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Possible significance of small numbers of functional platelets in a population of aspirin-treated platelets in vitro and in vivo
THROMBOSIS RESEARCH (c) Pergamon Press
18; Ltd.
389-397 1980.
Printed in the U.S.A. 0049-3848/80/100389-09$02.00/0
POSSIBLE SIGNIFICANCE OF SMALL NUMBERS OF FUNCTIONAL PLATELETS IN A POPULATION OF ASPIRIN-TREATED PLATELETS IN L'IZ'ROAND IN VIVO
A.L. Cerskus, M. Ali, B.J. Davies and J.W.D. McDonald Department of Medicine, University of Western Ontario University Hospital, London, Ontario. Canada. N6A 5A5 in revised form 1.2.1980. (Received 3.10.1979; Accepted by Editor M.A. Packham. Received by Executive Editorial Office 10.3.1980)
ABSTRACT
Recent evidence has suggested that platelets capable of thromboxane synthesis enter the human circulation at the rate of 10% per day after a single oral dose of aspirin (ASA). We investigated the possible importance of a similar proportion of functional rabbit platelets in vitro and in the circulation of ASA-treated rabbits. Functional platelets obtained from untreated rabbits and ASA-treated rabbits wert combined in a ratio of 1:9 respectively. Arachidonate-induced platelet aggregation and serotonin release were measured in vitro. The small proportion of functional platelets fully restored platelet aggregation and partially restored serotonin release in ASA-treated platelets. Untreated rabbits infused with arachidonic acid exhibited profound in vivo platelet aggregation and hypotension. These changes were associated with elevated thromboxane BP (TXB,) levels. Rabbits pretreated with ASA were protected from these effects and the elevation in plasma TXB2 was blocked. When ASA-treated rabbits were transfused prior to arachidonate infusion with a sufficient number of functional platelets to contribute 10% of the platelet population, the protective effect of ASA was reversed. These results suggest that 10% functional platelets can remove the protective effect of ASA on platelet aggregation both in vitro and in vivo. INTRODUCTION
Clinical trials in patients with cerebral vascular disease (1,2) and deep veir. thrombosis '(3) have demonstrated the efficacy of ASA as an antithrombotic
Key Words:
platelets, aspirin, thromboxane B2, platelet aggregation
* Please Mail Reprint Requests to: Dr. of Medicine, University Hospital, P.O. don, Ontario, Canada N6A 5A5. 380
J.W.D. McDonald, Department Box 5339, Terminal A, Lon-
.
FL-NCTIONAL, PLATELETS
v01.18,\0.~~/-+
agent. ASA acetylates prostaglandin fatty acid cyclooxygenase in platelets (4), inhibiting the synthesis of the pro-aggregatory thromboxane A2 (TXA2). This antiplate~letaction probably accounts for the antithrombotic effect. In the above clinical trials, ASA was administered to patients two to four times ,!aily. However, the ASA effect on platelets is irreversible (4) and can only je overcome by the emergence of new platelets into the circulation. It has jeen reported that ASA may also act on megakaryocyte cyclooxygenase (5). This "ay account for a 48 hour lag phase during which platelets harvested from subjects who had ingested ASA are not capable of being acetylated in ;(~Itmby radiolabelled ASA (5). For this reason, some investigators have suggested that effective antithrombotic therapy could be achieved using a once-daily regimen (6,7). On the basis of a.~ oiso tests we reported that the ability of washed human platelet suspensions to convert 14C-arachidonate to 14Cprostaglandin D2 (PGD2) and 14C-TXB2 was more than 95% inhibited 45 minutes after a single oral 650 mg dose of ASA (8,9). We observed that approximately 10% of this activity returned within 24 hours of the ASA dose and we interpreted this observation as indicating that functional platelets enter the circulation within a few hours of an ASA dose. We have investigated the possible significance of 10% functional platelets in a population of ASAinactivated platelets. METHODS ~vr Vitro Platelet Aggregation Rabbits (2.5 kg) were either untreated (source of functional platelets) or treated with 250 mg/kg of ASA by intraperitoneal injection 45 minutes prior to exsanguination (source of ASA-treated platelets). The rabbits, anesthetized with sodium thiopentol(30 mg/kg), were exsanguinated by withdrawal of blood via a cannula inserted into the carotid artery. Blood was anticoagulated by mixing with 3.7% sodium citrate (9 parts blood to 1 part sodium citrate) and platelet rich-plasma (PRP) was prepared by centrifugation at 500 x g for 10 minutes. The PRP was removed from the red cell layer and the red cells were recentrifuged for 15 minutes at 1000 x g to obtain platelet-poor-plasma (PPP). The platelet count in both untreated and ASA-treated plasma was adjusted to 3OO,OOO/ul by dilution with autologous PPP. Platelet aggregation was measureti in samples composed of: 1) 1 ml PRP from an untreated rabbit; 2) 1 ml PRP from an ASA-treated rabbit; or 3) a mixture of 0.9 ml PRP from an ASA-treated rabbit and 0.1 ml PRP from an untreated rabbit. Aggregation was initiated by the addition of 100 ~1 of the appropriate stock solutions of sodium arachidonate required to give a final concentration of 0.03 to 0.3 mM. 'Y-Serotonin Release PRP from u,ltreatedand ASA-treated rabbits was prepared as described above. Each batch of PRP(30 ml) was incubated for 30 minutes at room temperature with 0.05 uCi/ml of '"C-serotonin (14C-5-hydroxytryptamine-creatinine-sulphate, 55 mCi/mmol, Amersham Searle, Oakville, Ontario). Labelled PRP was then pipe:,;& into aggregation cuvettes to obtain the three experimental groups described above. Aggregation was initiated by the addition of 0.03 to 0.3 mM arachii;onate (final concentration). Aggregation was stopped three minutes after the addition of the aggregating agent by adding 100 ii1of ice cold 300 mM i\lazECTA followed by separation of the platelets from plasma in an Eppendorf 3,720i:icrocentrifudc (8000 x g for 0.5 minutes). Uptake and release of
