .01 0 .1 1 10
20 20 20 20 20
(16) (8) (8) (8) (8)
71,000 + 61,500'; 51,300 + 43,200 + 40,600 +
2,200 2,800 2,300 1,700 1,100
0 1
40 40
(96) (96)
66,500 + 2,300 62,600 + 2,600
< 0 .2
0 10
40 40
(16) (16)
69,000 + 2,700 70,400 + 6,200
< 0 .9
< 0 .03 < 0 .001 < 0 .001 < 0 .1 < < < <
0 .02 0 .001 0 .001 0 .001
Platelets were resuspended in Eagle's medium containing 4% albumin, ypyrase, 5 mM Nepes buffer and 40% hematocrit . Platelet count was 300,000/mm . The probes were rotated at 200 rpm for 10 min in the platelet red cell suspension as described in "Materials and Methods ." * **
Number of segments Significance of difference between the means using an unpaired two-tailed t test
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O j~'I1-L?f'I\, AND PL_ATELPTS
Since the number of platelets adherent to the s
_ _ ., this test system
increased with the time of rotation until a maximum was reached, we also examined whether the effect of ASA on adherence was related to the time of rotation (Table it) . At a 40% hematocrit, ASA had no effect on adherence when examined at different times over a thirty minute period . Table IT also shows, however, that at a hematocrit of 107,, the inhibiting effect of aspirin was apparent after either 10 min or 30 min of rotation .
TABLE II Effect of ASA on the adherence of rabbit platelets to a collagen-coated glass surface when tested after various times of rotation
cxp .
ASA (mM)
Her
0 0 .1 0 0 .1
10
0 0 .1 0 0 .1 0 0 .1 0
40
Time of rotation (min)
0 .1
(n)*
I Number of platelets per mm (mean + S .E .)
10 10 30 30
(8) (8) (8) (8)
45,500 24,300 75,000 40,400
+ + + +
2,600 2,200 7,700 2,800
2 2
(8) (8) (8) (8) (8) (8) (8) (8)
10,200 13, ;00 29,500 32,900 5 ,, ,400 5,', ;00 i0 ,B00 12',290
+ + + + + +
2,100 900 1,600 1,900 4,500 2,600 7,400
5 5 10 10 30 30
+ 4,300
< 0 .001 < 0 .001
< 0 .2 < 0,2 C 0 .975 C 0,2
For legend see Table I in contrast to ASA, indomethacin inhibited piscelet adherence to the surface at all the hematocrits tested (Table Lit) . 'dhen tested at a 20% hematocrit, maximum inhibition of platelet adherence to collagen was obtained at a concentration of 0 .5 pM and further inhibition was not observed at higher concentrations (Table IV) . In a separate experiment it was observed that at concentrations of 5 pM or greater, indomethacin completely inhibited malondialdehyde formation when platelets were exposed to 1 U/ml of thrombin (Table IV) . Table V shows results of experiments in which the platelets were preincubated with either ASA or indomethacin, washed and resuspended in fresh medium before being added to the test system with the 47 . albumin and the 40% hematocrit . Pretreatment of the platelets with ASA did not affect their ability to adhere to the collagen-coated glass surface . In contrast, the adherence of platelets pretreated with indomethacin was less than that of untreated platelets and subjected to the same incubation, washing and resuspending procedures .
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TABLE III
Effect of indomethacin on the adherence of platelets to a collagen-coated glass surface when tested in the presence of various red blood cell concentrations
Indomethacin (pM)
Rct b
(n)*
Exp .
1.
0 100 0 100 0 100 0 100
0 0 10 10 20 20 40 40
(8) (8) (8) (8) (8) (8) (8) (8)
4,300 3,100 30,300 19,100 61,100 47,200 79,000 54,600
+ + + + + + + +
300 300 2,400 2,200 2,200 1,300 800 700
0 0 .1 0 .5 1 5 10 100
40
(64) (8) (8) (16) (8) (8) (16)
101,700 86,400 69,200 77,300 84,400 83,400 73,200
+ + + + + + +
2,500 6,700 1,400 900 2,500 1,400 1 600
2.
3.
0 20
40
(24) (24)
2P**
Number of platelets per mm2 (mean + S .E .)
97,900 + 2,200 68,200 + 4,400
< 0 .025 < 0 .005 < 0 .001 < 0 .001
< < < < < <
0 .05 0 .001 0 .001 0 .001 0 .001 0 .001
< 0 .001
For legend see Table I
In experiments in which platelet adherence to the subendothelium of the aorta was measured, the effects of the drugs were similar to the effects with the collagen-coated surface .
Although ASA inhibited platelet adherence to the subendotheliam at low hematocrits, at a 40% hematocrit no significant inhibition was apparent (Table VI) . In contrast, indomethacin was inhibitory at a 40% hematocrit . Figure 1 shows scanning electron micrographs of the subendothelial surface of a rabbit aorta following exposure to platelet suspensions with and without aspirin (40Z hematocrit) . There appeared to be similar numbers of platelets on the surfaces and no platelet aggregates were seen . Neither ASA nor indomethacin appeared to cause any detectable morphological effect on spreading or pseudopod formation .