FUNCTIONAL
PLATELETS
391
l'+C-serotoninby Platelets was determined as previously describeti ( 10). IYL viva Platelet Aggregation In vivo platelet aggregation was induced by intravenous arachidonate infusion in either untreated rabbits or rabbits treated intraperitoneally with 250 mg/kg of ASA, 45 minutes previously. This large dose was selected because work of others (11) had shown a requirement for such doses to prevent death from experimental pulmonary embolism in rabbits. We have no information on plasma FSA levels or the rate of hydrolysis of ASA injected intraperitoneally into the rabbit. Plasma concentrations of acetylsalicylate were not determined. .hlonitoring of blood pressure and sampling of blood for the determination of platelet count and plasma level of TXB2 were facilitated by the insertion of a carotid cannula. These parameters were measured irrrnediately prior to and immediately following arachidonate infusion. The dose of arachidonate used (1.4 mg/kg) had previously been shown to cause massive in vivo platelet aggregation and death in rabbits (12,13). Details of the infusion techniques and methods for radioimmunoassay (RIA) determination of TXB2 and 6-keto-PGF,cl have been described (14,15). The effect of 10% functional platelets in this animal model was determined as follows: ASA-treated rabbits (250 mg/kg ASA, 45 minutes previously) were transfused with a concentrate of platelets harvested from either an untreated donor rabbit or an ASA-treated donor rabbit. The number of platelets transfused was 5 x 10' which represented approximately 10% of the normal platelet population in rabbits of this size. Blood from donor rabbits was anticoagulated in acidcitrate-dextrose (1 part acid-citrate-dextrose to 6 parts blood) and PRP was prepared as described above. PRP was centrifuged at 1000 x g for 15 minutes to obtain a platelet pellet. Platelets were then resuspended in autologous PPP to obtain a concentration of 5 x 10' platelets/ml. Recipient rabbits were cannulated and a blood sample was removed prior to platelet transfusion for determination of platelet counts. One ml of platelet concentrate was infused into the marginal ear vein and five minutes later a second blood sample was removed for platelet count and plasma TXB2 determinations. The average increase in platelet count was found to be 10.2%. Arachidonate (1.4 mg/kg) was infused whil,emonitoring blood pressure and a third blood sample, immediately following the infusion, was taken.
RESULTS In Vitro Platelet Aggregation Platelets harvested from untreated rabbits demonstrated graded aggregation responses to added arachidonate (Figure 1). As expected, platelets from ASAtreated rabbits did not aggregate with any of the arachidonate concentrations used (Figure 1). When the two platelet populations were mixed in a ratio of 1 part functional platelets to 9 parts ASA-treated platelets, the aggregation response elicited by 0.15 and 0.3 mM arachidonate was equivalent to the aggregation response elicited by these concentrations of arachidonate in functional platelets alone. No aggregation was observed in the mixed platelets at lower concentrations of arachidonate (Figure 1). Typical aggregation curves observed with 0.3 ml? qrachidonate are shown in Fiyure 2.
FT.-NCTIONAL PLATELETS
100
is 5
.o u g
50
<
L 0
Tl 0.15
0.0 75 ARACHIOONIC
FIG.
ACID
0 225
a30
(mM1
1
Arachidonate-induced aggregation of functional platelets (w), ASA-treated platelets (M) and a mixture of 10% functional platelets and 90% ASA-treated platelets (WI).. Aggregation was quantified by measuring the Each point represents maximum recorder-pen displacement. a mean5.E.M. of four experinents. The dearee of aggregation elicited with 0.3 mM arachidonate in functional platelets was considered the control or 100% aggregation value. All other results are expressed as the per cent of control.
FIG. Typical aggregation arachidonate.
2
curves obtained
with 0.3 mM
Vo1.18,No.3/4
FUNCTIONAL
393
PLATELETS
0.075
0.225
ARACHIDONICACIO
0.3m
(mM)
FIG. 3 Arachidonate-induced 'Y-serotonin release from functional platelets (O-o), ASA-treated platelets (ti) and a mixture of 10% functional platelets and 90% ASA-treated platelets (042). Each point rapresents a mean+S.E.M. of four experiments. The dashed line indicates the expected release from the mixed platelets in the absence of any interaction.
TABLE
I
Effect of Pretreatment with 250 mg/kg ASA on Arachidonic Acid-Induced Changes in Blood Pressure (B.P.), Platelet Count (P.C.) and Plasma TXB2
Treatment
n
Control 1.4 mg/kg Arachidonic Acid
10
250 mg/kg ASA 1.4 mg/kg Arachidonic Acid
7
+ Plasma TXB2 q/ml Mean 2 S.E.M.
% + B.P. Mean + S.E.M.
% + P.C. Mean + S.E.M.
99.5 +
0.5
80.6 * 5.2
142.3 + 41.1
17.1 + 10.0 *
16.7 k 5.0 *
10.3
* significantly differclntfrom control (p < 0.05) by Student's t-test
FUNCTIONAL
39’c
PLBTELEIS
"'C-Sero-Lenin Release release from prelabelled functional platelets was The degree of "C-serotonin dependent on the concentration of arachidonate used. Maximal release (30.2 + 2.1%) was observed with 0.3 mM arachidonate (Figure 3). No serotonin release was observed in ASA-treated platelets irrespective of the arachidonate Using the observed values, the expected "C-serotonin release concentration. in a mixture of 1 part functional platelets and 9 parts ASA-treated platelets !as calculated. When 14C-serotonin release was observed in the platelet iixture, the degree of release was approximately twice the expected value {Figure 3).
_r,,[/iv0 Platelet
Aggregation
Table 1 shows the effect of intravenous infusion of arachidonate on platelet Untreated rabbits count, blood pressure and plasma TXB;! levels in rabbits. subjected to intravenous arachidonate responded with pronounced thrombocyto?enia (80.6 f 5.2% decrease in platelet count) and complete irreversible circulatory collapse. Plasma TXBz, undetectable prior to infusion, increased to 142.3 k 41.1 ng/ml. In contrast, blood pressure and platelet count in most ,\SA-treated rabbits were relatively unaffected by arachidonate infusion. Similarly, TXBZ synthesis in ASA-treated rabbits was blocked. Table 2 summarizes the results obtained when arachidonate was infused into ASA-treated rabbits, transfused with either functional or ASA-treated platelets in amounts representing roughly 10% of the average normal platelet population. When the source of transfused platelets was an ASA-treated rabbit, the effects of arachidonate infusion were similar to those observed in ASA-treated, untransfused rabbits. However, when ASA-treated rabbits were transfused with a small proportion of functional platelets the protective effect of ASApretreatment was reversed. Platelet count and blood pressure decreased by an average of 6"5.3 4 14.2% and 64.0 + 7.3%, respectively. The average plasma level of TXB2 observed in these rabbits was 17.8 i 5.1%, a value which, as expected, was roughly 10% of the plasma TXB2 level observed in untreated, untransfused rabbits.
TABLE
II
Effect of Transfusion with ei-Lher Functional or ASA-Treated Platelets on ASA-Inhibition of Arachidonic-Acid Induced Changes in Platelet Count (P.C.), Blood Pressure (B.P.) and TXB2
Source of Platelets for Transfusion (5 x log Platelets)
I n
% + P.C. Mean k S.E.M.
ASA-Treated Rabbit (Control)
10
16.1 ? 10.5
Untreated
Rabbit
10
65.3 i 14.2 *
-‘- significantly
different
/
“6 + B.P. Mean k S.E.M.
:
Plasma TXBZ ng/ml Mean + S.E.M.
29.4 + 9.4
1.4 ? 0.9
64.0 + 7.3 *
17.8 r 5.1 *
1 from control (p c 0.05) by Student's --~-~.-.-__-___
t-test.
vo1.18,No.3/4
FUNCTIONAL
PLATELETS
395
DISCUSSIOi’d Interaction between two populations of platelets iy~Ktro has been described previously (16). Mixtures of equal numbers of ASA-treated platelets and storage-pool deficient platelets were shown to undergo normal second phase aggregation in response to aggregating agents, while the individual platelet populations did not (16). In the experiments outlined in this report, we have shown that a similar type of interaction between ASA-treated and normal rabbit platelets exists. A small proportion (10%) of functional platelets was able to completely restore platelet aggregation induced by high concentrations of arachidonate in ASA-treated platelets. l'+C-serotoninrelease in the platelet mixtures, although not restored as completely as the aggregation response, was twice the value expected in the absence of platelet interaction. IYZviva, intravenous arachidonate elicited severe thrombocytopenia and irreversible hypotension associated with a marked increase in plasma TXB2. Pretreatment with ASA protected the rabbits from the effects of arachidonate infusion.- TXB2 synthesis was also blocked by ASA. ASA-treated rabbits, transfused with ASAtreated platelets and subjected to intravenous arachidonate, behaved as UtItranSfUSed, ASA-treated rabbits. In contrast, transfusion of functional platelets significantly reversed the protective action of ASA. TXB;! synthesis in this group was, as expected, 10% of the synthesis seen in untreated rabbits. Synthesis of TXA2 by only a small number of functional platelets appears to cause release and aggregation in ASA-treated platelets circulating in the same animal. The in vivo synthesis of TXB2 in untreated rabbits exposed to arachidonate infusion showed wide variation. In individual rabbits pronounced hypotension and platelet aggregation were associated with plasma TXBZ levels as low as 12 ng/ml and as high as 428 ng/ml. It has been suggested that adequate antithrombotic therapy with ASA could be achieved by once-daily administration of the drug (6,7). In human subjects the interval following ASA ingestion which lapses before a significant number of functional platelets is present in the circulation is ilOt certain. Majerus and colleagues (4,5) reported that there is an interval of approximately 48 hours following ASA ingestion during which platelets do not undergo in vitro acetylation by radioactively-labelled ASA. It is assumed that platelets which do not accept the acetyl residue in vitro were acetylated in vivo and are nonfunctional. Other studies have also shown a delay of several days following ASA before platelet aggregation is restored to normal Certain other studies which depended on measurements of cyclooxygenase &!&e activity rather than acetylation or platelet aggregation (8,18,19) ihaveindicated a more rapid return of activity. In these reports, cyclooxygenase activity increased to approximately 10% pf control values within 24 hours of a dose of ASA. Since 10% functional platelets restored aggregation in our experiments only with high concentrations of arachidonate, it appears that rather intense TXA2 synthesis by functional platelets may be required to exert effects on inactivated platelets. Such high rates of TXA2 synthesis may not be achieved in actoreoation studies in which aoents other than arachidonate are used. Therefore;-in"vitro aggregation studie; would not necessarily :eflect the presence of 10% functional platelets. rurther study is required to resolve the apparent differences in time required for emergence of functional platelets in these studies. Regardless of the interval required, the emergence of a small proportion of functional platelets following a dose of ASA may have clinical significance.
386
FUNCTIONAL
PLATELETS
Vo1.18,No.3/~
;ioIia1 In our studies of rabbit plateiets, in vitpu anu ia vivc, l’J’); fi;riC platelets have the capacity to restore aggregation to ASA-inactivated platelets. Because of species differences and differences between artificiallyinduced and natural thrombotic stimuli, it is difficult to extrapolate results obtained from animal experiments to the clinical situation. However, Valeri and Feingold (20) previously showed that a relatively small proportion of functional transfused platelets was sufficient to correct the prolonged bleeding time caused by ASA ingestion. A small number of functional platelets appearing in the circulation between infrequent doses of ASA may be sufficient to permit platelet-mediated thrombosis or vasoconstriction to occur.