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TABLE IV Effect of indomethacin on the inhibition of adherence of platelets to a collagen-coated glass surface and on the inhibition of malondialdehyde ('[DA) formation*
Exp .
Indomethacin (PM)
1 2 3 4 5 6 7 B
0 0 .5 1 2 5 10 50 100
(n)**
(32) (8) (8) (8) (16) (16) (8) (8)
Number of platelets per mm` (mean + S .E .)
60,500 42,200 38,400 40,000 40,200 43,200 42,800 42,900
+ + + + + + + +
2,300 1,200 1,800 700 1,600 1,700 1,600 800
2P***
< < < < < < <
0 .001 0 .001 0 .001 0 .001 0 .001 0 .001 0 .001
MDA**** formation (%)
100 82 32 4 .5 0 0 0 0
Platelets were resuspended in Tyrode solution containing 4% albumin, apyrase, 5 3mM Hepes buffer and 20% hematocrit . Platelet count was 300,000/mm . ** Number of segments *** Compared with control . 2, 3, 4, 5, 6, 7, and B were not significantly different from each other, p < 0 .30 . **** MIA formation was measured on a sample of platelet suspension after incubation with the inhibitor or its solvent and addition of I U/ml thrombin . 100% corresponds to approximately 1 .5 nmol MDA/10 9 platelets .
TABLE V Effect of pretreatment of platelets with ASA or indomethacin and resuspension in fresh medium on the inhibition of platelet adherence to a collagen-coated surface
Pretreatment
(n)*
Number of platelets per mm2 (mean + S .E .)
2P**
Control ASA 0 .5 mM
(8) (8)
68,300 62,600
+ 2,000 ++ 2,000
C 0 .1
Control Indomethacin 100 yM
(8) (8)
89,400 69,100
+ 2,600 + 1,300
< 0 .001
51
Cr-labeled rabbit platelets were incubated in the second washing fluid for 15 minutes with 0 .5 mM ASA, 100 pM indomethacin or the appropriate solvent . Then the platelets were resuspended in fresh Tyrode solution containing 4% albumin, apyrase, 5 mM Hepes buffer and a 40% hematocrit . For legend see Table I
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TABLE VI Effect of hematocrit on the inhibition by ASA or indomethacin of platelet adherence to the subendothelium Exp .
Drug
(4m)
Hct I
(n)*
1.
ASA
0 100
0
(20) (20)
9,900 5,800
0 10 100 250
10
(30) (30) (30) (30)
57,300 24,200 22,900 17,200
0 100
20
(20) (20)
153,100 81,400
+ 16,300 + 15,100
< 0 .05
0 100 250
40
(80) (80) (80)
112,200 114,800 100,400
+ 9,400 + 6,900 + 10,600
< 0 .10 < 0 .10
0 20
40
(30) (30)
78,900 65,300
+ 4,400 + 3,800
< 0 .025
2.
Indomethacin
Number of platelets per mm2 (mean + S .E .)
2P**
+ 2,100 + 1,500
< 0 .10
+ + + T
< 0 .001 < 0 .001 < 0 .001
7,900 2,200 1,200 1,700
Platelet count was 700,000/mm 3 in Exp . 1 and 300,000/mm 3 i For legend see Table I
Exp . 2
Figure 1 . Scanning electron micrograph of platelets adherent to the subendothelium of a rabbit thoracic aorta . The endothelium had been removed with a balloon catheter and the aorta segment had been everted on a probe and rotated in a platelet suspension containing apyrase, 4% albumin and 40% red blood cells at a platelet count of 500,000/mm and the surface was rinsed with EDTA (as described in materials and methods) . A monolayer of platelets covered the surfaces and platelet aggregates were not present . There appeared to be similar number of platelets on the surfaces whether the platelets had not been exposed (control, A) or had been exposed to 1 mM ASA (B) .
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DISCUSSION It appears that under the conditions of flow used in this system, the hematocrit determines whether or not an inhibitory effect of ASA on rabbit platelet adherence is demonstrable . As observed in the present study and in earlier experiments (1,2), in the presence of a low hematocrit, ASA can be
shown to inhibit platelet adherence to surfaces ; at a 40% hematocrit ASA is not inhibitory, even at very high concentrations . In vivo, a concentration of 8 uM ASA can be achieved in rabbits 5 min after an intravenous injection of 100 mg ASA/kg (Dr . M . R . Buchanan, personal communication) . In contrast, indomethacin does show a definite inhibitory effect on rabbit platelet adhesion to the surfaces even in the presence of a 40% hematocrit . At the higher concentrations used, both drugs completely block the arachidonate pathway in platelets as indicated by prevention of detectable malondialdehyde formation in response to a high concentraton of thrombin . It seems unlikely, therefore, that this pathway plays a major role in platelet adherence . However, both drugs may have an inhibitory effect on the formation of platelet thrombi because of their ability to inhibit the cyclo-oxygenase of the arachidonate pathway and thus prevent the formation of the prostaglandin endoperoxides and thromboxane A2 .
Indomethacin, but not ASA, may have an additional inhibitory effect on
the adherence of the initial platelet layer at an injury site on a vessel wall . The apparently mild inhibitory effect of indomethacin may be related to an effect on the availability of cellular calcium, as has been suggested by Northover (9) . The method used in these studies measures the adherence of individual platelets to the surfaces
(4,5) .
The presence of apyrase in the suspending
medium, and the EDTA rinse of the surface to which the rabbit platelets have adhered, ensures that platelet aggregates do not remain on the surface
(4,5) .
Thus, when inhibition occurs, it must be attributable to inhibition of adherence and not to inhibition of aggregate formation . Under conditions in which both adherence of single platelets and thrombus formation are measured, aspirin inhibits the accumulation of platelet thrombi on the subendothelium (10) . The results of the present studies are in agreement with those of Baumgartner and his colleagues (3,10,11,12) who have shown that when rabbit or
human platelet adherence to the rabbit subendothelium is tested in the presence of a 40% hematocrit, ASA does not inhibit the adherence of single platelets although it inhibits thrombus formation . However, our results also show that the test conditions appear to be an important determinant of whether an effect
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of aspirin on adherence can be demonstrated . When the physical forces on the platelets are not high, ASA has some effect on platelet adherence and this may be of some significance at sites in the vascular tree where flow is slow such as in veins .
The recent report that aspirin reduces the incidence of venous
thrombosis in men undergoing hip surgery could be due to an effect of aspirin on platelet adherence as well as on the arachidonate pathway (13) . ACKNOWLEDGEMENTS We wish to thank Dr . Mary Richardaon for the scanning electron microscopy and Mrs . D . Blondowska for excellent technical assistance . This work was supported by grants from the Medical Research Council of Canada (MT 1309 and MT 2629) and by Senior Research Fellowships of the Ontario Heart Foundation to J .-P . Cazenave and R .L . Kinlough-Rathbone . REFERENCES 1 . CAZENAVE, J .-P ., PACKHAM, M .A ., GUCCIONE, M .A . and MUSTARD, J .F . Inhibition of platelet adherence to a collagen-coated surface by nonsteroidal anti-inflammatory drugs, pyrimido-pyrimidine and tricyclic compounds, and lidocaine . J . Lab . Clin . Med . 83, 797, 1974 . 2 . CAZENAVE, J .-P ., PACKHAM, M .A ., GUCCIONE, M .A . and MUSTARD, J .F . Inhibition of platelet adherence to damaged surface of rabbit aorta . J . Lab . Clin . Med . 86, 551, 1975 . 3.
TSCHOPP, T .B . Aspirin inhibits platelet aggregation on, but not adhesion to, collagen fibrils : an assessmeDt of platelet adhesion and deposited platelet mass by morphometry and 71 Cr-labeling . Thromb . Res . 11, 619, 1977 .
4 . CAZENAVE, J .-P . Platelet adherence to collagen containing surfaces . Thesis, Ph .D, McMaster University, Hamilton, 1977 . 5.
CAZENAVE, J .-P ., RICHARDSON, M ., KINLOUGH-RATHBONE, R .L ., PACKHAM, M .A . and MUSTARD, J .F . Quantitative radioisotopic measurement and scanning electronmicroscopic study of platelet adherence to collagen-coated surface and to subendothelium with a rotating probe device . J . Lab . Clin . Med . Submitted for publication .
6.
CAZENAVE, J .-P ., PACKHAM, M .A . and MUSTARD, J .F . Adherence of platelets to a collagen-coated surfaces development of a quantitative method . J . Lab . Clin . Med . 82, 978, 1973 .
7.
SMITH, J .B ., INGERMAN, C .M . and SILVER, M .J . Malondialdehyde formation as an indicator of prostaglandin production by human platelets . J . Lab . Clin . Med . 88, 167, 1976 .
8 . GERRITY, R .G ., RICHARDSON, M ., SOMER, J .8 ., 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, 1977 .
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9.
NORTHOVER, B .J . 293, 1977 .
INDOMETH CZN, AND PLATELETS
Indomethacin .
A calcium antagonist .
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Gen . Pha nacol . 8,
10, BAUMGARTNER, H .R . and MUGGLI, R . Effect of acetylsalicylic acid on platelet adhesion to subendothelium and on the formation of mural platelet thrombi in vivo . Thromb . Diath . Haemorrh . Suppl . 60, 345, 1974, 11, WEISS, H,J ., TSCHOPP, T,B, and BAUMGARTNER, H .R . Impaired interaction (adhesion-aggregation) of platelets with the subendothelium in storageN . Engl . J . Med . 293, 619, pool disease and after aspirin ingestion . 1975 . 12 . BAUMGARTNER, H .R ., TSCHOPP, T .B . and WEISS, H .J . Platelet interaction with collagen fibrils in flowing blood . II . Impaired adhesion-aggregation in bleeding disorders . A comparison with subendothelium . Thromb . Haemostas . 37, 17, 1977 . 13 . HARRIS, W .H ., SALZMAN, E .W ., ATHANASOULIS, C,A„ WALTMAN, A .C . and DESANCTIS, R .W . Aspirin prophylaxis of venous thromboembolism after total N . Engl . J . Med . 297, 1246, 1977 . hip replacement .