ACKNOWLEDGEMENTS The authors gratefully acknowledge Dr. J.B. Smith of the Cardeza Foundation, Philadelphia, Pennsylvania, for kindly providing anti-TXB:!antiserum, Dr. John E. Pike of the Upjohn Company, Kalamazoo, Michigan, for his gift of TXB2 and 6-keto-PGF,a, and Miss Marion Marchant for her expert technical assistance. This work was supported by grants from the Ontario Heart Foundation and the Medical Research Council of Canada. REFERENCES 1.
THE CANADIAN CO-OPERATIVE STUDY GROUP. A randomized trial of aspirin and sulfinpyrazone in threatened stroke. N. Enql. J. Med. 299, 53-59, 197".
2.
Controlled FIELDS, W.S., LEMAK, N.A., FRANKOWSKI, R.F. and HARDY, R.J. trial of aspirin in cerebral ischemia. -_ Stroke. 8_, 301-314, 1977.
7 J-
HARRIS, W.H., SALZMAN, E.W., ATHANASOULIS, C,A., WALTMAN, A.C. and DESANCTIS, R.W. Aspirin prophylaxis of venous thromboembolism after total hip replacement. N. Enql. J. Med. 297, 1246-1249, 1977.
‘j~
ROTH, G.J., STANFORD, N. and MAJERUS, P.W. Acetylation of prostaglandin synthase by aspirin. Proc. Nat. Acad. Sci. USA. --72, 3073-3076, 1975.
5.
BURCH, J.W., STANFORD, N. and MAJERUS, P.W. Inhibition of platelet synthetase by oral aspirin. J. Clin. Invest. 6_l, 314-319, 1978. -_-
J.
JAFFE, E.A. and WEKSLER, B.B. Recovery of endothelial cell prostacyclin production after inhibition by low doses of aspirin. J. Clin. Invest. 63, 532-535, 1979.
i 1.
AMEZCUA, J-L., O'GRADY, J., SALMON, J.A. and MONCADA, S. Prolonged peridoxical effect of aspirin on platelet behaviour and bleeding time in man. Thrombosis Research. -16, 69-79, 1979.
I. ALI, M., MCDONALD, J.W.D. and THIESSEN, J.J., Plasma COATES, P.E acetylsalicylate and platelet cyclooxygenase activity foliowing plain and enteric-coated aspirin. Stroke. 11, 9-13, 1980.
VOI.I~,NO. 3/4
9.
FUNCTIONAL
PLATELETS
397
CERSKUS, A.L., ALI, i,!. ani i;ciA,ALti, J.W.ti. %ss~L~‘I~ siI;ilifica;:ce of functional platelets circulating between doses of aspirin. m. 52, 160(#256), 1978.
70.
ALI, M. and MCDONALD, J.W.D. Effects of sulfinpyrazone on platelet prostaglandin synthesis and platelet release of serotonin. J. Lab. Clin. Med. -89, 868-875, 1977.
11.
TODD, M.H., CRAGG, D.B., FOREST, J.B. and HIRSH, J.H. synthetase inhibitors in pulmonary embolism. Blood.
Prostaglandin
5tJ 284(#690),1977
1' L.
CERSKUS, A.L., ALI, M., ZAMECNIK, J and MCDONALD, J.W.D. Effects of indomethacin and sulfinpyrazone on in viuo formation of thromboxane B2 and prostaglandin D2 during arachidonate infusion in rabbits. Thromb. Res. l& 549-553,1978.
13.
SILVER, M-J., HOCH, W., KOCSIS, J.J., INGERMAN, C.M. and SMITH, J.B. Arachidonic acid causes sudden death in rabbits. Science. 183 1085-1087, 1974. &
/0il. .
CERSKUS, A.L., ALI, M. and MCDONALD, J.W.D. Thromboxane B2 and 6_ketoPGF,a synthesis during infusion of collagen and arachidonic acid in rabbits: Inhibition by aspirin and sul,finpyrazone. ’ Thrombosis Research. IN PRESS.
_ 15.
ALI, M. and MCDONALD, J.W.D. Synthesis of thromboxane B, and 6-keto-PGF,o by bovine gastric mucosal and muscle microsomes. Prostaglandins. IN PRESS. -_
16.
WHITE, J.G. and WITKOP, C.J. Effects of normal and aspirin platelets on defective secondary aggregation in the Hermansky-Pudlak syndrome. Am. J. Path. 68: 57-66, 1972.
17.
KOCSIS, J.J., HERNANDOVICH, J., SILVER, M.J., SMITH, J.B. and INGERMAN, C Duration of inhibition of platelet prostaqlandin formation and aggregation by ingested aspirin or indomethacin. Prostaqlandins. & 141-144, 1973.
18.
STUART, M.J., MURPHY, S., OSKI, F.A. A simple nonradioisotope technic for the determination of platelet life-span. N. Engl. J. Med. z, 1310-1313, 1975.
19.
RONCUCCI, R., DEPERON, R., LANSEN, ,I., DESTAILLEUR, J., VERHAEGHE, R., DOUMONT, J., LAMBELIN, G., VAN STALLE, F. Nonradioactive semi-automated determination of platelet survival time in man. Thrombosis and Hemostasis. 389, 92, 1977.
20.
VALERI, C.R. and FEINGOLD, H. Hemostatic effectiveness of liquidpreserved platelets stored at 4°C or 22 C and freeze-preserved platelets stored with 5 percent DMSO at -150°C or stored with 6 percent DMSO at -80°C. In: Platelets: Production, Function, Transfusionand Storage. M.G. Baldini and S. Ebbe (Eds.) New York. Grune & Stratton, Inc. 1974. pp. 377-391, 1974.
18; Ltd.
389-397 1980.
Printed in the U.S.A. 0049-3848/80/100389-09$02.00/0
POSSIBLE SIGNIFICANCE OF SMALL NUMBERS OF FUNCTIONAL PLATELETS IN A POPULATION OF ASPIRIN-TREATED PLATELETS IN L'IZ'ROAND IN VIVO
A.L. Cerskus, M. Ali, B.J. Davies and J.W.D. McDonald Department of Medicine, University of Western Ontario University Hospital, London, Ontario. Canada. N6A 5A5 in revised form 1.2.1980. (Received 3.10.1979; Accepted by Editor M.A. Packham. Received by Executive Editorial Office 10.3.1980)
ABSTRACT
Recent evidence has suggested that platelets capable of thromboxane synthesis enter the human circulation at the rate of 10% per day after a single oral dose of aspirin (ASA). We investigated the possible importance of a similar proportion of functional rabbit platelets in vitro and in the circulation of ASA-treated rabbits. Functional platelets obtained from untreated rabbits and ASA-treated rabbits wert combined in a ratio of 1:9 respectively. Arachidonate-induced platelet aggregation and serotonin release were measured in vitro. The small proportion of functional platelets fully restored platelet aggregation and partially restored serotonin release in ASA-treated platelets. Untreated rabbits infused with arachidonic acid exhibited profound in vivo platelet aggregation and hypotension. These changes were associated with elevated thromboxane BP (TXB,) levels. Rabbits pretreated with ASA were protected from these effects and the elevation in plasma TXB2 was blocked. When ASA-treated rabbits were transfused prior to arachidonate infusion with a sufficient number of functional platelets to contribute 10% of the platelet population, the protective effect of ASA was reversed. These results suggest that 10% functional platelets can remove the protective effect of ASA on platelet aggregation both in vitro and in vivo. INTRODUCTION
Clinical trials in patients with cerebral vascular disease (1,2) and deep veir. thrombosis '(3) have demonstrated the efficacy of ASA as an antithrombotic
Key Words:
platelets, aspirin, thromboxane B2, platelet aggregation
* Please Mail Reprint Requests to: Dr. of Medicine, University Hospital, P.O. don, Ontario, Canada N6A 5A5. 380
J.W.D. McDonald, Department Box 5339, Terminal A, Lon-
.
FL-NCTIONAL, PLATELETS
v01.18,\0.~~/-+
agent. ASA acetylates prostaglandin fatty acid cyclooxygenase in platelets (4), inhibiting the synthesis of the pro-aggregatory thromboxane A2 (TXA2). This antiplate~letaction probably accounts for the antithrombotic effect. In the above clinical trials, ASA was administered to patients two to four times ,!aily. However, the ASA effect on platelets is irreversible (4) and can only je overcome by the emergence of new platelets into the circulation. It has jeen reported that ASA may also act on megakaryocyte cyclooxygenase (5). This "ay account for a 48 hour lag phase during which platelets harvested from subjects who had ingested ASA are not capable of being acetylated in ;(~Itmby radiolabelled ASA (5). For this reason, some investigators have suggested that effective antithrombotic therapy could be achieved using a once-daily regimen (6,7). On the basis of a.~ oiso tests we reported that the ability of washed human platelet suspensions to convert 14C-arachidonate to 14Cprostaglandin D2 (PGD2) and 14C-TXB2 was more than 95% inhibited 45 minutes after a single oral 650 mg dose of ASA (8,9). We observed that approximately 10% of this activity returned within 24 hours of the ASA dose and we interpreted this observation as indicating that functional platelets enter the circulation within a few hours of an ASA dose. We have investigated the possible significance of 10% functional platelets in a population of ASAinactivated platelets. METHODS ~vr Vitro Platelet Aggregation Rabbits (2.5 kg) were either untreated (source of functional platelets) or treated with 250 mg/kg of ASA by intraperitoneal injection 45 minutes prior to exsanguination (source of ASA-treated platelets). The rabbits, anesthetized with sodium thiopentol(30 mg/kg), were exsanguinated by withdrawal of blood via a cannula inserted into the carotid artery. Blood was anticoagulated by mixing with 3.7% sodium citrate (9 parts blood to 1 part sodium citrate) and platelet rich-plasma (PRP) was prepared by centrifugation at 500 x g for 10 minutes. The PRP was removed from the red cell layer and the red cells were recentrifuged for 15 minutes at 1000 x g to obtain platelet-poor-plasma (PPP). The platelet count in both untreated and ASA-treated plasma was adjusted to 3OO,OOO/ul by dilution with autologous PPP. Platelet aggregation was measureti in samples composed of: 1) 1 ml PRP from an untreated rabbit; 2) 1 ml PRP from an ASA-treated rabbit; or 3) a mixture of 0.9 ml PRP from an ASA-treated rabbit and 0.1 ml PRP from an untreated rabbit. Aggregation was initiated by the addition of 100 ~1 of the appropriate stock solutions of sodium arachidonate required to give a final concentration of 0.03 to 0.3 mM. 'Y-Serotonin Release PRP from u,ltreatedand ASA-treated rabbits was prepared as described above. Each batch of PRP(30 ml) was incubated for 30 minutes at room temperature with 0.05 uCi/ml of '"C-serotonin (14C-5-hydroxytryptamine-creatinine-sulphate, 55 mCi/mmol, Amersham Searle, Oakville, Ontario). Labelled PRP was then pipe:,;& into aggregation cuvettes to obtain the three experimental groups described above. Aggregation was initiated by the addition of 0.03 to 0.3 mM arachii;onate (final concentration). Aggregation was stopped three minutes after the addition of the aggregating agent by adding 100 ii1of ice cold 300 mM i\lazECTA followed by separation of the platelets from plasma in an Eppendorf 3,720i:icrocentrifudc (8000 x g for 0.5 minutes). Uptake and release of