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THE EFFECT OF ACETYLSALICYLIC ACID AND INDOMETHACIN ON RABBIT PLATELET ADHERENCE TO COLLAGEN AND THE SUBENDOTHELIUM IN THE PRESENCE OF A LOW OR HIGH HEMATOCRIT
Jean-Pierre Cazenave, Raelene L . Kinlough-Rathbone, Marian A . Packham and J . Fraser Mustard Department of Pathology, McMaster University, Hamilton and Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada (Received 13 .6 .1978 ; in revised form 6 .9 .1978 . Accepted by Editor K .M . Brinkhous)
ABSTRACT We have examined the effect of ASA and indomethacin on rabbit platelet adherence to collagen or the subendothelium over a range of hematocrits . Platelet adherence to the surfaces was measured in vitro using a rooting probe . Washed rabbit platelets were 5 prelabeled with Cr and resuspended in media containing 4% albumin, apyrase and physiological concentrations of calcium and magnesium . ASA, indomethacin or their solvents were incubated for 10 min with the platelet suspensions before washed rabbit red blood cells were added . Collagen-coated glass cylinders or everted segments of de-endothelialized rabbit thoracic aorta were mounted on probes and rotated for 10 min at 200 rpm at 37 ° C in a suspension of washed platelets containing various concentrations of red blood cells . The surfaces were rinsed in EDTA to remove platelet aggregates and the radioactivity associated with the surfaces was measured . Under the conditions of flow used in this system the hematocrit determined whether or not an inhibitory effect of ASA on ASA inhibited the adherence of rabbit platelets was demonstrable . platelet adherence to collagen and to the subendothelium at a low hematocrit, but at a 40% hematocrit it did not . In contrast, indomethacin showed an inhibitory effect on platelet adherence to the surfaces even at a 40% hematocrit . ASA and indomethacin were tested at concentrations that were higher than those required to block the arachidonate pathway, as measured by malondialdehyde formation . Under these conditions, ASA did not inhibit platelet adherence, indicating that the arachidonate pathway does not play a major role in platelet adherence at a 40% hematocrit . Indomethacin inhibited adherence by a mechanism that was probably independent of the These results show that inhibition of the platelet cyclo-oxygenase . the magnitude of the physical force imposed on the platelets by the red cells determines the extent of platelet adherence and modifies the effects of drugs on adherence to the surfaces .
971
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INTRODUCTION We have previously reported that when acetylsalicylic acid (ASA) is tested in a system without red cells it causes some inhibition of rabbit platelet adherence to collagen-containing surfaces (1,2) .
Tschopp (3) has shown that
ASA does not inhibit the adhesion of rabbit platelets to the subendothelium in the presence of red blood cells, although it inhibits the formation of plateler thrombi . The object of the experiments reported in this paper was to examine the effect of ASA and indomethacin on rabbit platelet adherence to collagen and the subendothelium in the presence of a 40% hematocrit and 4Z albumin . We ha ve • shown elsewhere that with this test system the adherence of individual platelets can be determined and platelet aggregates are not present on the surfaces
(4,5) . MATERIALS AND METHODS
Preparation of washed
rabbit platelets labeled with
51
Cr . 51 Suspension of washed rabbit platelets labeled with Cr were prepared as
described previously (5) .
The final resuspending medium was Tyrode solution or
Eagle's medium (GIBCO, Grand Island,
N.Y .,
to which was added 2 .2 g sodium
bicarbonate per liter) containing 4% bovine albumin (Pentax, Fraction V, Miles Laboratories, Kankakee, I11 .), apyrase and 5 mM Hepes buffer (N-2-hydroxethylpiperazine-N'2-ethane sulfonic acid, Sigma Chemical Co ., St . Louis, Mo .) to maintain the pH of the medium at 7 .35 . Platelet counts were adjusted to 300,000 to 700,000/mm3 in the total volume of the suspension, including the volume occupied by the washed red blood cells (see below) . The platelet suspensions were stored at 37 ° C . Preparation of washed rabbit red blood cells . Rabbit red blood cells were washed twice as described previously (5) and finally resuspended in the same medium as the platelets, except that the pH was adjusted to 9 before the addition of the red blood cells . Within 5 min of the addition of the red blood cells, the pH fell to 7 .3 and remained at this value . Just before the surfaces on the probes were to be rotated in the platelet suspension, the red blood cells were packed by centrifugation and the packed cells were added to the platelet Suspension to obtain the required hematocrit . The pH was 7 .35 and remained at this value during the rotation of the probes in the suspension . Preparation of collagen-coated segment& of glass rods . The preparation of acid-soluble collagen from bovine tendon collagen (type I collagen, reference C9879, Sigma) and the coating of 1 cm glass cylinders have been described previously (5,6) .
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Preparation of segments of aorta damaged by a balloon catheter . Immediately after exsanguination of the rabbits, the thoracic aorta was zxposed, rinsed with modified Tyrode solution (no calcium, magnesium or ;lucose), and the endothelium was removed by 5 passages through the aorta of an inflated balloon catheter (5F, Edwards Laboratories, Santa Anna,
Ca .) .
The
,~e-endothellalized aortas were everted, mounted on metal rods and cut into 1 cm segments as described previously
(5) .
Adherence of platelets to collagen-coated glass
or to damaged aorta
segments (5) . Two metal probes were fitted with collagen-coated glass cylinders or with everted segments of rabbit aorta and rotated simultaneously at 200 rpm for 10 min in two siliconized glass test tubes, each containing 10 ml of platelet and red blood cell suspension at 37 ° C . In some experiments, washed red cells were omitted . After rotation in the platelet suspension, the segments of aorta or of collagen-coated glass were rotated in fresh modified Tyrode solution containing 10 mM EDTA, pH 7 .35 at 200 rpm for 5 min at 37 ° C . This was done to ensure that only single platelets and not platelet aggregates remained adherent to the surface . The radioactivity associated with segments of aorta or collagen-coated glass was measured in a well-type gamma-scintillation counter . The number the platelets adherent to the surfaces was calculated as described previously (2) . Treatment of platelets with drugs . Acetylsalicylic acid (ASA, British Drug Houses, Toronto) was prepared in modified Tyrode solution immediately before use . Indomethacin (Merck, Sharp and Dohme, West Point, Pa .) was prepared as a stock solution (10 2M) in 951 ethanol and further diluted with modified Tyrode solution . The drugs, or their appropriate solvents, were incubated for 10 min at 37°C with the platelet suspensions before the addition of red blood cells . In some experiments the platelets were incubated with the drugs, washed and resuspended in fresh medium before the addition of red blood cells . For each experiment appropriate controls were run so that day-to-day variations would not obscure the effects of the drugs on platelet adherence . Malondialdehyde formation . Malondialdehyde (MDA) formed by platelets exposed to 1 U/ml of thrombin (topical bovine thrombin, Parke, Davis and Co ., Detroit, Mich .) was assayed by the thiobarbituric acid method of Smith et al (7) in which the optical density at 532 nm is measured . A standard curve was constructed using malondialdehyde bis-(dimethyl acetal)(Aldrich Chemical Company Inc ., Milwaukee, Wis .) dissolved in a mixture of equal parts absolute alcohol and 0 .2 N HC1 .
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Scanningelectron microscopy . Segments of aorta were examined by scanning electron microscopy by the method of Gerrity et al (8) . RESULTS Table I shows the effect of the hematocrit on platelet adherence and on As the hematocrit was increased up to 20% the
the inhibitory effect of ASA .
number of platelets adhering to the collagen-coated surface also increased .
At
hematocrits of 10% or 20%, the presence of ASA at a concentration of 0 .25 mM significantly reduced the number of platelets adherent to the surface . However, at a 40% hematocrit, ASA (even at a concentration of 10 mM) did not have a significant effect on adherence .
In a separate experiment, it was shown
that concentrations of ASA of 0 .25 mM and greater blocked MDA formation in response to 1 U/ml of thrombin . TABLE I Effect of ASA on the adherence of rabbit platelets to a collagen-coated glass surface when tested in the presence of various red blood cell concentrations .
Exp . 1.
2.
3. 4.
ASA (MM)
Uct (%)
(n)*
Number of platelets per mm 2 (mean + S .E .)
2P**
0 0 .25 0 0 .25 0 0 .25 0 0 .25
0 0 10 10 20 20 40 40
(16) (16) (16) (16) (16) (16) (16) (16)
5,400 6,400 25,600 14,800 63,700 48,200 65,300 60,400
+ + + + + + + +
600 500 1,300 1,200 2,500 1,800 1,900 1,700
0 0 .01 0 .1 1 10
20 20 20 20 20
(16) (8) (8) (8) (8)
71,000 + 61,500'; 51,300 + 43,200 + 40,600 +
2,200 2,800 2,300 1,700 1,100
0 1
40 40
(96) (96)
66,500 + 2,300 62,600 + 2,600
< 0 .2
0 10
40 40
(16) (16)
69,000 + 2,700 70,400 + 6,200
< 0 .9
< 0 .03 < 0 .001 < 0 .001 < 0 .1 < < < <
0 .02 0 .001 0 .001 0 .001
Platelets were resuspended in Eagle's medium containing 4% albumin, ypyrase, 5 mM Nepes buffer and 40% hematocrit . Platelet count was 300,000/mm . The probes were rotated at 200 rpm for 10 min in the platelet red cell suspension as described in "Materials and Methods ." * **
Number of segments Significance of difference between the means using an unpaired two-tailed t test
3 .No .b
ASA, IL
a-5
O j~'I1-L?f'I\, AND PL_ATELPTS
Since the number of platelets adherent to the s
_ _ ., this test system
increased with the time of rotation until a maximum was reached, we also examined whether the effect of ASA on adherence was related to the time of rotation (Table it) . At a 40% hematocrit, ASA had no effect on adherence when examined at different times over a thirty minute period . Table IT also shows, however, that at a hematocrit of 107,, the inhibiting effect of aspirin was apparent after either 10 min or 30 min of rotation .