FUNCTIONAL
PLATELETS
391
l'+C-serotoninby Platelets was determined as previously describeti ( 10). IYL viva Platelet Aggregation In vivo platelet aggregation was induced by intravenous arachidonate infusion in either untreated rabbits or rabbits treated intraperitoneally with 250 mg/kg of ASA, 45 minutes previously. This large dose was selected because work of others (11) had shown a requirement for such doses to prevent death from experimental pulmonary embolism in rabbits. We have no information on plasma FSA levels or the rate of hydrolysis of ASA injected intraperitoneally into the rabbit. Plasma concentrations of acetylsalicylate were not determined. .hlonitoring of blood pressure and sampling of blood for the determination of platelet count and plasma level of TXB2 were facilitated by the insertion of a carotid cannula. These parameters were measured irrrnediately prior to and immediately following arachidonate infusion. The dose of arachidonate used (1.4 mg/kg) had previously been shown to cause massive in vivo platelet aggregation and death in rabbits (12,13). Details of the infusion techniques and methods for radioimmunoassay (RIA) determination of TXB2 and 6-keto-PGF,cl have been described (14,15). The effect of 10% functional platelets in this animal model was determined as follows: ASA-treated rabbits (250 mg/kg ASA, 45 minutes previously) were transfused with a concentrate of platelets harvested from either an untreated donor rabbit or an ASA-treated donor rabbit. The number of platelets transfused was 5 x 10' which represented approximately 10% of the normal platelet population in rabbits of this size. Blood from donor rabbits was anticoagulated in acidcitrate-dextrose (1 part acid-citrate-dextrose to 6 parts blood) and PRP was prepared as described above. PRP was centrifuged at 1000 x g for 15 minutes to obtain a platelet pellet. Platelets were then resuspended in autologous PPP to obtain a concentration of 5 x 10' platelets/ml. Recipient rabbits were cannulated and a blood sample was removed prior to platelet transfusion for determination of platelet counts. One ml of platelet concentrate was infused into the marginal ear vein and five minutes later a second blood sample was removed for platelet count and plasma TXB2 determinations. The average increase in platelet count was found to be 10.2%. Arachidonate (1.4 mg/kg) was infused whil,emonitoring blood pressure and a third blood sample, immediately following the infusion, was taken.
RESULTS In Vitro Platelet Aggregation Platelets harvested from untreated rabbits demonstrated graded aggregation responses to added arachidonate (Figure 1). As expected, platelets from ASAtreated rabbits did not aggregate with any of the arachidonate concentrations used (Figure 1). When the two platelet populations were mixed in a ratio of 1 part functional platelets to 9 parts ASA-treated platelets, the aggregation response elicited by 0.15 and 0.3 mM arachidonate was equivalent to the aggregation response elicited by these concentrations of arachidonate in functional platelets alone. No aggregation was observed in the mixed platelets at lower concentrations of arachidonate (Figure 1). Typical aggregation curves observed with 0.3 ml? qrachidonate are shown in Fiyure 2.
FT.-NCTIONAL PLATELETS
100
is 5
.o u g
50
<
L 0
Tl 0.15
0.0 75 ARACHIOONIC
FIG.
ACID
0 225
a30
(mM1
1
Arachidonate-induced aggregation of functional platelets (w), ASA-treated platelets (M) and a mixture of 10% functional platelets and 90% ASA-treated platelets (WI).. Aggregation was quantified by measuring the Each point represents maximum recorder-pen displacement. a mean5.E.M. of four experinents. The dearee of aggregation elicited with 0.3 mM arachidonate in functional platelets was considered the control or 100% aggregation value. All other results are expressed as the per cent of control.
FIG. Typical aggregation arachidonate.
2
curves obtained
with 0.3 mM
Vo1.18,No.3/4
FUNCTIONAL
393
PLATELETS
0.075
0.225
ARACHIDONICACIO
0.3m
(mM)
FIG. 3 Arachidonate-induced 'Y-serotonin release from functional platelets (O-o), ASA-treated platelets (ti) and a mixture of 10% functional platelets and 90% ASA-treated platelets (042). Each point rapresents a mean+S.E.M. of four experiments. The dashed line indicates the expected release from the mixed platelets in the absence of any interaction.
TABLE
I
Effect of Pretreatment with 250 mg/kg ASA on Arachidonic Acid-Induced Changes in Blood Pressure (B.P.), Platelet Count (P.C.) and Plasma TXB2
Treatment
n
Control 1.4 mg/kg Arachidonic Acid
10
250 mg/kg ASA 1.4 mg/kg Arachidonic Acid
7
+ Plasma TXB2 q/ml Mean 2 S.E.M.
% + B.P. Mean + S.E.M.
% + P.C. Mean + S.E.M.
99.5 +
0.5
80.6 * 5.2
142.3 + 41.1
17.1 + 10.0 *
16.7 k 5.0 *
10.3
* significantly differclntfrom control (p < 0.05) by Student's t-test
FUNCTIONAL
39’c
PLBTELEIS
"'C-Sero-Lenin Release release from prelabelled functional platelets was The degree of "C-serotonin dependent on the concentration of arachidonate used. Maximal release (30.2 + 2.1%) was observed with 0.3 mM arachidonate (Figure 3). No serotonin release was observed in ASA-treated platelets irrespective of the arachidonate Using the observed values, the expected "C-serotonin release concentration. in a mixture of 1 part functional platelets and 9 parts ASA-treated platelets !as calculated. When 14C-serotonin release was observed in the platelet iixture, the degree of release was approximately twice the expected value {Figure 3).
_r,,[/iv0 Platelet
Aggregation
Table 1 shows the effect of intravenous infusion of arachidonate on platelet Untreated rabbits count, blood pressure and plasma TXB;! levels in rabbits. subjected to intravenous arachidonate responded with pronounced thrombocyto?enia (80.6 f 5.2% decrease in platelet count) and complete irreversible circulatory collapse. Plasma TXBz, undetectable prior to infusion, increased to 142.3 k 41.1 ng/ml. In contrast, blood pressure and platelet count in most ,\SA-treated rabbits were relatively unaffected by arachidonate infusion. Similarly, TXBZ synthesis in ASA-treated rabbits was blocked. Table 2 summarizes the results obtained when arachidonate was infused into ASA-treated rabbits, transfused with either functional or ASA-treated platelets in amounts representing roughly 10% of the average normal platelet population. When the source of transfused platelets was an ASA-treated rabbit, the effects of arachidonate infusion were similar to those observed in ASA-treated, untransfused rabbits. However, when ASA-treated rabbits were transfused with a small proportion of functional platelets the protective effect of ASApretreatment was reversed. Platelet count and blood pressure decreased by an average of 6"5.3 4 14.2% and 64.0 + 7.3%, respectively. The average plasma level of TXB2 observed in these rabbits was 17.8 i 5.1%, a value which, as expected, was roughly 10% of the plasma TXB2 level observed in untreated, untransfused rabbits.
TABLE
II
Effect of Transfusion with ei-Lher Functional or ASA-Treated Platelets on ASA-Inhibition of Arachidonic-Acid Induced Changes in Platelet Count (P.C.), Blood Pressure (B.P.) and TXB2
Source of Platelets for Transfusion (5 x log Platelets)
I n
% + P.C. Mean k S.E.M.
ASA-Treated Rabbit (Control)
10
16.1 ? 10.5
Untreated
Rabbit
10
65.3 i 14.2 *
-‘- significantly
different
/
“6 + B.P. Mean k S.E.M.
:
Plasma TXBZ ng/ml Mean + S.E.M.
29.4 + 9.4
1.4 ? 0.9
64.0 + 7.3 *
17.8 r 5.1 *
1 from control (p c 0.05) by Student's --~-~.-.-__-___
t-test.
vo1.18,No.3/4
FUNCTIONAL
PLATELETS
395
DISCUSSIOi’d Interaction between two populations of platelets iy~Ktro has been described previously (16). Mixtures of equal numbers of ASA-treated platelets and storage-pool deficient platelets were shown to undergo normal second phase aggregation in response to aggregating agents, while the individual platelet populations did not (16). In the experiments outlined in this report, we have shown that a similar type of interaction between ASA-treated and normal rabbit platelets exists. A small proportion (10%) of functional platelets was able to completely restore platelet aggregation induced by high concentrations of arachidonate in ASA-treated platelets. l'+C-serotoninrelease in the platelet mixtures, although not restored as completely as the aggregation response, was twice the value expected in the absence of platelet interaction. IYZviva, intravenous arachidonate elicited severe thrombocytopenia and irreversible hypotension associated with a marked increase in plasma TXB2. Pretreatment with ASA protected the rabbits from the effects of arachidonate infusion.- TXB2 synthesis was also blocked by ASA. ASA-treated rabbits, transfused with ASAtreated platelets and subjected to intravenous arachidonate, behaved as UtItranSfUSed, ASA-treated rabbits. In contrast, transfusion of functional platelets significantly reversed the protective action of ASA. TXB;! synthesis in this group was, as expected, 10% of the synthesis seen in untreated rabbits. Synthesis of TXA2 by only a small number of functional platelets appears to cause release and aggregation in ASA-treated platelets circulating in the same animal. The in vivo synthesis of TXB2 in untreated rabbits exposed to arachidonate infusion showed wide variation. In individual rabbits pronounced hypotension and platelet aggregation were associated with plasma TXBZ levels as low as 12 ng/ml and as high as 428 ng/ml. It has been suggested that adequate antithrombotic therapy with ASA could be achieved by once-daily administration of the drug (6,7). In human subjects the interval following ASA ingestion which lapses before a significant number of functional platelets is present in the circulation is ilOt certain. Majerus and colleagues (4,5) reported that there is an interval of approximately 48 hours following ASA ingestion during which platelets do not undergo in vitro acetylation by radioactively-labelled ASA. It is assumed that platelets which do not accept the acetyl residue in vitro were acetylated in vivo and are nonfunctional. Other studies have also shown a delay of several days following ASA before platelet aggregation is restored to normal Certain other studies which depended on measurements of cyclooxygenase &!&e activity rather than acetylation or platelet aggregation (8,18,19) ihaveindicated a more rapid return of activity. In these reports, cyclooxygenase activity increased to approximately 10% pf control values within 24 hours of a dose of ASA. Since 10% functional platelets restored aggregation in our experiments only with high concentrations of arachidonate, it appears that rather intense TXA2 synthesis by functional platelets may be required to exert effects on inactivated platelets. Such high rates of TXA2 synthesis may not be achieved in actoreoation studies in which aoents other than arachidonate are used. Therefore;-in"vitro aggregation studie; would not necessarily :eflect the presence of 10% functional platelets. rurther study is required to resolve the apparent differences in time required for emergence of functional platelets in these studies. Regardless of the interval required, the emergence of a small proportion of functional platelets following a dose of ASA may have clinical significance.
386
FUNCTIONAL
PLATELETS
Vo1.18,No.3/~
;ioIia1 In our studies of rabbit plateiets, in vitpu anu ia vivc, l’J’); fi;riC platelets have the capacity to restore aggregation to ASA-inactivated platelets. Because of species differences and differences between artificiallyinduced and natural thrombotic stimuli, it is difficult to extrapolate results obtained from animal experiments to the clinical situation. However, Valeri and Feingold (20) previously showed that a relatively small proportion of functional transfused platelets was sufficient to correct the prolonged bleeding time caused by ASA ingestion. A small number of functional platelets appearing in the circulation between infrequent doses of ASA may be sufficient to permit platelet-mediated thrombosis or vasoconstriction to occur.