TABLE II Effect of ASA on the adherence of rabbit platelets to a collagen-coated glass surface when tested after various times of rotation
cxp .
ASA (mM)
Her
0 0 .1 0 0 .1
10
0 0 .1 0 0 .1 0 0 .1 0
40
Time of rotation (min)
0 .1
(n)*
I Number of platelets per mm (mean + S .E .)
10 10 30 30
(8) (8) (8) (8)
45,500 24,300 75,000 40,400
+ + + +
2,600 2,200 7,700 2,800
2 2
(8) (8) (8) (8) (8) (8) (8) (8)
10,200 13, ;00 29,500 32,900 5 ,, ,400 5,', ;00 i0 ,B00 12',290
+ + + + + +
2,100 900 1,600 1,900 4,500 2,600 7,400
5 5 10 10 30 30
+ 4,300
< 0 .001 < 0 .001
< 0 .2 < 0,2 C 0 .975 C 0,2
For legend see Table I in contrast to ASA, indomethacin inhibited piscelet adherence to the surface at all the hematocrits tested (Table Lit) . 'dhen tested at a 20% hematocrit, maximum inhibition of platelet adherence to collagen was obtained at a concentration of 0 .5 pM and further inhibition was not observed at higher concentrations (Table IV) . In a separate experiment it was observed that at concentrations of 5 pM or greater, indomethacin completely inhibited malondialdehyde formation when platelets were exposed to 1 U/ml of thrombin (Table IV) . Table V shows results of experiments in which the platelets were preincubated with either ASA or indomethacin, washed and resuspended in fresh medium before being added to the test system with the 47 . albumin and the 40% hematocrit . Pretreatment of the platelets with ASA did not affect their ability to adhere to the collagen-coated glass surface . In contrast, the adherence of platelets pretreated with indomethacin was less than that of untreated platelets and subjected to the same incubation, washing and resuspending procedures .
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TABLE III
Effect of indomethacin on the adherence of platelets to a collagen-coated glass surface when tested in the presence of various red blood cell concentrations
Indomethacin (pM)
Rct b
(n)*
Exp .
1.
0 100 0 100 0 100 0 100
0 0 10 10 20 20 40 40
(8) (8) (8) (8) (8) (8) (8) (8)
4,300 3,100 30,300 19,100 61,100 47,200 79,000 54,600
+ + + + + + + +
300 300 2,400 2,200 2,200 1,300 800 700
0 0 .1 0 .5 1 5 10 100
40
(64) (8) (8) (16) (8) (8) (16)
101,700 86,400 69,200 77,300 84,400 83,400 73,200
+ + + + + + +
2,500 6,700 1,400 900 2,500 1,400 1 600
2.
3.
0 20
40
(24) (24)
2P**
Number of platelets per mm2 (mean + S .E .)
97,900 + 2,200 68,200 + 4,400
< 0 .025 < 0 .005 < 0 .001 < 0 .001
< < < < < <
0 .05 0 .001 0 .001 0 .001 0 .001 0 .001
< 0 .001
For legend see Table I
In experiments in which platelet adherence to the subendothelium of the aorta was measured, the effects of the drugs were similar to the effects with the collagen-coated surface .
Although ASA inhibited platelet adherence to the subendotheliam at low hematocrits, at a 40% hematocrit no significant inhibition was apparent (Table VI) . In contrast, indomethacin was inhibitory at a 40% hematocrit . Figure 1 shows scanning electron micrographs of the subendothelial surface of a rabbit aorta following exposure to platelet suspensions with and without aspirin (40Z hematocrit) . There appeared to be similar numbers of platelets on the surfaces and no platelet aggregates were seen . Neither ASA nor indomethacin appeared to cause any detectable morphological effect on spreading or pseudopod formation .
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TABLE IV Effect of indomethacin on the inhibition of adherence of platelets to a collagen-coated glass surface and on the inhibition of malondialdehyde ('[DA) formation*
Exp .
Indomethacin (PM)
1 2 3 4 5 6 7 B
0 0 .5 1 2 5 10 50 100
(n)**
(32) (8) (8) (8) (16) (16) (8) (8)
Number of platelets per mm` (mean + S .E .)
60,500 42,200 38,400 40,000 40,200 43,200 42,800 42,900
+ + + + + + + +
2,300 1,200 1,800 700 1,600 1,700 1,600 800
2P***
< < < < < < <
0 .001 0 .001 0 .001 0 .001 0 .001 0 .001 0 .001
MDA**** formation (%)
100 82 32 4 .5 0 0 0 0
Platelets were resuspended in Tyrode solution containing 4% albumin, apyrase, 5 3mM Hepes buffer and 20% hematocrit . Platelet count was 300,000/mm . ** Number of segments *** Compared with control . 2, 3, 4, 5, 6, 7, and B were not significantly different from each other, p < 0 .30 . **** MIA formation was measured on a sample of platelet suspension after incubation with the inhibitor or its solvent and addition of I U/ml thrombin . 100% corresponds to approximately 1 .5 nmol MDA/10 9 platelets .
TABLE V Effect of pretreatment of platelets with ASA or indomethacin and resuspension in fresh medium on the inhibition of platelet adherence to a collagen-coated surface
Pretreatment
(n)*
Number of platelets per mm2 (mean + S .E .)
2P**
Control ASA 0 .5 mM
(8) (8)
68,300 62,600
+ 2,000 ++ 2,000
C 0 .1
Control Indomethacin 100 yM
(8) (8)
89,400 69,100
+ 2,600 + 1,300
< 0 .001
51
Cr-labeled rabbit platelets were incubated in the second washing fluid for 15 minutes with 0 .5 mM ASA, 100 pM indomethacin or the appropriate solvent . Then the platelets were resuspended in fresh Tyrode solution containing 4% albumin, apyrase, 5 mM Hepes buffer and a 40% hematocrit . For legend see Table I
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TABLE VI Effect of hematocrit on the inhibition by ASA or indomethacin of platelet adherence to the subendothelium Exp .
Drug
(4m)
Hct I
(n)*
1.
ASA
0 100
0
(20) (20)
9,900 5,800
0 10 100 250
10
(30) (30) (30) (30)
57,300 24,200 22,900 17,200
0 100
20
(20) (20)
153,100 81,400
+ 16,300 + 15,100
< 0 .05
0 100 250
40
(80) (80) (80)
112,200 114,800 100,400
+ 9,400 + 6,900 + 10,600
< 0 .10 < 0 .10
0 20
40
(30) (30)
78,900 65,300
+ 4,400 + 3,800
< 0 .025
2.
Indomethacin
Number of platelets per mm2 (mean + S .E .)
2P**
+ 2,100 + 1,500
< 0 .10
+ + + T
< 0 .001 < 0 .001 < 0 .001
7,900 2,200 1,200 1,700
Platelet count was 700,000/mm 3 in Exp . 1 and 300,000/mm 3 i For legend see Table I
Exp . 2
Figure 1 . Scanning electron micrograph of platelets adherent to the subendothelium of a rabbit thoracic aorta . The endothelium had been removed with a balloon catheter and the aorta segment had been everted on a probe and rotated in a platelet suspension containing apyrase, 4% albumin and 40% red blood cells at a platelet count of 500,000/mm and the surface was rinsed with EDTA (as described in materials and methods) . A monolayer of platelets covered the surfaces and platelet aggregates were not present . There appeared to be similar number of platelets on the surfaces whether the platelets had not been exposed (control, A) or had been exposed to 1 mM ASA (B) .
Vo1 .13 .No .b
ASA, INDOKETHACIN, AND PLATELETS
0'P
DISCUSSION It appears that under the conditions of flow used in this system, the hematocrit determines whether or not an inhibitory effect of ASA on rabbit platelet adherence is demonstrable . As observed in the present study and in earlier experiments (1,2), in the presence of a low hematocrit, ASA can be
shown to inhibit platelet adherence to surfaces ; at a 40% hematocrit ASA is not inhibitory, even at very high concentrations . In vivo, a concentration of 8 uM ASA can be achieved in rabbits 5 min after an intravenous injection of 100 mg ASA/kg (Dr . M . R . Buchanan, personal communication) . In contrast, indomethacin does show a definite inhibitory effect on rabbit platelet adhesion to the surfaces even in the presence of a 40% hematocrit . At the higher concentrations used, both drugs completely block the arachidonate pathway in platelets as indicated by prevention of detectable malondialdehyde formation in response to a high concentraton of thrombin . It seems unlikely, therefore, that this pathway plays a major role in platelet adherence . However, both drugs may have an inhibitory effect on the formation of platelet thrombi because of their ability to inhibit the cyclo-oxygenase of the arachidonate pathway and thus prevent the formation of the prostaglandin endoperoxides and thromboxane A2 .
Indomethacin, but not ASA, may have an additional inhibitory effect on
the adherence of the initial platelet layer at an injury site on a vessel wall . The apparently mild inhibitory effect of indomethacin may be related to an effect on the availability of cellular calcium, as has been suggested by Northover (9) . The method used in these studies measures the adherence of individual platelets to the surfaces
(4,5) .