ACKNOWLEDGEMENTS The authors gratefully acknowledge Dr. J.B. Smith of the Cardeza Foundation, Philadelphia, Pennsylvania, for kindly providing anti-TXB:!antiserum, Dr. John E. Pike of the Upjohn Company, Kalamazoo, Michigan, for his gift of TXB2 and 6-keto-PGF,a, and Miss Marion Marchant for her expert technical assistance. This work was supported by grants from the Ontario Heart Foundation and the Medical Research Council of Canada. REFERENCES 1.
THE CANADIAN CO-OPERATIVE STUDY GROUP. A randomized trial of aspirin and sulfinpyrazone in threatened stroke. N. Enql. J. Med. 299, 53-59, 197".
2.
Controlled FIELDS, W.S., LEMAK, N.A., FRANKOWSKI, R.F. and HARDY, R.J. trial of aspirin in cerebral ischemia. -_ Stroke. 8_, 301-314, 1977.
7 J-
HARRIS, W.H., SALZMAN, E.W., ATHANASOULIS, C,A., WALTMAN, A.C. and DESANCTIS, R.W. Aspirin prophylaxis of venous thromboembolism after total hip replacement. N. Enql. J. Med. 297, 1246-1249, 1977.
‘j~
ROTH, G.J., STANFORD, N. and MAJERUS, P.W. Acetylation of prostaglandin synthase by aspirin. Proc. Nat. Acad. Sci. USA. --72, 3073-3076, 1975.
5.
BURCH, J.W., STANFORD, N. and MAJERUS, P.W. Inhibition of platelet synthetase by oral aspirin. J. Clin. Invest. 6_l, 314-319, 1978. -_-
J.
JAFFE, E.A. and WEKSLER, B.B. Recovery of endothelial cell prostacyclin production after inhibition by low doses of aspirin. J. Clin. Invest. 63, 532-535, 1979.
i 1.
AMEZCUA, J-L., O'GRADY, J., SALMON, J.A. and MONCADA, S. Prolonged peridoxical effect of aspirin on platelet behaviour and bleeding time in man. Thrombosis Research. -16, 69-79, 1979.
I. ALI, M., MCDONALD, J.W.D. and THIESSEN, J.J., Plasma COATES, P.E acetylsalicylate and platelet cyclooxygenase activity foliowing plain and enteric-coated aspirin. Stroke. 11, 9-13, 1980.
VOI.I~,NO. 3/4
9.
FUNCTIONAL
PLATELETS
397
CERSKUS, A.L., ALI, i,!. ani i;ciA,ALti, J.W.ti. %ss~L~‘I~ siI;ilifica;:ce of functional platelets circulating between doses of aspirin. m. 52, 160(#256), 1978.
70.
ALI, M. and MCDONALD, J.W.D. Effects of sulfinpyrazone on platelet prostaglandin synthesis and platelet release of serotonin. J. Lab. Clin. Med. -89, 868-875, 1977.
11.
TODD, M.H., CRAGG, D.B., FOREST, J.B. and HIRSH, J.H. synthetase inhibitors in pulmonary embolism. Blood.
Prostaglandin
5tJ 284(#690),1977
1' L.
CERSKUS, A.L., ALI, M., ZAMECNIK, J and MCDONALD, J.W.D. Effects of indomethacin and sulfinpyrazone on in viuo formation of thromboxane B2 and prostaglandin D2 during arachidonate infusion in rabbits. Thromb. Res. l& 549-553,1978.
13.
SILVER, M-J., HOCH, W., KOCSIS, J.J., INGERMAN, C.M. and SMITH, J.B. Arachidonic acid causes sudden death in rabbits. Science. 183 1085-1087, 1974. &
/0il. .
CERSKUS, A.L., ALI, M. and MCDONALD, J.W.D. Thromboxane B2 and 6_ketoPGF,a synthesis during infusion of collagen and arachidonic acid in rabbits: Inhibition by aspirin and sul,finpyrazone. ’ Thrombosis Research. IN PRESS.
_ 15.
ALI, M. and MCDONALD, J.W.D. Synthesis of thromboxane B, and 6-keto-PGF,o by bovine gastric mucosal and muscle microsomes. Prostaglandins. IN PRESS. -_
16.
WHITE, J.G. and WITKOP, C.J. Effects of normal and aspirin platelets on defective secondary aggregation in the Hermansky-Pudlak syndrome. Am. J. Path. 68: 57-66, 1972.
17.
KOCSIS, J.J., HERNANDOVICH, J., SILVER, M.J., SMITH, J.B. and INGERMAN, C Duration of inhibition of platelet prostaqlandin formation and aggregation by ingested aspirin or indomethacin. Prostaqlandins. & 141-144, 1973.
18.
STUART, M.J., MURPHY, S., OSKI, F.A. A simple nonradioisotope technic for the determination of platelet life-span. N. Engl. J. Med. z, 1310-1313, 1975.
19.
RONCUCCI, R., DEPERON, R., LANSEN, ,I., DESTAILLEUR, J., VERHAEGHE, R., DOUMONT, J., LAMBELIN, G., VAN STALLE, F. Nonradioactive semi-automated determination of platelet survival time in man. Thrombosis and Hemostasis. 389, 92, 1977.
20.
VALERI, C.R. and FEINGOLD, H. Hemostatic effectiveness of liquidpreserved platelets stored at 4°C or 22 C and freeze-preserved platelets stored with 5 percent DMSO at -150°C or stored with 6 percent DMSO at -80°C. In: Platelets: Production, Function, Transfusionand Storage. M.G. Baldini and S. Ebbe (Eds.) New York. Grune & Stratton, Inc. 1974. pp. 377-391, 1974.