The presence of apyrase in the suspending
medium, and the EDTA rinse of the surface to which the rabbit platelets have adhered, ensures that platelet aggregates do not remain on the surface
(4,5) .
Thus, when inhibition occurs, it must be attributable to inhibition of adherence and not to inhibition of aggregate formation . Under conditions in which both adherence of single platelets and thrombus formation are measured, aspirin inhibits the accumulation of platelet thrombi on the subendothelium (10) . The results of the present studies are in agreement with those of Baumgartner and his colleagues (3,10,11,12) who have shown that when rabbit or
human platelet adherence to the rabbit subendothelium is tested in the presence of a 40% hematocrit, ASA does not inhibit the adherence of single platelets although it inhibits thrombus formation . However, our results also show that the test conditions appear to be an important determinant of whether an effect
ASA, INDOMETHACIN, AND PLATELETS
980
Vol .13,No .6
of aspirin on adherence can be demonstrated . When the physical forces on the platelets are not high, ASA has some effect on platelet adherence and this may be of some significance at sites in the vascular tree where flow is slow such as in veins .
The recent report that aspirin reduces the incidence of venous
thrombosis in men undergoing hip surgery could be due to an effect of aspirin on platelet adherence as well as on the arachidonate pathway (13) . ACKNOWLEDGEMENTS We wish to thank Dr . Mary Richardaon for the scanning electron microscopy and Mrs . D . Blondowska for excellent technical assistance . This work was supported by grants from the Medical Research Council of Canada (MT 1309 and MT 2629) and by Senior Research Fellowships of the Ontario Heart Foundation to J .-P . Cazenave and R .L . Kinlough-Rathbone . REFERENCES 1 . CAZENAVE, J .-P ., PACKHAM, M .A ., GUCCIONE, M .A . and MUSTARD, J .F . Inhibition of platelet adherence to a collagen-coated surface by nonsteroidal anti-inflammatory drugs, pyrimido-pyrimidine and tricyclic compounds, and lidocaine . J . Lab . Clin . Med . 83, 797, 1974 . 2 . CAZENAVE, J .-P ., PACKHAM, M .A ., GUCCIONE, M .A . and MUSTARD, J .F . Inhibition of platelet adherence to damaged surface of rabbit aorta . J . Lab . Clin . Med . 86, 551, 1975 . 3.
TSCHOPP, T .B . Aspirin inhibits platelet aggregation on, but not adhesion to, collagen fibrils : an assessmeDt of platelet adhesion and deposited platelet mass by morphometry and 71 Cr-labeling . Thromb . Res . 11, 619, 1977 .
4 . CAZENAVE, J .-P . Platelet adherence to collagen containing surfaces . Thesis, Ph .D, McMaster University, Hamilton, 1977 . 5.
CAZENAVE, J .-P ., RICHARDSON, M ., KINLOUGH-RATHBONE, R .L ., PACKHAM, M .A . and MUSTARD, J .F . Quantitative radioisotopic measurement and scanning electronmicroscopic study of platelet adherence to collagen-coated surface and to subendothelium with a rotating probe device . J . Lab . Clin . Med . Submitted for publication .
6.
CAZENAVE, J .-P ., PACKHAM, M .A . and MUSTARD, J .F . Adherence of platelets to a collagen-coated surfaces development of a quantitative method . J . Lab . Clin . Med . 82, 978, 1973 .
7.
SMITH, J .B ., INGERMAN, C .M . and SILVER, M .J . Malondialdehyde formation as an indicator of prostaglandin production by human platelets . J . Lab . Clin . Med . 88, 167, 1976 .
8 . GERRITY, R .G ., RICHARDSON, M ., SOMER, J .8 ., 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, 1977 .
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9.
NORTHOVER, B .J . 293, 1977 .
INDOMETH CZN, AND PLATELETS
Indomethacin .
A calcium antagonist .
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Gen . Pha nacol . 8,
10, BAUMGARTNER, H .R . and MUGGLI, R . Effect of acetylsalicylic acid on platelet adhesion to subendothelium and on the formation of mural platelet thrombi in vivo . Thromb . Diath . Haemorrh . Suppl . 60, 345, 1974, 11, WEISS, H,J ., TSCHOPP, T,B, and BAUMGARTNER, H .R . Impaired interaction (adhesion-aggregation) of platelets with the subendothelium in storageN . Engl . J . Med . 293, 619, pool disease and after aspirin ingestion . 1975 . 12 . BAUMGARTNER, H .R ., TSCHOPP, T .B . and WEISS, H .J . Platelet interaction with collagen fibrils in flowing blood . II . Impaired adhesion-aggregation in bleeding disorders . A comparison with subendothelium . Thromb . Haemostas . 37, 17, 1977 . 13 . HARRIS, W .H ., SALZMAN, E .W ., ATHANASOULIS, C,A„ WALTMAN, A .C . and DESANCTIS, R .W . Aspirin prophylaxis of venous thromboembolism after total N . Engl . J . Med . 297, 1246, 1977 . hip replacement .